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Allergy is one of the world's most common chronic conditions. The state of the art method for allergy testing is the skin prick test (SPT) where different allergens are introduced into the skin of the tested person. This method is at best semi- quantitative, a relatively low number of tests can be performed and patients might experience unpleasant reactions. This invention is a novel allergy test providing fast, accurate and quantitative monitoring of allergic reactions in the skin. As a novelty, microneedle-based electrochemical sensor arrays are used to introduce allergens in the skin and perform electrochemical detection of biomarkers released from activated mast cells in the interstitial fluid.

Recruitment to the local tissue and alerted phenotype are the hallmarks of basophils in chronic urticaria (CU). Chemokine receptors such as chemokine (C-C motif) receptor 4 (CCR4) or CCR8 have been studied in skin diseases, e.g., atopic dermatitis, but not in CU. In this study, we aimed to define CU's basophil homing potential and receptor profile and the effect of Omalizumab treatment on these. Unstimulated and activated (anti-IgE, fMLP, C5a, and Substance P) whole blood basophils from 11 Omalizumab-treated CU patients and 10 healthy subjects were investigated with flow cytometry. Unstimulated basophils in CU showed higher expression of the skin-associated (CCR8) and scavenger (CCX-CKR) receptors and lower expression of the lung-associated (CCR3) receptor in contrast to healthy ones. IgE-mediated activation increased the percentage of CCR8 and CCX-CKR in CU compared to healthy group and elevated the expression of the lung-associated chemokine receptor, XCR1, in all groups. A trend of augmented expression of the coagulation cascade (CD87) and fMLP (FPR1) receptors was seen on basophils in CU, while a tendency of reduced expression was seen for itch (IL-31RA) and immunotolerance (CD109) receptors. fMLP and C5a increased the expression of CCR4, CCR8, CCX-CKR, and CD87 and decreased CCR2 and CCR3, though no changes between the groups were found. In conclusion, CU basophils exhibit skin-homing potential amplified by IgE-mediated stimulation.

Background: The basophil histamine release (HR) assay can be used for allergy diagnosis in addition to the conventional measurement of allergen-specific IgE (sIgE). Passive sensitization of basophils increases the versatility and allows testing the biological relevance of allergen-induced IgE cross-linking in any serum unbiased by the cellular component. However, not all the patient sera perform equally well and we hypothesized that the absolute level and fraction of sIgE affect the performance. Choosing birch pollen allergy as a model, we investigated the concentration of sIgE needed for successful passive sensitization using soluble- or matrix-fixed Bet v 1.Methods: Twenty-eight sera with Bet v 1 sIgE [7 sera within each allergy class (1: 0.1-0.70 kUA/L, 2: 0.71-3.50 kUA/L, 3: 3.51-17.50 kUA/L, and 4+: >17.50 kUA/L)] and a negative control serum pool were used to passively sensitize donor basophils, obtained from buffy coat blood ( n = 3). The cells were incubated (30 min) with a soluble allergen (rBet v 1 from 0.2 to 50 ng/ml), matrix-fixed allergen (ImmunoCAP™ containing recombinant Bet v 1), or phorbol 12-myristate 13-acetate (PMA)/ionomycin mixture (maximal HR) and released histamine was quantified fluorometrically. Results: The lowest level of Bet v 1 sIgE generating a detectable HR (HR > 10% of maximal release) in all the 3 runs was found to be 1.25 kUA/L (corresponding to allergy class 2, 0.71-3.50 kUA/L). Furthermore, sera from allergy classes 3 and 4+ ascertained a significant reproducible HR: 42/42 vs. 5/21 in allergy class 1 and 15/21 in allergy class 2. Using ImmunoCAP™s containing Bet v 1 as a matrix-fixed allergen system, similar results were obtained where the lowest sIgE concentration mediating an HR was 1.68 kUA/L and 7/7 for both allergy classes 3 and 4+.Conclusion: The results demonstrate that the IgE titer is strikingly robust in predicting the ability to sensitize basophils and produce a measurable HR.

Background: The basophil histamine release (HR) assay can be used for allergy diagnosis in addition to the conventional measurement of allergen-specific IgE (sIgE). Passive sensitization of basophils increases the versatility and allows testing the biological relevance of allergen-induced IgE cross-linking in any serum unbiased by the cellular component. However, not all the patient sera perform equally well and we hypothesized that the absolute level and fraction of sIgE affect the performance. Choosing birch pollen allergy as a model, we investigated the concentration of sIgE needed for successful passive sensitization using soluble- or matrix-fixed Bet v 1.Methods: Twenty-eight sera with Bet v 1 sIgE [7 sera within each allergy class (1: 0.1-0.70 kUA/L, 2: 0.71-3.50 kUA/L, 3: 3.51-17.50 kUA/L, and 4+: >17.50 kUA/L)] and a negative control serum pool were used to passively sensitize donor basophils, obtained from buffy coat blood ( n = 3). The cells were incubated (30 min) with a soluble allergen (rBet v 1 from 0.2 to 50 ng/ml), matrix-fixed allergen (ImmunoCAP™ containing recombinant Bet v 1), or phorbol 12-myristate 13-acetate (PMA)/ionomycin mixture (maximal HR) and released histamine was quantified fluorometrically. Results: The lowest level of Bet v 1 sIgE generating a detectable HR (HR > 10% of maximal release) in all the 3 runs was found to be 1.25 kUA/L (corresponding to allergy class 2, 0.71-3.50 kUA/L). Furthermore, sera from allergy classes 3 and 4+ ascertained a significant reproducible HR: 42/42 vs. 5/21 in allergy class 1 and 15/21 in allergy class 2. Using ImmunoCAP™s containing Bet v 1 as a matrix-fixed allergen system, similar results were obtained where the lowest sIgE concentration mediating an HR was 1.68 kUA/L and 7/7 for both allergy classes 3 and 4+.Conclusion: The results demonstrate that the IgE titer is strikingly robust in predicting the ability to sensitize basophils and produce a measurable HR.

