Equine CD4(+) CD25(high) T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro

Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4(+) Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4(+) CD25(+) T cells, mainly in the CD4(+) CD25(high) subpopulation. Proliferation of CD4(+) CD25(-) sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co-culture with CD4(+) CD25(high) sorted cells in a dose-dependent manner. The mechanism of suppression by the CD4(+) CD25(high) cell population is mediated by close contact as well as interleukin (IL)-10 and transforming growth factor-beta1 (TGF-beta1) and probably other factors. In addition, we studied the in vitro induction of CD4(+) Treg and their characteristics compared to those of freshly isolated CD4(+) Treg cells. Upon stimulation with a combination of concanavalin A, TGF-beta1 and IL-2, CD4(+) CD25(+) T cells which express FoxP3 and have suppressive capability were induced from CD4(+) CD25(-) cells. The induced CD4(+) CD25(high) express higher levels of IL-10 and TGF-beta1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4(+) CD25(high) subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4(+) CD25(+) cells in horses and offers insights into ex vivo manipulation of Treg cells.

Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.

Molecular cloning and characterization of equine thymic stromal lymphopoietin

Thymic stromal lymphopoietin (TSLP) is a novel cytokine that plays a central role in T helper 2 (Th2) cell differentiation and allergic inflammation. It is predominantly expressed by epithelial cells, and its expression is increased in patients with atopic dermatitis and asthma. Mice overexpressing TSLP in the skin develop allergic dermatitis and mice overexpressing TSLP in lungs develop asthma-like disease. However, it is not known whether TSLP plays an important role in equine allergies. Therefore, we cloned and sequenced the complete translated region of equine TSLP gene and measured its expression in various tissues. The equine TSLP gene is organized in 4 exons and encodes a protein of 143 amino acids, which has 62% amino acid identity with human TSLP.

Update in clinical allergy and immunology

In the recent years, a tremendous body of studies has addressed a broad variety of distinct topics in clinical allergy and immunology. In this update, we discuss selected recent data that provide clinically and pathogenetically relevant insights or identify potential novel targets and strategies for therapy. The role of the microbiome in shaping allergic immune responses and molecular, as well as cellular mechanisms of disease, is discussed separately and in the context of atopic dermatitis, as an allergic model disease. Besides summarizing novel evidence, this update highlights current areas of uncertainties and debates that, as we hope, shall stimulate scientific discussions and research activities in the field.

Granulocyte death regulation by naturally occurring autoantibodies

Programmed cell death (PCD) plays a central role in the regulation of granulocytes that are key effector cells of the innate immune system. Granulocytes are produced in high amounts in the bone marrow. A safe elimination of granulocytes by cell death (apoptosis) is essential to maintain the numbers of these cells balanced. In many acute and chronic inflammatory diseases, delayed apoptosis is one mechanism that contributes to accumulation of neutrophil and eosinophil granulocytes at the site of inflammation. On the other hand, a safe elimination of granulocytes by cell death is required to avoid unwanted tissue damage for instance by secretion of toxic products from these cells. Recent evidence shows that humans produce an array of naturally occurring autoantibodies (NAbs) with the capacity to regulate granulocyte death, including agonistic and antagonistic NAbs that bind to the receptors Fas, Siglec-8, and Siglec-9. Together with other factors, these various NAbs exhibit different properties in terms of the form of cell death they induce, the molecular signaling pathways they engage, as well as the efficacy or potency by which they induce cell death. Moreover, several regulatory mechanisms seem to exist that control their biological activity. Novel insights support the concept of granulocyte death regulation by NAbs, which might have important implications for our understanding of the pathogenesis and treatment of inflammatory diseases, including many autoimmune and allergic disorders.

Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease

Eosinophil extracellular traps (EETs) are part of the innate immune response and are seen in multiple infectious, allergic, and autoimmune eosinophilic diseases. EETs are composed of a meshwork of DNA fibers and eosinophil granule proteins, such as major basic protein (MBP) and eosinophil cationic protein (ECP). Interestingly, the DNA within the EETs appears to have its origin in the mitochondria of eosinophils, which had released most their mitochondrial DNA, but were still viable, exhibiting no evidence of a reduced life span. Multiple eosinophil activation mechanisms are represented, whereby toll-like, cytokine, chemokine, and adhesion receptors can all initiate transmembrane signal transduction processes leading to the formation of EETs. One of the key signaling events required for DNA release is the activation of the NADPH oxidase. Here, we review recent progress made in the understanding the molecular mechanisms involved in DNA and granule protein release, discuss the presence of EETs in disease, speculate on their potential role(s) in pathogenesis, and compare available data on other DNA-releasing cells, particularly neutrophils.

