Current and future therapies for the treatment of histamine-induced angioedema.

INTRODUCTION: Angioedema, a sudden, self-limited swelling of localized areas of any part of the body that may or may not be associated with urticaria, is thought to be the result of a mast-cell mediated process versus a bradykinin etiology. Understanding the mechanism is key in determining the proper treatment. Areas Covered: Clinical presentation of varying angioedema types may be similar; however, the appropriate treatment algorithm is dependent upon clinicians' knowledge of the underlying pathophysiology and classification of angioedema. Literature review of recent guidelines, available medications, and alternative therapies was completed to provide an overview of options. CONCLUSION: There are no formal guidelines for treatment of acute or chronic histamine-mediated angioedema, and therefore, algorithms for the treatment of acute and chronic urticaria should be followed until such information becomes available. Differentiating histamine-mediated versus bradykinin mediated angioedema is essential, as treatments and treatment responses are quite different. Further research is needed to better understand idiopathic angioedema that is unresponsive to H1/H2 antagonists, LTMAs, or medications designed to treat bradykinin-mediated angioedema.

A designer cell-based histamine-specific human allergy profiler.

Allergic disorders are markedly increasing in industrialized countries. The identification of compounds that trigger the immunoglobulin E-dependent allergic reaction remains the key to limit patients' exposure to critical allergens and improve their quality of life. Here we use synthetic biology principles to design a mammalian cell-based allergy profiler that scores the allergen-triggered release of histamine from whole-blood-derived human basophils. A synthetic signalling cascade engineered within the allergy profiler rewires histamine input to the production of reporter protein, thereby integrating histamine levels in whole-blood samples with remarkable sensitivity and a wide dynamic range, allowing for rapid results or long-term storage of output, respectively. This approach provides non-intrusive allergy profiles for the personalized medicine era.

Adjuvant effects of aluminium hydroxide-adsorbed allergens and allergoids - differences in vivo and in vitro.

Allergen-specific immunotherapy (SIT) is a clinically effective therapy for immunoglobulin (Ig)E-mediated allergic diseases. To reduce the risk of IgE-mediated side effects, chemically modified allergoids have been introduced. Furthermore, adsorbance of allergens to aluminium hydroxide (alum) is widely used to enhance the immune response. The mechanisms behind the adjuvant effect of alum are still not completely understood. In the present study we analysed the effects of alum-adsorbed allergens and allergoids on their immunogenicity in vitro and in vivo and their ability to activate basophils of allergic donors. Human monocyte derived dendritic cells (DC) were incubated with native Phleum pratense or Betula verrucosa allergen extract or formaldehyde- or glutaraldehyde-modified allergoids, adsorbed or unadsorbed to alum. After maturation, DC were co-cultivated with autologous CD4(+) T cells. Allergenicity was tested by leukotriene and histamine release of human basophils. Finally, in-vivo immunogenicity was analysed by IgG production of immunized mice. T cell proliferation as well as interleukin (IL)-4, IL-13, IL-10 and interferon (IFN)-γ production were strongly decreased using glutaraldehyde-modified allergoids, but did not differ between alum-adsorbed allergens or allergoids and the corresponding unadsorbed preparations. Glutaraldehyde modification also led to a decreased leukotriene and histamine release compared to native allergens, being further decreased by adsorption to alum. In vivo, immunogenicity was reduced for allergoids which could be partly restored by adsorption to alum. Our results suggest that adsorption of native allergens or modified allergoids to alum had no consistent adjuvant effect but led to a reduced allergenicity in vitro, while we observed an adjuvant effect regarding IgG production in vivo.

Vigna angularis inhibits mast cell-mediated allergic inflammation.

The aim of the present study was to elucidate whether extracts of Vigna angularis (EVA) inhibit allergic inflammatory reactions and to elucidate the possible mechanisms of action. For the assessment of allergic inflammatory response, histamine release and the expression of pro-inflammatory cytokines from human mast cells (HMC-1) were examined. To identify the underlying mechanisms of action, intracellular calcium and the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) were assayed. To confirm the effects of EVA in vivo, systemic and local allergic reaction mouse models were employed. EVA dose-dependently reduced phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced histamine release from mast cells. The inhibitory effects of EVA on the release of histamine from mast cells were mediated by the reduction of intracellular calcium levels. EVA decreased the PMACI-stimulated gene expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6. The inhibitory effects of EVA on pro-inflammatory cytokines were NF-κB- and MAPK-dependent. In addition, EVA inhibited compound 48/80-induced systemic anaphylaxis and immunoglobulin E (IgE)-mediated cutaneous anaphylaxis. Our findings provide evidence that EVA inhibits mast cell-derived allergic inflammation, and suggest the possible therapeutic application of EVA in allergic inflammatory disorders.