BACKGROUND: Polyethylene glycols (PEGs) are polymers of varying molecular weight (MW) used widely as excipients in drugs and products, including the mRNA vaccines against COVID-19. Allergy to PEGs is very rare. Skin testing and graded challenge carries a high risk of inducing systemic reactions.OBJECTIVE: To evaluate skin prick test (SPT) and in-vitro reactivity over time to different MW PEGs and assess cross-sensitization patterns in PEG allergy.METHODS: Ten patients with previously diagnosed PEG allergy were skin prick tested twice with PEGs 26 months apart. Lower MW (PEG 300, 3000, 6000) were tested, followed by PEG 20,000 in stepwise, increasing concentrations. Cross-sensitization to polysorbate 80 and poloxamer 407 was assessed. SPT was performed in 16 healthy controls. In vitro Basophil Histamine Release (HR) test and passive sensitization HR test were performed in patients and controls.RESULTS: Patients previously testing positive on SPT to PEG 3000 and/or 6000 also tested positive to PEG 20,000. Patients with longer interval since diagnosis tested negative to lower MW PEGs and positive mainly to higher concentrations of PEG 20,000. Three patients developed systemic urticaria during SPT. Eight patients showed cross-sensitization to poloxamer 407 and three to polysorbate 80. All controls tested negative. In-vitro tests showed limited usefulness.CONCLUSION: Skin test reactivity to PEG can decrease over time, but titrated SPT with increasing concentrations of PEG 20,000 can be diagnostic when lower MW PEGs test negative. To avoid systemic reactions stepwise SPT is mandatory.CLINICAL IMPLICATIONS: An algorithm using a stepwise approach of skin prick testing to PEGs of increasing molecular weights and concentrations can be used to diagnose allergy to PEGs.

BACKGROUND: Polyethylene glycols (PEGs) are polymers of varying molecular weight (MW) used widely as excipients in drugs and products, including the mRNA vaccines against COVID-19. Allergy to PEGs is very rare. Skin testing and graded challenge carries a high risk of inducing systemic reactions.OBJECTIVE: To evaluate skin prick test (SPT) and in-vitro reactivity over time to different MW PEGs and assess cross-sensitization patterns in PEG allergy.METHODS: Ten patients with previously diagnosed PEG allergy were skin prick tested twice with PEGs 26 months apart. Lower MW (PEG 300, 3000, 6000) were tested, followed by PEG 20,000 in stepwise, increasing concentrations. Cross-sensitization to polysorbate 80 and poloxamer 407 was assessed. SPT was performed in 16 healthy controls. In vitro Basophil Histamine Release (HR) test and passive sensitization HR test were performed in patients and controls.RESULTS: Patients previously testing positive on SPT to PEG 3000 and/or 6000 also tested positive to PEG 20,000. Patients with longer interval since diagnosis tested negative to lower MW PEGs and positive mainly to higher concentrations of PEG 20,000. Three patients developed systemic urticaria during SPT. Eight patients showed cross-sensitization to poloxamer 407 and three to polysorbate 80. All controls tested negative. In-vitro tests showed limited usefulness.CONCLUSION: Skin test reactivity to PEG can decrease over time, but titrated SPT with increasing concentrations of PEG 20,000 can be diagnostic when lower MW PEGs test negative. To avoid systemic reactions stepwise SPT is mandatory.CLINICAL IMPLICATIONS: An algorithm using a stepwise approach of skin prick testing to PEGs of increasing molecular weights and concentrations can be used to diagnose allergy to PEGs.

A highly sensitive amperometric Prussian blue-based hydrogen peroxide sensor was developed using 3D pyrolytic carbon microelectrodes. A 3D printed multielectrode electrochemical cell enabled simultaneous highly reproducible Prussian blue modification on multiple carbon electrodes. The effect of oxygen plasma pre-treatment and deposition time on Prussian blue electrodeposition was studied. The amperometric response of 2D and 3D sensors to the addition of hydrogen peroxide in µM and sub-µM concentrations in phosphate buffer was investigated. A high sensitivity comparable to flow injection systems and a detection limit of 0.16 µM was demonstrated with 3D pyrolytic carbon microelectrodes at stirred batch condition.

Human anaphylactic reactions largely involve an increase in vascular permeability, which is mainly controlled by endothelial cells (ECs). Due to the acute and serious nature of human anaphylaxis, in vivo studies of blood vessels must be replaced or supplemented with in vitro models. Therefore, we used a macromolecular tracer assay (MMTA) to investigate the EC permeability of three phenotypes of human ECs: artery (HAECs), vein (HSVECs) and microvessels from lung (HMLECs). ECs were stimulated with two fast-acting anaphylactic mediators (histamine and platelet-activating factor (PAF)) and one longer-lasting mediator (thrombin). At steady-state conditions, HSVEC monolayers were the most permeable and HMLEC the least (15.8% and 8.3% after 60 min, respectively). No response was found in ECs from artery or vein to any stimuli. ECs from microvessels reacted to stimulation with thrombin and also demonstrated a tendency of increased permeability for PAF. There was no reaction for histamine. This was not caused by missing receptor expression, as all three EC phenotypes expressed receptors for both PAF and histamine. The scarce response to fast-acting mediators illustrates that the MMTA is not suitable for investigating EC permeability to anaphylactic mediators.

Human anaphylactic reactions largely involve an increase in vascular permeability, which is mainly controlled by endothelial cells (ECs). Due to the acute and serious nature of human anaphylaxis, in vivo studies of blood vessels must be replaced or supplemented with in vitro models. Therefore, we used a macromolecular tracer assay (MMTA) to investigate the EC permeability of three phenotypes of human ECs: artery (HAECs), vein (HSVECs) and microvessels from lung (HMLECs). ECs were stimulated with two fast-acting anaphylactic mediators (histamine and platelet-activating factor (PAF)) and one longer-lasting mediator (thrombin). At steady-state conditions, HSVEC monolayers were the most permeable and HMLEC the least (15.8% and 8.3% after 60 min, respectively). No response was found in ECs from artery or vein to any stimuli. ECs from microvessels reacted to stimulation with thrombin and also demonstrated a tendency of increased permeability for PAF. There was no reaction for histamine. This was not caused by missing receptor expression, as all three EC phenotypes expressed receptors for both PAF and histamine. The scarce response to fast-acting mediators illustrates that the MMTA is not suitable for investigating EC permeability to anaphylactic mediators.