Workshop report from the National Institutes of Health Taskforce on the Research Needs of Eosinophil-Associated Diseases (TREAD)

Eosinophils are blood cells that are often found in high numbers in the tissues of allergic conditions and helminthic parasite infections. The pathophysiologic roles that eosinophils may serve in other human "eosinophil-associated" diseases remain obscure.

Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor

IgE antibodies bind the high-affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response. Inhibitors of IgE-FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma. However, preformed IgE-FcεRI complexes that prime cells before allergen exposure dissociate extremely slowly and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms. Here we demonstrate that an engineered protein inhibitor, DARPin E2_79 (refs 9, 10, 11), acts through a non-classical inhibition mechanism, not only blocking IgE-FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79-IgE-Fc(3-4) complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE-FcεRI complex, with site 1 distant from the receptor and site 2 exhibiting partial steric overlap. Although the structure is indicative of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modelling indicate that E2_79 acts through a facilitated dissociation mechanism at site 2 alone. These results demonstrate that high-affinity IgE-FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein-protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.

An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, nonreceptor binding conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

Pediatric severe asthma is characterized by eosinophilia and remodeling without T(H)2 cytokines

BACKGROUND: The pathology of pediatric severe therapy-resistant asthma (STRA) is little understood. OBJECTIVES: We hypothesized that STRA in children is characterized by airway eosinophilia and mast cell inflammation and is driven by the T(H)2 cytokines IL-4, IL-5, and IL-13. METHODS: Sixty-nine children (mean age, 11.8 years; interquartile range, 5.6-17.3 years; patients with STRA, n = 53; control subjects, n = 16) underwent fiberoptic bronchoscopy, bronchoalveolar lavage (BAL), and endobronchial biopsy. Airway inflammation, remodeling, and BAL fluid and biopsy specimen T(H)2 cytokines were quantified. Children with STRA also underwent symptom assessment (Asthma Control Test), spirometry, exhaled nitric oxide and induced sputum evaluation. RESULTS: Children with STRA had significantly increased BAL fluid and biopsy specimen eosinophil counts compared with those found in control subjects (BAL fluid, P < .001; biopsy specimen, P < .01); within the STRA group, there was marked between-patient variability in eosinophilia. Submucosal mast cell, neutrophil, and lymphocyte counts were similar in both groups. Reticular basement membrane thickness and airway smooth muscle were increased in patients with STRA compared with those found in control subjects (P < .0001 and P < .001, respectively). There was no increase in BAL fluid IL-4, IL-5, or IL-13 levels in patients with STRA compared with control subjects, and these cytokines were rarely detected in induced sputum. Biopsy IL-5(+) and IL-13(+) cell counts were also not higher in patients with STRA compared with those seen in control subjects. The subgroup (n = 15) of children with STRA with detectable BAL fluid T(H)2 cytokines had significantly lower lung function than those with undetectable BAL fluid T(H)2 cytokines. CONCLUSIONS: STRA in children was characterized by remodeling and variable airway eosinophil counts. However, unlike in adults, there was no neutrophilia, and despite the wide range in eosinophil counts, the T(H)2 mediators that are thought to drive allergic asthma were mostly absent.

Etiology and pathogenesis of adverse drug reactions

In clinical routine, adverse drug reactions (ADR) are common, and they should be included in the differential diagnosis in all patients undergoing drug treatment. Only part of those ADR are immune-mediated hypersensitivity reactions and thus true drug allergies. Far more common are non-immune-mediated ADR, e.g. due to the pharmacological properties of the drug or to the individual predisposition of the patient (enzymopathies, cytokine dysbalance, mast cell hyperreactivity). In true drug allergiesT cell- and immunoglobulin E (lgE)-mediated reactions dominate the clinical presentation. T cell-mediated ADR usually have a delayed appearance and include skin eruptions in most cases. Nevertheless, it should not be forgotten that they may involve systemic T cell activation and thus take a severe, sometimes lethal turn. Clinical danger signs are involvement of mucosal surfaces, blistering within the exanthematous skin areas and systemic symptoms, e.g. fever or malaise. Drug presentation via antigen-presenting cells to T cells can either involve the classical pathway of haptenization of endogenous proteins or be directly mediated via noncovalent binding to immune receptors (MHC molecules or T cell receptors), the so-called p-i concept. Flare-up reactions during the acute phase of T cell-mediated ADR should not be mistaken for true drug allergies, as they only occur in the setting of a highly activated T cell pool. IgE-mediated ADR are less frequent and involve mast cells and/or basophils as peripheral effector cells. Recent data suggest that certain patients with drug allergy have a preexistent sensitization although they have never been exposed to the culprit drug, probably due to cross-reactivity. Thus, allergic drug reactions on first encounter are possible. In general, the extent of cross-reactivity is higher in IgE-compared to T cell-mediated ADR. Based on a specific ethnic background and only for severe T cell-mediated ADR to certain drugs, a strong HLA association has been established recently.