Immunoglobulin E Reactivity and Allergenic Potency of Morus papyrifera (Paper Mulberry) Pollen

Antecedentes: El polen de la morera del papel se considera uno de los aeroalérgenos más relevantes en Pakistán, cuyas propiedades alergénicas no han sido estudiadas hasta el momento actual. Objetivo: El objetivo de este estudio fue caracterizar el perfil de sensibilización de los pacientes alérgicos a las proteínas de este polen que contribuye a la polinosis en Pakistán. Métodos: La extracción de las proteínas de este polen fue realizada mediante diferentes protocolos. La unión de la IgE a proteínas del polen de la morera del papel, perteneciente a la familia de las moráceas fue determinada mediante InmunoCAP e Inmunoblotting utilizando suero de 29 pacientes alérgicos a este polen con prueba cutánea positiva. Se realizó test de liberación de histamina in vitro para determinar la potencia alergénica de los extractos de polen y de un alérgeno parcialmente purificado. Se secuenciaron la N-terminal y MALDI-TOF/TOF para identificar la proteína. Resultados: En cuanto a los resultados obtenidos se confirmó la sensibilización a dicho polen mediante ImmunoCAP frente a polen de Morus alba en 23 de los 29 pacientes alérgicos al polen de morera del papel. Una proteína de 10 kDa del extracto de dicho polen se consideró como el alérgeno mayor sobre el resto de las proteínas reactivas a la IgE. El suero del 79% de los pacientes reaccionó con este alérgeno de 10 kDa, el cual mostró capacidad para liberar histamina in vitro en 3 de 4 pacientes. La secuenciación N-terminal y MALDI-TOF/TOF arrojó una secuencia de aminoácidos con ausencia de homología con otras proteínas conocidas. Conclusiones: En conclusión, los pacientes alérgicos al polen de morera del papel están sensibilizados a múltiples alérgenos de este polen. Se identifica una nueva proteína de 10 kDa como alérgeno mayoritario que deberá ser investigado con fines diagnósticos y terapéuticos (AU)

Background: Paper mulberry (Morus papyrifera) pollen is considered to be one of the most clinically relevant aeroallergens in Pakistan. To date, the allergenicity of the pollen has not been investigated. Objective: To characterize the sensitization profile of mulberry-allergic patients and the proteins of paper mulberry pollen contributing to pollinosis in the Pakistani population. Methods: Proteins were extracted from mulberry pollen using different protocols. Immunoglobulin (Ig) E binding proteins to mulberry pollen was determined by ImmunoCAP testing and immunoblotting using sera from 29 mulberry pollen–allergic patients with positive skin prick test results to mulberry pollen antigens. The histamine release assay was performed in vitro to determine the allergenic potency of pollen extracts and a partially purified mulberry pollen allergen. The protein was identified using N-terminal sequencing and matrix-assisted laser desorption/ionization–time of flight spectrometry (MALDI-TOF/TOF). Results: IgE sensitization to mulberry pollen was confirmed by positive ImmunoCAP results to pollen from Morus alba (white mulberry) in 23 out of 29 mulberry pollen–allergic patients. A 10-kDa protein from the paper mulberry pollen extract was considered a major allergen, along with additional IgE-reactive proteins. Sera from 79% of the patients reacted to the 10-kDa allergen, which showed substantial capacity to trigger histamine release in 3 out of 4 patients. N-terminal sequencing and MALDI-TOF/TOF yielded an amino acid sequence with no homology to known proteins. Conclusions: Mulberry-allergic patients are sensitized to multiple mulberry pollen allergens. We identified a novel 10-kDa protein that was a major allergen and should be further investigated for diagnostic and therapeutic purposes (AU)

Hypoallergenicity and immunological characterization of enzyme-treated royal jelly from Apis mellifera.