BACKGROUND: Anaphylaxis includes mast cell (MC) activation, but less is known about downstream mechanisms (ie, vascular permeability controlled by endothelial cells [ECs]). The TNF-like weak inducer of apoptosis (TWEAK) and its sole receptor, fibroblast growth factor-inducible molecule 14 (Fn14), belong to the TNF superfamily and are involved in proinflammatory responses.OBJECTIVE: We sought to investigate the role of TWEAK/Fn14 axis in anaphylaxis.METHODS: In vivo vascular permeability and mouse models of passive systemic anaphylaxis (PSA) and active systemic anaphylaxis were applied to wild-type (WT), TWEAK- and Fn14-deficient mice (TWEAK-/- and Fn14-/-, respectively). Primary bone marrow-derived mast cells (BMMCs) and ECs from WT and Fn14-/- or TWEAK-/- mice were studied. The TWEAK/Fn14 axis was also investigated in human samples.RESULTS: Mice with PSA and active systemic anaphylaxis had increased Fn14 and TWEAK expression in lung tissues and increased serum soluble TWEAK concentrations. TWEAK and Fn14 deficiencies prevent PSA-related symptoms, resulting in resistance to decreased body temperature, less severe reactions, and maintained physical activity. Numbers of MCs after PSA are similar between genotypes in different tissue regions, such as ear skin and the trachea, tongue, peritoneum, lungs, and bone marrow. Moreover, in vitro studies revealed no differences in degranulation or mediator release between WT and Fn14-/- BMMCs after IgE-FcεRI stimulation. In vivo and in vitro histamine and platelet-activating factor administration increases Fn14 receptor expression in lungs and ECs. Moreover, Fn14 deficiency in ECs maintained in vitro impermeability when stimulated by mediators or activated BMMCs but not by TWEAK-/- BMMCs, indicating that Fn14 is crucial for endothelial barrier function. TWEAK/Fn14 deletion or TWEAK-blocking antibody prevented histamine/platelet-activating factor-induced vascular subcutaneous permeability. Circulating sol

Basophils have been suggested to express low quantities of RNA, challenging the study of gene expression within these cells. However, the purification technique employed might have an impact on the quantity and quality of RNA purified from basophils. This chapter describes a method which gives an optimal RNA output using a TRIzol-based method in contrast to a commercial kit.

SCOPE: Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy.METHODS AND RESULTS: Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild-type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long-term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE-binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross-inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate-based adjuvant formulation in a mouse model.CONCLUSIONS: An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.

SCOPE: Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy.METHODS AND RESULTS: Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild-type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long-term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE-binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross-inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate-based adjuvant formulation in a mouse model.CONCLUSIONS: An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.

INTRODUCTION: Mast cells are the primary effector cells of allergy. This study aimed at characterizing human peripheral blood-derived mast cells (PBdMC) from peanut allergic and non-allergic subjects by investigating whether the molecular and stimulus-response profile of PBdMC discriminate between peanut allergic and healthy individuals.METHODS: PBdMC were generated from eight peanut allergic and 10 non-allergic subjects. The molecular profile (cell surface receptor expression) was assessed using flow cytometry. The stimulus-response profile (histamine release induced by secretagogues, secretion of cytokines/chemokines and changes in miRNA expression following anti-IgE activation) was carried out with histamine release test, luminex multiplex assay and miRNA arrays.RESULTS: Expression of activating receptors (FcϵRI, CD48, CD88, CD117, and C3aR) on PBdMC was not different among peanut allergic and non-allergic subjects. Likewise, inhibitory receptors (CD32, CD200R, CD300a, and siglec-8) displayed comparable levels of expression. Both groups of PBdMC were unresponsive to substance P, compound 48/80 and C5a but released comparable levels of histamine when stimulated with anti-IgE and C3a. Interestingly, among the secreted cytokines/chemokines (IL-8, IL-10, IL-13, IL-23, IL-31, IL-37, MCP-1, VEGF, GM-CSF) PBdMC from peanut allergic subjects showed a different secretion pattern of IL-31 compared to non-allergic subjects. Investigating miRNA expression from resting or activated PBdMC revealed no significantly difference between peanut allergic and non-allergic subjects.CONCLUSION: The molecular and stimulus-response profile revealed that PBdMC from peanut allergic subjects differently express IL-31 compared to non-allergic subjects. However, since only one altered parameter was found among 893 investigated, it is still questionable if the pathophysiological mechanisms of peanut allergy are revealed in PBdMC.

Anaphylaxis, the most serious and life-threatening allergic reaction, produces the release of inflammatory mediators by mast cells and basophils. Regulator of calcineurin 1 (Rcan1) is a negative regulator of mast-cell degranulation. The action of mediators leads to vasodilation and an increase in vascular permeability, causing great loss of intravascular volume in a short time. Nevertheless, the molecular basis remains unexplored on the vascular level. We investigated Rcan1 expression induced by histamine, platelet-activating factor (PAF), and epinephrine in primary human vein (HV)-/artery (HA)-derived endothelial cells (ECs) and human dermal microvascular ECs (HMVEC-D). Vascular permeability was analyzedin vitroin human ECs with forced Rcan1 expression using Transwell migration assays andin vivousing Rcan1 knockout mice. Histamine, but neither PAF nor epinephrine, induced Rcan1-4 mRNA and protein expression in primary HV-ECs, HA-ECs, and HMVEC-D through histamine receptor 1 (H1R). These effects were prevented by pharmacological inhibition of calcineurin with cyclosporine A. Moreover, intravenous histamine administration increased Rcan1 expression in lung tissues of mice undergoing experimental anaphylaxis. Functionalin vitroassays showed that overexpression of Rcan1 promotes barrier integrity, suggesting a role played by this molecule in vascular permeability. Consistent with these findings,in vivomodels of subcutaneous and intravenous histamine-mediated fluid extravasation showed increased response in skin, aorta, and lungs ofRcan1-deficient mice compared with wild-type animals. These findings reveal that endothelial Rcan1 is synthesized in response to histamine through a calcineurin-sensitive pathway and may reduce barrier breakdown, thus contributing to the strengthening of the endothelium and resistance to anaphylaxis. These new insights underscore its potential role as a regulator of sensitivity to anaphylaxis in humans.