[House dust mite allergy]

House dust mites can be found all over the world where human beings live independent from the climate. Proteins from the gastrointestinal tract- almost all known as enzymes - are the allergens which induce chronic allergic diseases. The inhalation of small amounts of allergens on a regular base all night leads to a slow beginning of the disease with chronically stuffed nose and an exercise induced asthma which later on persists. House dust mites grow well in a humid climate - this can be in well isolated dwellings or in the tropical climate - and nourish from human skin dander. Scales are found in mattresses, upholstered furniture and carpets. The clinical picture with slowly aggravating complaints leads quite often to a delayed diagnosis, which is accidently done on the occasion of a wider spectrum of allergy skin testing. The beginning of a medical therapy with topical steroids as nasal spray or inhalation leads to a fast relief of the complaints. Although discussed in extensive controversies in the literature - at least in Switzerland with the cold winter and dry climate - the recommendation of house dust mite avoidance measures is given to patients with good clinical results. The frequent ventilation of the dwelling with cold air in winter time cause a lower indoor humidity. Covering encasings on mattresses, pillow, and duvets reduces the possibility of chronic contact with mite allergens as well as the weekly changing the bed linen. Another option of therapy is the specific immunotherapy with extracts of house dust mites showing good results in children and adults. Using recombinant allergens will show a better quality in diagnostic as well as in therapeutic specific immunotherapy.

Impaired innate interferon induction in severe therapy resistant atopic asthmatic children

Deficient type I interferon-β and type III interferon-λ induction by rhinoviruses has previously been reported in mild/moderate atopic asthmatic adults. No studies have yet investigated if this occurs in severe therapy resistant asthma (STRA). Here, we show that compared with non-allergic healthy control children, bronchial epithelial cells cultured ex vivo from severe therapy resistant atopic asthmatic children have profoundly impaired interferon-β and interferon-λ mRNA and protein in response to rhinovirus (RV) and polyIC stimulation. Severe treatment resistant asthmatics also exhibited increased virus load, which negatively correlated with interferon mRNA levels. Furthermore, uninfected cells from severe therapy resistant asthmatic children showed lower levels of Toll-like receptor-3 mRNA and reduced retinoic acid inducible gene and melanoma differentiation-associated gene 5 mRNA after RV stimulation. These data expand on the original work, suggesting that the innate anti-viral response to RVs is impaired in asthmatic tissues and demonstrate that this is a feature of STRA.

Equine insect bite hypersensitivity: immunoblot analysis of IgE and IgG subclass responses to Culicoides nubeculosus salivary gland extract

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to these proteins. Individual IgE and IgG subclass responses to proteins of C. nubeculosus SGE were evaluated by immunoblot in 42 IBH-affected and 26 healthy horses belonging to different groups (Icelandic horses born in Iceland, Icelandic horses and horses from different breeds born in mainland Europe). Additionally, the specific antibody response was studied before exposure to bites of Culicoides spp. and over a period of 3 years in a cohort of 10 Icelandic horses born in Iceland and imported to Switzerland. Ten IgE-binding protein bands with approximate molecular weights of 75, 66, 52, 48, 47, 32, 22/21, 19, 15, 13/12 kDa were found in the SGE. Five of these bands bound IgE from 50% or more of the horse sera. Thirty-nine of the 42 IBH-affected horses but only 2 of the 26 healthy horses showed IgE-binding to the SGE (p<0.000001). Similarly, more IBH-affected than healthy horses had IgGa ab binding to the Culicoides SGE (19/22 and 9/22, respectively, p<0.01). Sera of IBH-affected horses contained IgE, IgGa and IgGT but not IgGb ab against significantly more protein bands than the sera of the healthy horses. The cohort of 10 Icelandic horses confirmed these results and showed that Culicoides SGE specific IgE correlates with onset of IBH. IBH-affected horses that were born in Iceland had IgGa and IgGT ab (p< or =0.01) as well as IgE ab (p=0.06) against a significantly higher number of SGE proteins than IBH-affected horses born in mainland Europe. The present study shows that Culicoides SGE contains at least 10 potential allergens for IBH and that IBH-affected horses show a large variety of IgE-binding patterns in immunoblots. These findings are important for the future development of a specific immunotherapy with recombinant salivary gland allergens.