Royal jelly (RJ), the exclusive food for queen bees, is taken as a dietary supplement because it is highly rich in nutrients. However, RJ is known to induce an anaphylactic response in some individuals. We evaluated in the present study the hypoallergenicity of alkaline protease-treated RJ in vitro and in vivo. We first confirmed that this treated RJ contained the same levels of vitamins, minerals and specific fatty acid as in untreated RJ. We then showed that the IgE-binding capacity of the treated RJ was very significantly reduced by conducting in vitro assays of the blood from RJ-sensitive patients. An in vivo skin-prick test on the RJ-sensitive patients also showed that, in the majority of the patients (3 out of 4 tested), the treated RJ did not evoke any allergenic response. It is thus advantageous to prepare hypoallergenic RJ by a protease enzyme treatment for its safe consumption.

Blockade of peanut allergy with a novel Ara h 2-Fcγ fusion protein in mice.

BACKGROUND: Conventional immunotherapy for peanut allergy using crude peanut extracts is not recommended because of the unacceptably high risk of anaphylaxis. Allergen-specific immunotherapy is not currently undertaken for peanut allergy. OBJECTIVES: The objective of this study was to develop a novel peanut-human fusion protein to block peanut-induced anaphylaxis. METHODS: We genetically designed and expressed a novel plant-human fusion protein composed of the major peanut allergen Ara h 2 and human IgG Fcγ1. We tested the Ara h 2-Fcγ fusion protein (AHG2)'s function in purified human basophils. Transgenic mice expressing human FcεRIα and a murine peanut allergy model were used. RESULTS: AHG2 inhibited histamine release induced by whole peanut extract (WPE) from basophils of patients with peanut allergy, whereas the fusion protein itself did not induce mediator release. AHG2 inhibited the WPE-induced, peanut-specific, IgE-mediated passive cutaneous anaphylaxis in hFcεRIα transgenic mice. AHG2 also significantly inhibited acute anaphylactic reactivity, including the prototypical decrease in body temperature in WPE-sensitized mice challenged with crude peanut extract. Histologic evaluation of the airways showed that AHG2 decreased peanut-induced inflammation, whereas the fusion protein itself did not induce airway inflammation in peanut-sensitized mice. AHG2 did not exert an inhibitory effect in mice lacking FcγRII. CONCLUSION: AHG2 inhibited peanut-specific IgE-mediated allergic reactions in vitro and in vivo. Linking specific peanut allergen to Fcγ can provide a new approach for the allergen immunotherapy of peanut allergy.

Inhibitory effects of panduratin A on allergy-related mediator production in rat basophilic leukemia mast cells.

Immediate-type hypersensitivity is characterized by elevated levels of immunoglobulin E (IgE) and activated mast cell plays a crucial role by releasing granule contents, lipid-derived mediators, cytokines, and chemokines. To evaluate the antiallergic effects of panduratin A isolated from Boesenbergia pandurata Roxb., we determined its effects on calcium (Ca(2+)) influx, degranulation, and inflammatory mediators in calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA)-stimulated rat basophilic leukemia (RBL-2H3) cells. Panduratin A (20 µM) inhibited secretion of ß-hexosaminidase (46.69 ± 9.6 %), histamine (34.32 ± 2.1 %), and Ca(2+) influx (43.84 %). Panduratin A reduced the production of prostaglandin E(2) (PGE(2), 47.58 ± 3.4 %), leukotriene B(4) (LTB(4), 98.15 ± 1.6 %), and the mRNA expression of cyclooxygenase-2, 5-lipoxygenase, interleukin (IL)-4, IL-13, and tumor necrosis factor-α. Furthermore, panduarin A attenuated phosphorylation of Akt, the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) expression. These results indicate that panduratin A might be useful as an agent against immediate-type hypersensitivity.

Isolation and characterization of a 28 kDa major allergen from blackgram (Phaseolus mungo).