Anaphylaxis in humans is inherently difficult to study due to the acuteness of symptoms and the lack of biomarkers serving as risk predictors. Most cases are related to IgE sensitizations to foods, insect venoms, and drugs with mastocytosis patients forming a smaller risk group. However, identifying the relatively small fraction of persons at risk has been exceedingly difficult. In this review, we propose to describe anaphylaxis in a broader context than defined by IgE sensitization alone. Exposure to a trigger, such as an allergen, may lead to anaphylaxis, but in particular, the internal dose sensed by the immune system needs to be established. Moreover, intrinsic patient factors as well as the specific circumstances of the exposure, i.e., the extrinsic factors, need to be thoroughly accounted for. More controversially, other triggers of anaphylaxis, such as increased sensitivity to or reduced catabolism of histamine ("histamine intolerance") or mast cell activation syndrome also named mast cell activation disorder have been suggested, but still with very limited epidemiological evidence that a significant proportion of the observed reactions are caused by these alleged conditions. Thus, when all conditions are considered, it seems as if IgE-mediated reactions are responsible for the vast majority of anaphylactic conditions.

PURPOSE: To assess the effect of elevated core body temperature on temporal and spatial contrast sensitivity and retinal vessel diameters.METHODS: The study included 13 healthy volunteers aged 20-37 years. Core body temperature elevation (target +1.1°C) was induced by wrapping the participants in cling film, tinfoil and warming blankets. Subsequent cooling was achieved by undressing. Flicker sensitivity (critical flicker fusion frequency) was chosen to assess temporal resolution, while the Freiburg Vision Test was used to determine spatial contrast sensitivity at 1.5 cycles per degree. Scanning laser ophthalmoscopy was used to measure retinal trunk vessel diameters. Assessment was made at baseline, during hyperthermia and after cooling.RESULTS: The induction of a mean increase in core body temperature of 1.02°C was associated with a 7.15-mmHg mean reduction in systolic blood pressure (p < 0.01), a 10.6-mmHg mean reduction in diastolic blood pressure (p < 0.01), a mean increase in pulse rate of 36.3 bpm (p < 0.0001), a 2.66% improvement in flicker sensitivity (CI95 1.37-3.94, p < 0.001), a 2.80% increase in retinal artery diameters (CI95 1.09-4.51, p < 0.01) and a 2.95% increase in retinal vein diameters (CI95 0.96-4.94, p < 0.01). There was no detectable effect of temperature on spatial contrast sensitivity. All ocular test parameters returned to baseline levels after cooling.CONCLUSION: Increased core body temperature was accompanied by improved temporal visual resolution and retinal trunk vessel dilation. The results suggest that hyperthermia is associated with enhanced retinal function and increased retinal metabolism.

Anaphylaxis in humans is inherently difficult to study due to the acuteness of symptoms and the lack of biomarkers serving as risk predictors. Most cases are related to IgE sensitizations to foods, insect venoms, and drugs with mastocytosis patients forming a smaller risk group. However, identifying the relatively small fraction of persons at risk has been exceedingly difficult. In this review, we propose to describe anaphylaxis in a broader context than defined by IgE sensitization alone. Exposure to a trigger, such as an allergen, may lead to anaphylaxis, but in particular, the internal dose sensed by the immune system needs to be established. Moreover, intrinsic patient factors as well as the specific circumstances of the exposure, i.e., the extrinsic factors, need to be thoroughly accounted for. More controversially, other triggers of anaphylaxis, such as increased sensitivity to or reduced catabolism of histamine ("histamine intolerance") or mast cell activation syndrome also named mast cell activation disorder have been suggested, but still with very limited epidemiological evidence that a significant proportion of the observed reactions are caused by these alleged conditions. Thus, when all conditions are considered, it seems as if IgE-mediated reactions are responsible for the vast majority of anaphylactic conditions.

Anaphylaxis, the most serious and life-threatening allergic reaction, produces the release of inflammatory mediators by mast cells and basophils. Regulator of calcineurin 1 (Rcan1) is a negative regulator of mast-cell degranulation. The action of mediators leads to vasodilation and an increase in vascular permeability, causing great loss of intravascular volume in a short time. Nevertheless, the molecular basis remains unexplored on the vascular level. We investigated Rcan1 expression induced by histamine, platelet-activating factor (PAF), and epinephrine in primary human vein (HV)-/artery (HA)-derived endothelial cells (ECs) and human dermal microvascular ECs (HMVEC-D). Vascular permeability was analyzedin vitroin human ECs with forced Rcan1 expression using Transwell migration assays andin vivousing Rcan1 knockout mice. Histamine, but neither PAF nor epinephrine, induced Rcan1-4 mRNA and protein expression in primary HV-ECs, HA-ECs, and HMVEC-D through histamine receptor 1 (H1R). These effects were prevented by pharmacological inhibition of calcineurin with cyclosporine A. Moreover, intravenous histamine administration increased Rcan1 expression in lung tissues of mice undergoing experimental anaphylaxis. Functionalin vitroassays showed that overexpression of Rcan1 promotes barrier integrity, suggesting a role played by this molecule in vascular permeability. Consistent with these findings,in vivomodels of subcutaneous and intravenous histamine-mediated fluid extravasation showed increased response in skin, aorta, and lungs ofRcan1-deficient mice compared with wild-type animals. These findings reveal that endothelial Rcan1 is synthesized in response to histamine through a calcineurin-sensitive pathway and may reduce barrier breakdown, thus contributing to the strengthening of the endothelium and resistance to anaphylaxis. These new insights underscore its potential role as a regulator of sensitivity to anaphylaxis in humans.

Diamine oxidase (DAO) is the main histamine metabolizing enzyme performing its function extracellularly. Its activity can be affected by multiple factors like the presence of enzyme inhibitors, lack of enzyme cofactors or existing intestinal damage. Low DAO activity can lead to histamine accumulation in the organism that may cause detrimental histamine-mediated allergy-like symptoms. Histaminosis describes this state of imbalance between histamine and the capacity of histamine metabolizing enzymes to reduce histamine levels. DAO belongs to the family of copper-containing amine oxidases (AOCs) and is encoded by the AOC1 gene. DAO is expressed mainly by kidney and intestinal epithelial cells. High levels of DAO can be detected in placenta during pregnancy. Eosinophils, Neutrophils and Platelets have been reported to possess histaminase activity, though only very few studies have researched this more. Other AOCs belonging to this family are Retina specific amine oxidase encoded by AOC2 gene and Vascular adhesion protein encoded by AOC3. It is unknown whether the native form of these proteins can metabolize histamine as well and therefore could be contributing to histaminase activity in the circulation as well. In this study we challenged previous findings and tested the presence of AOC1, AOC2 and AOC3 on mRNA and protein levels in the blood, zooming in on different blood cell types step by step. We also analyzed plasma and serum DAO concentrations from various donors and analyzed DAO activity in Platelets. Analysis of DAO concentration is serum and plasma showed that, when the blood was drawn into heparin blood tubes, DAO levels from the same individual were twice as higher as when the blood was drawn into EDTA or serum tubes. We also demonstrated that AOC1 mRNA was mainly contained in Granulocytes, particularly in Eosinophils. Protein analysis showed that Platelets seemed to comprise large amounts of DAO. mRNA analysis of AOC2 and AOC3 demonstrated

