Bettina M. Jensen

IL-33 induces IL-9 production in human CD4+ T cells and basophils

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4+CD45RA+CD45RO−CD25− T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.

Fast: Towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies

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.

Generation, Isolation, and Maintenance of Human Mast Cells and Mast Cell Lines Derived from Peripheral Blood or Cord Blood

Antigen-mediated mast cell activation is a pivotal step in the initiation of allergic disorders including anaphylaxis and atopy. To date, studies aimed at investigating the mechanisms regulating these responses, and studies designed to identify potential ways to prevent them, have primarily been conducted in rodent mast cells. However, to understand how these responses pertain to human disease, and to investigate and develop novel therapies for the treatment of human mast cell-driven disease, human mast cell models may have greater relevance. Recently, a number of systems have been developed to allow investigators to readily obtain sufficient quantities of human mast cells to conduct these studies. These mast cells release the appropriate suite of inflammatory mediators in response to known mast cell activators including antigen. These systems have also been employed to examine the signaling events regulating these responses. Proof of principle studies has also demonstrated utility of these systems for the identification of potential inhibitors of mast cell activation and growth. In this unit, techniques for the development and culture of human mast cells from their progenitors and the culture of human mast cell lines are described. The relative merits and drawbacks of each model are also described.

Targeting Kit Activation: A Potential Therapeutic Approach in the Treatment of Allergic Inflammation

The prevalence of allergic diseases is increasing worldwide. Hence, there is continued need for novel pharmacological therapies for the treatment of these disorders. As the mast cell is one of the essential cells that contributes to the inflammation associated with allergic diseases, this cell type remains an attractive target for such pharmacological intervention. Mast cells are major players in the early phase of the allergic response since they generate and release a variety of inflammatory mediators following antigen-dependent aggregation of IgE-bound FcepsilonRI (high affinity IgE-receptor) on the cell surface. These mediators also contribute to the late and chronic stages of allergic inflammation. Thus, the IgE/antigen response has been a major focus in the development of new drugs targeting mast cells. The essential role that stem cell factor (SCF) and its receptor, Kit, play in mast cell biology, however, may provide us with an alternative or adjunct therapy. SCF is necessary for mast cell development, proliferation and survival, but it is also known to play a role in homing and adhesion of mast cells. Furthermore, there is an increasing amount of literature demonstrating that SCF is necessary for optimal IgE/antigen-induced mast cell degranulation and cytokine production. Several drug candidates targeting SCF and/or Kit have been studied for their anti-allergic properties. These include anti-SCF antibodies, antisense oligonucleotides, Kit inhibitors, and inhibitors of downstream signaling molecules. In this review, we provide an overview of the role of SCF and Kit in mast cell activation and discuss potential drug candidates for targeting this response.

Concurrent inhibition of Kit- and FcεRI-mediated signaling: Coordinated suppression of mast cell activation

Although primarily required for the growth, differentiation, and survival of mast cells, Kit ligand (stem cell factor) is also required for optimal antigen-mediated mast cell activation. Therefore, concurrent inhibition of Kit- and FcϵRI-mediated signaling would be an attractive approach for targeting mast cell-driven allergic reactions. To explore this concept, we examined the effects of hypothemycin, a molecule that we identified as having such properties, in human and mouse mast cells. Hypothemycin blocked Kit activation and Kit-mediated mast cell adhesion in a similar manner to the well characterized Kit inhibitor imatinib mesylate (imatinib). In contrast to imatinib, however, hypothemycin also effectively inhibited FcϵRI-mediated degranulation and cytokine production in addition to the potentiation of these responses via Kit. The effect of hypothemycin on Kit-mediated responses could be explained by its inhibition of Kit kinase activity, whereas the inhibitory effects on FcϵRI-dependent signaling were at the level of Btk activation. Because hypothemycin also significantly reduced the mouse passive cutaneous anaphylaxis response in vivo, these data provide proof of principle for a coordinated approach for the suppression of mast cell activation and provide a rationale for the development of compounds with a similar therapeutic profile.

Benzoxazinoids: Cereal phytochemicals with putative therapeutic and health-protecting properties.

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, has 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.

Absorption and metabolic fate of bioactive dietary benzoxazinoids in humans

SCOPE Benzoxazinoids, which are natural compounds recently identified in mature whole grain cereals and bakery products, have been suggested to have a range of pharmacological properties and health-protecting effects. There are no published reports concerned with the absorption and metabolism of bioactive benzoxazinoids in humans. METHODS AND RESULTS The absorption, metabolism, and excretion of ten different dietary benzoxazinoids were examined by LC-MS/MS by analyzing plasma and urine from 20 healthy human volunteers after daily intake of 143 μmol of total benzoxazinoids from rye bread and rye buns. The results showed that 2-β-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc) and its oxidized analog, 2-β-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc), were the major circulating benzoxazinoids. After consuming a benzoxazinoid diet for 1 week, morning urine contained eight benzoxazinoids with abundant HBOA-Glc (219 nmol × μmol⁻¹ of creatinine). The sulfate and glucuronide conjugates of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) were detected in plasma and urine, indicating substantial phase II metabolism. Direct absorption of lactam glycosides, the reduction of hydroxamic acid glycosides, glucuronidation, and sulfation were the main mechanisms of the absorption and metabolism of benzoxazinoids. CONCLUSION These results indicate that following ingestion in healthy humans, a range of unmetabolized bioactive dietary benzoxazinoids and their sulfate and glucuronide conjugates appear in circulation and urine.

Molecular and stimulus-response profiles illustrate heterogeneity between peripheral and cord blood-derived human mast cells

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, FcɛRIα, 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.

Authentication of food allergen quality by physicochemical and immunological methods

Purified allergens are required to detect cross‐contamination with other allergenic foods and to understand allergen interaction with other components of the food matrix. Pure allergens are also used for the diagnosis and treatment of food allergies. For example, serological methods are being developed to improve the quality of diagnosis, and to reduce the need for food challenge tests. In addition, recombinant allergens are being evaluated as candidate vaccines for safe and efficacious specific immunotherapy. Pure allergens are indispensable as reference materials for the calibration and standardization of methods between different laboratories and operators for risk assessment in the food industry. Therefore, there is a need for well‐defined purified food allergens. In this context, a panel of 46 food allergens from plant and animal sources has been purified, from either the food sources or as recombinant forms, within the EU‐funded EuroPrevall project. These allergens have been characterized by a battery of diagnostic tests demonstrating that they constitute an authentic, well‐defined library of comparable quality. The review summarizes the applications, potentials and limitations of key techniques used for the characterization and authentication of these allergen preparations, with a special emphasis on protein purity and identity, folding, post‐translational modifications and immunochemical properties. One key area identified is the development of powerful analytical techniques, such as mass spectrometry and nuclear magnetic resonance, to improve the authentication of allergens for routine applications in allergy management.

Dynamics of plasma levels of specific IgE in chlorhexidine allergic patients with and without accidental re-exposure.

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.