Legumes are the major elicitors of IgE-mediated food allergy in many countries of the world. Purified major allergens are prerequisite for component resolved diagnosis of allergy. The present study was aimed to isolate and characterize a major allergenic protein from blackgram (Phaseolus mungo). Respiratory allergy patients with history of blackgram allergy were skin prick tested (SPT) and sera were collected from SPT positive patients. The blackgram extract was fractionated using a combination of anion exchange and hydrophobic interaction chromatography. The purified protein was characterized by indirect ELISA, immunoblot, ELISA inhibition, SPTs, stripped basophil histamine release, lymphoproliferation assay and digestibility assay. The purified protein separated at 28 kDa on 12% gel and showed IgE binding with 81% of blackgram hypersensitive patients' sera on immunoblot indicating it to be a major allergen. Periodic Acid Schiff's and meta-periodate treatment staining detected it to be a glycoprotein. The 28 kDa protein recognized 7/9 (77.8%) of blackgram positive patients by SPT, where as all 9 patients showed significant histamine release on stimulation with protein as compared to controls. The 28 kDa protein remained stable up to 15 min on incubation with SGF. Bands of 14-16 kDa appeared after 15 min of pepsin digestion that remained stable up to 60 min of incubation. However, purified protein degraded within 5 min after incubation with SIF. The N-terminus-12 residues sequence of 28 kDa protein was GRREDDYDNLQL. A stretch of residues 'DDYDNLQL' showed homology with Rho-specific inhibitor of transcription termination (E=0.42, Identity=87%) and NBS-LRR type disease resistant protein from peanut (Arachis hypogaea) (E=2, Identity=77%). In conclusion, the purified 28 kDa protein is a potent major allergen that may have implication in diagnosis of blackgram allergy.

Pyeongwee-San extract (KMP6): a new anti-allergic effect.

OBJECTIVES: The prevalence of allergic diseases is increasing due to rapid industrialization and changes in lifestyle. Pyeongwee-San (KMP6) is a traditional Korean medicine that has been used as a basic prescription for digestive disorders. This study investigated the efficacy of KMP6 and its component hesperidin on experimental allergic models. METHODS: The anti-allergic effect of KMP6 was studied against a compound 48/80-induced systemic anaphylactic reaction and the ear swelling response. In addition, a human mast cell line (HMC-1) was used to analyze the activity of histidine decarboxylase. Passive cutaneous anaphylaxis (PCA) from immunoglobulin E (IgE) was used. KEY FINDINGS: KMP6 and hesperidin inhibited the compound 48/80-induced systemic anaphylactic reaction and the ear swelling response as well as histamine release, intracellular calcium levels and tryptase release from rat peritoneal mast cells. KMP6 inhibited histidine decarboxylase activity in stimulated HMC-1 cells and macrophages. In addition, KMP6 inhibited the PCA reaction induced by IgE as well as the levels of IgE, interleukin (IL)-4, IL-5, IL-6 and IL-13 in serum from mice. CONCLUSIONS: These results suggest that KMP6 may exert an anti-allergic effect through not only the inhibition of mast cell degranulation but also the inhibition of histamine synthesis.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

Fritillaria ussuriensis extract inhibits the production of inflammatory cytokine and MAPKs in mast cells.

Fritillaria ussuriensis (FU, derived from the bulbs of various species of the genus Fritillaria, including Fritillaria thunbergii Miq.) is used in herbal medicine to treat conditions such as eczema, skin burns, and frostbite. In this study, we investigated the mechanism of the anti-allergy effect of FU. FU extract (80 mg/kg), orally administered to Sprague-Dawley (SD) rats, significantly inhibited the passive cutaneous anaphylaxis (PCA) reaction. It inhibited the compound 48/80-induced release of histamine from rat peritoneal mast cells in a concentration-dependent manner. Significant inhibitory effects of the FU extract on IL-6, IL-8, and TNF-α (1, 10, and 100 µg/mL) were observed in HMC-1 cells. Treatment with FU attenuated PMA plus A23187-induced phosphorylation of all three MAPKs, especially at concentrations of 10 and 100 µg/mL. Further, it (80 mg/kg) led to significant inhibition of mast-cell accumulation in ear tissue at the chronic phase. These results indicate that it inhibits allergic reactions.

Clinical and immunobiochemical characterization of airborne Peltophorum pterocarpum (yellow gulmohar tree) pollen: a dominant avenue tree of India.