Diamine oxidase (DAO) is the main histamine metabolizing enzyme performing its function extracellularly. Its activity can be affected by multiple factors like the presence of enzyme inhibitors, lack of enzyme cofactors or existing intestinal damage. Low DAO activity can lead to histamine accumulation in the organism that may cause detrimental histamine-mediated allergy-like symptoms. Histaminosis describes this state of imbalance between histamine and the capacity of histamine metabolizing enzymes to reduce histamine levels. DAO belongs to the family of copper-containing amine oxidases (AOCs) and is encoded by the AOC1 gene. DAO is expressed mainly by kidney and intestinal epithelial cells. High levels of DAO can be detected in placenta during pregnancy. Eosinophils, Neutrophils and Platelets have been reported to possess histaminase activity, though only very few studies have researched this more. Other AOCs belonging to this family are Retina specific amine oxidase encoded by AOC2 gene and Vascular adhesion protein encoded by AOC3. It is unknown whether the native form of these proteins can metabolize histamine as well and therefore could be contributing to histaminase activity in the circulation as well. In this study we challenged previous findings and tested the presence of AOC1, AOC2 and AOC3 on mRNA and protein levels in the blood, zooming in on different blood cell types step by step. We also analyzed plasma and serum DAO concentrations from various donors and analyzed DAO activity in Platelets. Analysis of DAO concentration is serum and plasma showed that, when the blood was drawn into heparin blood tubes, DAO levels from the same individual were twice as higher as when the blood was drawn into EDTA or serum tubes. We also demonstrated that AOC1 mRNA was mainly contained in Granulocytes, particularly in Eosinophils. Protein analysis showed that Platelets seemed to comprise large amounts of DAO. mRNA analysis of AOC2 and AOC3 demonstrated

BACKGROUND: Chlorhexidine is an effective disinfectant, which may cause severe allergic reactions. Plasma level of specific IgE to chlorhexidine (ImmunoCAP(®) ) has high estimated sensitivity and specificity when measured within 6 months of allergic reaction, but knowledge of the dynamics over longer time periods is lacking and it is unknown whether levels fall below <0.35 kUA/L in patients with previously elevated levels. It is also unclear whether re-exposure influences levels of specific IgE.OBJECTIVE: To investigate the dynamics of specific IgE in chlorhexidine allergic patients with and without re-exposure.METHODS: All patients diagnosed with chlorhexidine allergy in the Danish Anaesthesia Allergy Centre January 1999 to March 2015 were invited to participate. The study included blood samples from the time of reaction and time of investigation and blood samples drawn prospectively over several years.RESULTS: Overall, 23 patients were included. Specific IgE within hours of reaction was available in eight patients and was >0.35 kUA/L in six of these. During allergy investigations, usually 2-4 months later, specific IgE was >0.35 kUA/L in 22 of 23 patients. In the following months/years specific IgE declined <0.35 kUA/L in 17 of 23 patients (most rapidly within 4 months). Nine re-exposures in the healthcare setting were reported by seven patients (35%). Most re-exposures caused symptoms and were followed by an increase in specific IgE. Two patients with specific IgE <0.35 kUA/L reacted upon re-exposure.CONCLUSIONS & CLINICAL RELEVANCE: Time from reaction should be considered when interpreting specific IgE results. Specific IgE is >0.35 kUA/L in most patients at time of reaction but should be repeated after a few weeks/months if negative. The optimal sampling time seems to be >1 month and <4 months. A value <0.35 kUA/L neither excludes allergy nor implies loss of reactivity in previously sensitized patien

Mast cells (MCs) are one of the key players in allergy and have been linked to many other pathological conditions. They reside in tissues throughout the body, from where they elicit their functions. MCs are a very heterogeneous population of cells with a unique activation profile. They are highly granulated and release a high variety of mediators, such as histamine, that causes different responses in the body, including bronchoconstriction and increased vascular permeability. MCs differentiate from CD34+ progenitor cells and mature in the tissue they home to, highly influenced by their surrounding microenvironment. This causes high heterogeneity among MCs located in different tissues. MCs have histologically been found in the human blood vessel wall, but both their function and profile here are unknown. Due to their location they have been speculated to be involved in anaphylaxis which is the most severe allergic reaction. However, the function of MCs in initiation and spread of the response remains unknown. In order to investigate this possible role of blood vessel MCs, they first have to be isolated and characterized. The aim of this thesis was to characterize MCs in human saphenous veins (HSVMC) both by histology, by isolation and by molecular and functional profiling. Two in vitro MC cultures, PBdMC and LAD2, were also profiled as possible models for future studies of HSVMC. The isolation protocol was developed by testing many different parameters and by evaluating the isolated HSVMC on the basis of morphological appearance and histamine content. The final protocol had a high number of morphologically round HSVMC with clear, visible granules. The isolated HSVMC were easily identified by flow cytometry as CD117+FcɛRI+ and demonstrated surprisingly high histamine content compared to both LAD2 and PBdMC. However, both flow cytometry and histamine release data indicated that HSVMC ha

Mast cells (MCs) are one of the key players in allergy and have been linked to many other pathological conditions. They reside in tissues throughout the body, from where they elicit their functions. MCs are a very heterogeneous population of cells with a unique activation profile. They are highly granulated and release a high variety of mediators, such as histamine, that causes different responses in the body, including bronchoconstriction and increased vascular permeability. MCs differentiate from CD34+ progenitor cells and mature in the tissue they home to, highly influenced by their surrounding microenvironment. This causes high heterogeneity among MCs located in different tissues. MCs have histologically been found in the human blood vessel wall, but both their function and profile here are unknown. Due to their location they have been speculated to be involved in anaphylaxis which is the most severe allergic reaction. However, the function of MCs in initiation and spread of the response remains unknown. In order to investigate this possible role of blood vessel MCs, they first have to be isolated and characterized. The aim of this thesis was to characterize MCs in human saphenous veins (HSVMC) both by histology, by isolation and by molecular and functional profiling. Two in vitro MC cultures, PBdMC and LAD2, were also profiled as possible models for future studies of HSVMC. The isolation protocol was developed by testing many different parameters and by evaluating the isolated HSVMC on the basis of morphological appearance and histamine content. The final protocol had a high number of morphologically round HSVMC with clear, visible granules. The isolated HSVMC were easily identified by flow cytometry as CD117+FcɛRI+ and demonstrated surprisingly high histamine content compared to both LAD2 and PBdMC. However, both flow cytometry and histamine release data indicated that HSVMC ha

The grey matter of the human brain is asymmetrically distributed between the cerebral hemispheres. This asymmetry includes visual areas, but its relevance to visual function is not understood. Voxel-based morphometry is a well-established technique for localization and quantification of cerebral grey matter on the basis of MR images. This method has been used previously for interhemispheric comparison, but without examining the functional implications of the identified anatomical asymmetries of the visual system. The aim of the present study was to relate anatomical interhemispheric asymmetries to asymmetries of visual function. We examined grey matter asymmetries of visual areas in a large population (n=56) of ophthalmologically and neurologically healthy individuals. We used state-of-the-art 3 T MRI and voxel-based morphometry to relate the visual parameters, (a) ocular dominance, (b) interocular difference in visual acuity and (c) visual attention (i.e. deviation on a line-bisection task), to interhemispheric differences in grey matter volume. As most visual input from one eye is processed in the contralateral hemisphere, ocular features may also depend on cerebral lateralization. Several lateralized visual areas were identified, both right>left and left>right. When correlating the asymmetries to the functional parameters, we found a significant correlation to ocular dominance (P<0.05), whereas visual acuity and visual attention showed no such relationship. The lateral occipital complex was identified to be significantly larger in the left hemisphere for right-eyed participants and vice versa. These results suggest a cerebral basis for ocular dominance.

Hemispheric asymmetry of a wide range of functions is a hallmark of the human brain. The visual system has traditionally been thought of as symmetrically distributed in the brain, but a growing body of evidence has challenged this view. Some highly specific visual tasks have been shown to depend on hemispheric specialization. However, the possible lateralization of cerebral responses to a simple checkerboard visual stimulation has not been a focus of previous studies. To investigate this, we performed two sessions of blood-oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) in 54 healthy subjects during stimulation with a black and white checkerboard visual stimulus. While carefully excluding possible non-physiological causes of left-to-right bias, we compared the activation of the left and the right cerebral hemispheres and related this to grey matter volume, handedness, age, gender, ocular dominance, interocular difference in visual acuity, as well as line-bisection performance. We found a general lateralization of cerebral activation towards the right hemisphere of early visual cortical areas and areas of higher-level visual processing, involved in visuospatial attention, especially in top-down (i.e., goal-oriented) attentional processing. This right hemisphere lateralization was partly, but not completely, explained by an increased grey matter volume in the right hemisphere of the early visual areas. Difference in activation of the superior parietal lobule was correlated with subject age, suggesting a shift towards the left hemisphere with increasing age. Our findings suggest a right-hemispheric dominance of these areas, which could lend support to the generally observed leftward visual attentional bias and to the left hemifield advantage for some visual perception tasks., Hemispheric asymmetry of a wide range of functions is a hallmark of the human brain. The visual system has traditionally been thought of as symmetrically distributed in the brain, but a growing body of evidence has challenged this view. Some highly specific visual tasks have been shown to depend on hemispheric specialization. However, the possible lateralization of cerebral responses to a simple checkerboard visual stimulation has not been a focus of previous studies. To investigate this, we performed two sessions of blood-oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) in 54 healthy subjects during stimulation with a black and white checkerboard visual stimulus. While carefully excluding possible non-physiological causes of left-to-right bias, we compared the activation of the left and the right cerebral hemispheres and related this to grey matter volume, handedness, age, gender, ocular dominance, interocular difference in visual acuity, as well as line-bisection performance. We found a general lateralization of cerebral activation towards the right hemisphere of early visual cortical areas and areas of higher-level visual processing, involved in visuospatial attention, especially in top-down (i.e., goal-oriented) attentional processing. This right hemisphere lateralization was partly, but not completely, explained by an increased grey matter volume in the right hemisphere of the early visual areas. Difference in activation of the superior parietal lobule was correlated with subject age, suggesting a shift towards the left hemisphere with increasing age. Our findings suggest a right-hemispheric dominance of these areas, which could lend support to the generally observed leftward visual attentional bias and to the left hemifield advantage for some visual perception tasks.

BACKGROUND: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1.OBJECTIVES: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1.METHODS: Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product.RESULTS: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability.CONCLUSION: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

Benzoxazinoids (BXs) are a group of natural chemical compounds with putative pharmacological and health-protecting properties. BXs were formerly identified in and isolated from selected dicot medicinal plants and young cereal plants. Recently, BXs were found to be present in mature cereal grains and bakery products, such that knowledge about the pharmacological properties of BXs, which until now have unknowingly been consumed through the daily bread and breakfast cereals, have come into new focus. This review discusses published results from in vitro studies and a few human and animal model studies on the health effects and pharmacological responses of various BX compounds. Many of these studies have reported antimicrobial, anticancer, reproductive system stimulatory, central nervous system stimulatory, immunoregulatory, and appetite- and weight-reducing effects of BXs and/or BX derivatives. The health benefits of wholegrain intake may be associated with the solitary and/or overlapping biological effects of fibers, lignans, phenolic acids, alkylresorcinols, BXs, and other bioactive compounds. In the context of BXs as dietary ingredients, further comprehensive investigations are required to understand their biological functions, to elucidate the underlying mechanisms, to explore their potential contribution on the health effects associated with wholegrain consumption, and to examine their potential as functional food ingredients.