BACKGROUND: Peltophorum pterocarpum (yellow gulmohar, PP) pollen is an important aeroallergen for type I hypersensitivity in the tropics. OBJECTIVE: To isolate and characterize the IgE-binding proteins of PP pollen for the first time. METHODS: Pollen extract was fractionated by a combination of Sephacryl S-200 column and diethylaminoethyl-Sephadex column. Allergen characterization was done by sodium dodecyl sulphate polyacrylamide gel electrophoresis, periodic acid-Schiff staining, enzyme-linked immunosorbent assay, and western blotting. Allergenic activities were determined by in vivo (skin prick test) and in vitro (enzyme-linked immunosorbent assay and histamine release) analyses. To determine whether the carbohydrate chains are involved in immunoreactivity, deglycosylation of PP pollen proteins was performed. RESULTS: SPT results on the respiratory allergic patients of Calcutta showed that 32.77% showed positivity with PP pollen. Eight IgE-reactive protein components were found in crude extract. Optimum IgE-reactive fraction 1 was resolved into five subfractions. The subfraction 1a showed maximum IgE reactivity containing the 28 kDa IgE-reactive component. Periodate oxidation showed that protein component was involved in its IgE binding. Twenty-eight kilodalton IgE reactive protein component was recognized by 75% of PP-sensitive patients in Western blotting. It also induced significant histamine release in sensitive patient sera. CONCLUSIONS: The purified 28 kDa protein is a clinically relevant allergen with a potential for diagnosis and therapy of patients susceptible to PP pollen.

Clinopodium gracile inhibits mast cell-mediated allergic inflammation: involvement of calcium and nuclear factor-kappaB.

Mast cell-mediated allergic disease is involved in many diseases such as anaphylaxis, rhinitis, asthma and atopic dermatitis. The discovery of drugs for the treatment of allergic disease is an important subject in human health. In this study, we investigated the effect of the water extract of Clinopodium gracile Matsum var. multicaule (WECG) on the mast cell-mediated allergic inflammation and studied the possible mechanism of action. WECG inhibited compound 48/80-induced systemic anaphylaxis and immunoglobulin E-mediated cutaneous anaphylaxis in a dose-dependent manner. WECG dose-dependently reduced histamine release from rat peritoneal mast cells and human mast cells. The inhibitory effect of WECG on histamine release was mediated by the modulation of intracellular calcium. In addition, WECG attenuated the phorbol 12-myristate 13-acetate and calcium ionophore A23187-stimulated gene expression and secretion of proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-6 in human mast cells. The inhibitory effect of WECG on these proinflammatory cytokines was nuclear factor-kappaB (NF-kappaB) dependent. Our findings provide evidence that WECG inhibits mast cell-derived allergic inflammation and involvement of calcium and NF-kappaB in these effects.

Forsythia suspensa extract alleviates hypersensitivity induced by soybean beta-conglycinin in weaned piglets.

AIM OF THE STUDY: Soybeans are known to stimulate food allergies; however, current therapies are lacking in alleviating hypersensitivity. The present study investigated whether Forsythia suspensa extract could attenuate purified soybean beta-conglycinin-induced anaphylactic reactions in weaned piglets. MATERIALS AND METHODS: Eighteen 14-day-old piglets were sensitized and boosted by oral administration of purified beta-conglycinin. Forsythia suspensa extract was supplemented in the diet after the initial sensitization and continued for the remainder of the experiment. Piglets were challenged on day 28, and anaphylactic symptoms, passive cutaneous anaphylaxis, plasma histamine and intestinal microbial flora were analyzed. T-cell proliferative responses and cytokine production were also determined. RESULTS: Forsythia suspensa extract alleviated beta-conglycinin-induced anaphylactic symptoms by markedly reducing anaphylactic antibodies, mast cell degranulation, and histamine release while improving intestinal microbial flora. Furthermore, Forsythia suspensa extract significantly suppressed beta-conglycinin-induced T lymphocyte proliferation and IL-4 synthesis. CONCLUSION: Forsythia suspensa extract protected beta-conglycinin-sensitized piglets from anaphylactic reactions. Forsythia suspensa extract may provide a novel effective therapy for soybean allergy.

Clinical and immunobiochemical characterization of airborne Delonix regia (Gulmohar tree) pollen and cross-reactivity studies with Peltophorum pterocarpum pollen: 2 dominant avenue trees from eastern India.