Different protocols exist for in vitro development of HuMCs from hematopoietic stem cells, which results in distinct mast cells regarding molecular markers and activation patterns. Here, we introduce a SR profile using immunological, neurogenic, and pharmacological stimuli to characterize cellular functionality. Mast cells were obtained from three culture protocols using two types of PBdMCs (CD34(+) PBdMC or CD133(+) PBdMC) and one type of CBdMC (CD133(+) CBdMC). We analyzed resting cells for specific mast cell markers at protein and mRNA levels, thereby creating a molecular profile. To characterize the SR profile, we stimulated cells with anti-IgE, C3a, C5a, Substance P, or Compound 48/80 and measured the release of histamine and cytokines (IL-10, IL-13, GM-CSF, TNF-α). Molecular profiling revealed that CD133(+) CBdMC expressed less chymase, FcεRIα, and CD203c but more CD117 compared with CD34(+) and CD133(+) PBdMC. The SR profile for histamine release illustrated a functional heterogeneity between PBdMC and CBdMC. PBdMC released >10% histamine upon stimulation with anti-IgE, C3a, Substance P, and Compound 48/80, whereas CBdMC only reacted to C3a. Cytokine secretion was only detected after anti-IgE stimulation. Here, the SR profile identified the CD133(+) PBdMC as the most active cells regarding secretion of IL-10, IL-13, GM-CSF, and TNF-α. Cells from all three culture protocols, however, produced IL-10 spontaneously at comparable levels. We recommend validating mast cell cultures by means of molecular and SR profiles to characterize the mast cells and enhance consensus among studies.

Soya and peanut are botanically closely related and share cross-reacting antigens, but compared to soya, peanut allergy has a higher prevalence with more severe allergic reactions. Furthermore, the threshold dose for eliciting reactions is higher for soya. A difference in undigested protein absorption between the two foods, might explain this diversity.In the current study the amount of soya protein absorbed after soya bean ingestion in healthy adults was estimated. Ten subjects ingested 100 grams of soya beans (40 grams of soya protein) and blood was drawn before and 1, 3 and 24 hours after administration. Serum was analysed by ELISA and histamine release (HR). In all serum samples the soya protein concentration was below quantification limit (1.6 ng/ml which corresponds to 4.8 mug or 0.12 parts per million absorbed soya protein.We could not detect any significant absorption of soya protein. While we cannot totally exlude technical reasons, it may also reflect a true poor absorption in healthy adult volunteers. This could, in turn, be relevant to the apparently weak allergenicity of soy protein by comparison with peanut protein in allergic subjects.

Soya and peanut are botanically closely related and share cross-reacting antigens, but compared to soya, peanut allergy has a higher prevalence with more severe allergic reactions. Furthermore, the threshold dose for eliciting reactions is higher for soya. A difference in undigested protein absorption between the two foods, might explain this diversity.In the current study the amount of soya protein absorbed after soya bean ingestion in healthy adults was estimated. Ten subjects ingested 100 grams of soya beans (40 grams of soya protein) and blood was drawn before and 1, 3 and 24 hours after administration. Serum was analysed by ELISA and histamine release (HR). In all serum samples the soya protein concentration was below quantification limit (1.6 ng/ml which corresponds to 4.8 mug or 0.12 parts per million absorbed soya protein.We could not detect any significant absorption of soya protein. While we cannot totally exlude technical reasons, it may also reflect a true poor absorption in healthy adult volunteers. This could, in turn, be relevant to the apparently weak allergenicity of soy protein by comparison with peanut protein in allergic subjects.

BACKGROUND. IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-?), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF-?/MMP-9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE-mediated activation of human basophils induces TNF-? and MMP-9 release. METHODS. Human peripheral blood mononuclear cells (PBMC), isolated basophils and monocytes were stimulated up to 21 h with anti-IgE. Mediator releases were assessed by ELISA, and surface expressions of mediators were detected by flow cytometry. Upregulation of cytokine production was detected by Western blot and polymerase chain reaction (PCR). RESULTS. IgE-mediated activation of basophils induced the synthesis and release of both TNF-? and MMP-9 from PBMC. In contrast, IgE-mediated activation of purified basophils induced the release and cellular expression of TNF-? but not MMP-9. Isolated monocytes did not release MMP-9 upon anti-IgE stimulation, but MMP-9 release was induced by stimulating monocytes with supernatants from activated basophils, and this release was inhibited by anti-TNF-? neutralizing antibodies. CONCLUSION. Our results strongly indicate that human basophils release TNF-? following IgE-dependent activation and that this cytokine subsequently stimulates MMP-9 release from monocytes. These findings support a direct involvement of basophils in inflammation as well as suggesting a role for the basophil in tissue remodelling.

BACKGROUND. IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-?), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF-?/MMP-9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE-mediated activation of human basophils induces TNF-? and MMP-9 release. METHODS. Human peripheral blood mononuclear cells (PBMC), isolated basophils and monocytes were stimulated up to 21 h with anti-IgE. Mediator releases were assessed by ELISA, and surface expressions of mediators were detected by flow cytometry. Upregulation of cytokine production was detected by Western blot and polymerase chain reaction (PCR). RESULTS. IgE-mediated activation of basophils induced the synthesis and release of both TNF-? and MMP-9 from PBMC. In contrast, IgE-mediated activation of purified basophils induced the release and cellular expression of TNF-? but not MMP-9. Isolated monocytes did not release MMP-9 upon anti-IgE stimulation, but MMP-9 release was induced by stimulating monocytes with supernatants from activated basophils, and this release was inhibited by anti-TNF-? neutralizing antibodies. CONCLUSION. Our results strongly indicate that human basophils release TNF-? following IgE-dependent activation and that this cytokine subsequently stimulates MMP-9 release from monocytes. These findings support a direct involvement of basophils in inflammation as well as suggesting a role for the basophil in tissue remodelling.

A debate has recently arisen as to whether murine basophils can function as antigen presenting cells in allergic inflammation. However, mouse and human basophils differ considerably, and the expression of MHC class II on human basophils has been investigated as a proxy for their capability of antigen presentation but conflicting results have emerged. In this technical note, we show that an antibody specific for all three MHC class II subtypes (HLA-DR, -DP, and -DQ), leads to a significantly higher amount of MHC class II+ basophils compared to antibodies specific for HLA-DR only. A significant difference was also observed between the HLA-DR specific antibodies, indicating that the choice of antibody is crucial. Furthermore, critical compensation was essential to avoid false HLA-DR+ basophils. Finally, we found that detection of MHC class II on human basophils was independent of atopic disposition.