BACKGROUND: Delonix regia and Peltophorum pterocarpum pollen are important aeroallergens for type 1 hypersensitivity in the tropics. The IgE-binding proteins of D regia and their cross-allergenity with P pterocarpum pollen have not been evaluated. OBJECTIVES: To isolate and characterize the IgE-binding proteins of D regia pollen for the first time and to investigate the cross-allergenity with P pterocarpum pollen belonging to the same family (Leguminosae). METHODS: Allergenic activities were determined by in vivo and in vitro analyses. Pollen extract was fractionated by a combination of 2 columns (diethyl amino ethyl Sephadex and Sephacryl S-200). Protein components were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, periodic acid-Schiff staining, and immunoblotting. In vitro inhibition tests were performed to evaluate the cross-reactivity. RESULTS: The skin prick test results of the patients with respiratory allergies in Calcutta, India, showed 31.1% positivity with D regia pollen. Nine IgE-reactive protein components were found in the crude extract. An optimum IgE-reactive fraction was resolved into 4 subfractions. Subfraction A, which showed maximum IgE reactivity, contained 2 (96- and 66-kDa) IgE-reactive protein components. The 66-kDa component was found to be glycoprotein. Remarkable cross-reactivity between D regia and P pterocarpum pollen was found on IgE enzyme-linked immunosorbent assay inhibition and dot blotting. Shared IgE-binding components (66, 56, 32, 28, 25, and 23 kDa) were observed between D regia and P pterocarpum pollen extracts, whereas the 96- and 43-kDa components were specific to D regia. CONCLUSION: The purification of the IgE-binding proteins and the identification of the shared/cross-reactive proteins in these taxonomically related pollen members should be helpful for the diagnosis and therapy of patients susceptible to these pollens.

Shikonin inhibits IgE-mediated histamine release by human basophils and Syk kinase activity.

OBJECTIVE: Shikonin, a component of the herbal medicine "Shikon", is known to suppress inflammatory reactions, but its molecular targets are not identified. This study examines the effect of shikonin on human basophil degranulation response and aims to identify its targets. MATERIALS: Human basophils in isolated leukocytes from healthy volunteers' peripheral blood; recombinant human Syk and Lyn tyrosine kinases. METHODS: Histamine release from basophils stimulated with anti-IgE antibody was analyzed fluorimetrically. Syk and Lyn kinase activities were tested in Vitro with recombinant proteins and analyzed by off-chip mobility shift assay. RESULTS: Shikonin dose-dependently inhibited the histamine release from basophils induced by anti-IgE antibody (IC50 = 2.6 +/- 1.0 microM; mean +/- SEM). A search for the target(s) of shikonin in the signal cascade of IgE-mediated activation showed that it strongly inhibits Syk (IC50 = 7.8 microM, in the recombinant kinase assay), which plays a pivotal role in the degranulation response. A less significant inhibition was found for Lyn, which phosphorylates FcepsilonRI-betagamma subunits and also Syk. CONCLUSIONS: These results indicate that the inhibition of Syk-dependent phosphorylation events might underlie the blocked histamine release from human basophils, thus contributing to the anti-inflammatory effects of shikonin.

The anti-allergic activity of the acetate fraction of Schinus terebinthifolius leaves in IgE induced mice paw edema and pleurisy.

Schinus is a genus of the Anacardiaceae family and contains Schinus terebinthifolius, the Brazilian pepper tree that is widely used in folk medicine. We investigate the anti-allergic activity of the ethyl acetate fraction of S. terebinthifolius Raddi (ST fraction). HPLC analysis reveled that gallic acid, methyl gallate and 1,2,3,4,6-pentagalloylglucose are the major aromatic components of the fraction. Oral pre-treatment with the ST fraction (100 mg/kg) significantly inhibited paw edema induced by compound 48/80 (100 ng/paw) and to a lesser extent, the allergic paw edema (OVA, 3 microg/paw). The ST fraction (100 and 200 mg/kg) also inhibited the edema induced by histamine (100 microg/paw), preventing mast cell degranulation and, consequently, histamine release in Wistar rat peritoneal mast cells induced by C 48/80 (5 microg/mL). This histamine inhibition was also observed after mast cell pre-treatment with both methyl gallate and 1,2,3,4,6-pentagalloylglucose (100 microg/mL), the isolated compounds from the ethyl acetate fraction. Pre-treatment with the ST fraction (100 mg/kg) significantly inhibited total leukocyte and eosinophil accumulation in pleural cavities 24 h after the intrathoracic injection of OVA (12.5 microg/cavity). This effect was related to the inhibition of CCL11/eotaxin and CCL5/RANTES in pleural lavage fluid. Pre-treatment with this fraction (100 mg/kg) failed to reduce the cell influx that was observed after LPS-injection into pleural cavity (250 ng/cavity). These findings demonstrate the anti-allergic effect of the ST fraction, which includes the inhibition of edema formation and histamine release caused by mast cell degranulation and eosinophil influx into the pleural cavity probably reflected by the decreased levels of chemokines in recovered pleural lavage fluid.