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

Background IL-9 has been suggested to play an important role in chronic inflammation due to its effect on remodeling and cell recruitment. Since basophils can be found in inflamed tissue, we wanted to test if human basophils are capable of secreting IL-9 under the influence of growth or tissue factors like IL-3 or IL-33, respectively. Methods Basophils, purified from buffy coat blood by negative selection (purity > 95%), were cultured for 24 hours in RPMI culture medium (1x106 cells/ml) supplemented with 5% human serum, and IL-3 (10 ng/ml) and/or IL-33 (50 ng/ml). The cells were then analysed by FACS for ST2 expression where basophils were gated as FceRIa+CD3-CD14-CD19-CD56-. The supernatant was tested for IL-9 using a multiplex magnetic bead assay. Results Human basophils have recently been described to express the IL-33 receptor ST2. In agreement with previous findings, our results showed increased surface expression of ST2 on basophils stimulated with IL-3 for 24 h, whereas IL-33 stimulation did not influence surface expression of its receptor. Nevertheless, basophils stimulated with IL-33 released IL-9 at a level comparable to what was found when stimulating with IL-3. Moreover, there was a marked synergy between IL-3 and IL-33 on the IL-9 release. Conclusion To our knowledge, this is the first report that human basophils secrete IL-9. A strong synergy between IL-3 and IL-33 was found on the IL-9 induction, likely to be explained by the IL-3 upregulated ST2 expression.

Background Human basophils are granulocytes with the capacity to play important roles in allergy for example by releasing histamine when activated by cross-linking of their high affinity IgE receptors (Fc¿RI). However, not all individuals have basophils responding with a histamine release after such activation and are therefore called nonreleasers. Intracellularly these basophils differ from responding cells by having a low level of the tyrosine kinase Syk, but studies of their surface profile are lacking. We have investigated the expression of Fc¿RI, HLA-DR, CD16, CD63, CD69, CD117, CD123, CD124, CD203c, C3aR, C5aR CCR3, FPR1, ST2, CRTH2 on anti-IgE respondsive and nonreleasing basophils by flow cytometry, thereby generating a surface profile of the two phenotypes. Methods Fresh buffy coat blood (< 6h) were screened for anti-IgE mediated histamine release and nonreleases were defined as having a maximum release < 12.7%. Periphereal blood mononuclear cells were purified by density centrifugation and using flow cytometry, basophils, defined as FceRIa+CD3-CD14-CD19-CD56-,were analysed for surface expression of relevant markers. All samples were compensated and analysed in logicle display. All gates were set on FMO (fluorescence minus one) controls and results are given in geoMFI normalised to FMO control. Releaser and nonreleasers were compared using unpaired t test with Welch’s correction. Results Twelve donors were included, three of which were nonreleasers and characterized in regard to surface expression of Fc¿RI, HLA-DR, CD16, CD63, CD69, CD117, CD123, CD124, CD203c, C3aR, C5aR CCR3, FPR1, ST2 and CRTH2. No significant difference was observed between normal and nonresponding basophils with any of the investigated receptors, even though a trend was found with CD203c indicating a lower expression on nonreleasers (p = 0.0609). Conclusion Surface profiling using 15 different receptors, illustrated n

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

Background IL-9 has been suggested to play an important role in chronic inflammation due to its effect on remodeling and cell recruitment. Since basophils can be found in inflamed tissue, we wanted to test if human basophils are capable of secreting IL-9 under the influence of growth or tissue factors like IL-3 or IL-33, respectively. Methods Basophils, purified from buffy coat blood by negative selection (purity > 95%), were cultured for 24 hours in RPMI culture medium (1x106 cells/ml) supplemented with 5% human serum, and IL-3 (10 ng/ml) and/or IL-33 (50 ng/ml). The cells were then analysed by FACS for ST2 expression where basophils were gated as FceRIa+CD3-CD14-CD19-CD56-. The supernatant was tested for IL-9 using a multiplex magnetic bead assay. Results Human basophils have recently been described to express the IL-33 receptor ST2. In agreement with previous findings, our results showed increased surface expression of ST2 on basophils stimulated with IL-3 for 24 h, whereas IL-33 stimulation did not influence surface expression of its receptor. Nevertheless, basophils stimulated with IL-33 released IL-9 at a level comparable to what was found when stimulating with IL-3. Moreover, there was a marked synergy between IL-3 and IL-33 on the IL-9 release. Conclusion To our knowledge, this is the first report that human basophils secrete IL-9. A strong synergy between IL-3 and IL-33 was found on the IL-9 induction, likely to be explained by the IL-3 upregulated ST2 expression.

Background Human basophils are granulocytes with the capacity to play important roles in allergy for example by releasing histamine when activated by cross-linking of their high affinity IgE receptors (Fc¿RI). However, not all individuals have basophils responding with a histamine release after such activation and are therefore called nonreleasers. Intracellularly these basophils differ from responding cells by having a low level of the tyrosine kinase Syk, but studies of their surface profile are lacking. We have investigated the expression of Fc¿RI, HLA-DR, CD16, CD63, CD69, CD117, CD123, CD124, CD203c, C3aR, C5aR CCR3, FPR1, ST2, CRTH2 on anti-IgE respondsive and nonreleasing basophils by flow cytometry, thereby generating a surface profile of the two phenotypes. Methods Fresh buffy coat blood (< 6h) were screened for anti-IgE mediated histamine release and nonreleases were defined as having a maximum release < 12.7%. Periphereal blood mononuclear cells were purified by density centrifugation and using flow cytometry, basophils, defined as FceRIa+CD3-CD14-CD19-CD56-,were analysed for surface expression of relevant markers. All samples were compensated and analysed in logicle display. All gates were set on FMO (fluorescence minus one) controls and results are given in geoMFI normalised to FMO control. Releaser and nonreleasers were compared using unpaired t test with Welch’s correction. Results Twelve donors were included, three of which were nonreleasers and characterized in regard to surface expression of Fc¿RI, HLA-DR, CD16, CD63, CD69, CD117, CD123, CD124, CD203c, C3aR, C5aR CCR3, FPR1, ST2 and CRTH2. No significant difference was observed between normal and nonresponding basophils with any of the investigated receptors, even though a trend was found with CD203c indicating a lower expression on nonreleasers (p = 0.0609). Conclusion Surface profiling using 15 different receptors, illustrated n