High affinity targeting of CD23 inhibits IgE synthesis in human B cells.

The low-affinity IgE receptor FcϵRII (CD23) is part of the regulatory system controlling IgE synthesis in human B cells and exists in membrane and soluble forms. Binding of IgE to CD23 has been described to have stabilizing effects and to prevent cleavage of CD23. Previous experiments using anti-CD23 antibodies reduced IgE synthesis but were difficult to interpret as the antibody Fc part might also mediate feedback mechanisms. The purpose of this study was to investigate the regulatory role of CD23, by using designed ankyrin repeat proteins (DARPins) that specifically recognize CD23. Anti-CD23 DARPins were isolated by ribosome display and were produced as monovalent and bivalent constructs. Affinities to CD23 were measured by surface plasmon resonance. IgE synthesis and up-regulation of CD23 in human peripheral B cells were induced by IL-4 and anti-CD40 antibody. We assessed CD23 expression and its stabilization by FACS and used an ELISA for detecting soluble CD23. IgE synthesis was measured by ELISA and real-time PCR. Surface plasmon resonance revealed affinities of the DARPins to CD23 in the pico-molar range. Anti-CD23 DARPins strongly inhibited binding of IgE to CD23 and share thus a similar binding epitope as IgE. The DARPins stabilized membrane CD23 and reduced IgE synthesis in an isotype specific manner. Furthermore, the anti-CD23 DARPins decreased IgE transcript through inhibition of mature Cϵ RNA synthesis suggesting a posttranscriptional control mechanism. This study demonstrates that targeting CD23 alone is sufficient to inhibit IgE synthesis and suggests that a negative signaling occurs directly through the CD23 molecule.

Drug hypersensitivity reactions involving skin

Immune reactions to drugs can cause a variety of diseases involving the skin, liver, kidney, lungs, and other organs. Beside immediate, IgE-mediated reactions of varying degrees (urticaria to anaphylactic shock), many drug hypersensitivity reactions appear delayed, namely hours to days after starting drug treatment, showing a variety of clinical manifestations from solely skin involvement to fulminant systemic diseases which may be fatal. Immunohistochemical and functional studies of drug-specific T cells in patients with delayed reactions confirmed a predominant role for T cells in the onset and maintenance of immune-mediated delayed drug hypersensitivity reactions (type IV reactions). In these reactions, drug-specific CD4+ and CD8+ T cells are stimulated by drugs through their T cell receptors (TCR). Drugs can stimulate T cells in two ways: they can act as haptens and bind covalently to larger protein structures (hapten-carrier model), inducing a specific immune response. In addition, they may accidentally bind in a labile, noncovalent way to a particular TCR of the whole TCR repertoire and possibly also major histocompatibility complex (MHC)-molecules - similar to their pharmacologic action. This seems to be sufficient to reactivate certain, probably in vivo preactivated T cells, if an additional interaction of the drug-stimulated TCR with MHC molecules occurs. The mechanism was named pharmacological interaction of a drug with (immune) receptor and thus termed the p-i concept. This new concept may explain the frequent skin symptoms in drug hypersensitivity to oral or parenteral drugs. Furthermore, the various clinical manifestations of T cell-mediated drug hypersensitivity may be explained by distinct T cell functions leading to different clinical phenotypes. These data allowed a subclassification of the delayed hypersensitivity reactions (type IV) into T cell reactions which, by releasing certain cytokines and chemokines, preferentially activate and recruit monocytes (type IVa), eosinophils (type IVb), or neutrophils (type IVd).

Human mast cells express intracellular TRAIL

Recently we demonstrated that human mast cells (MC) express functional TRAIL death receptors. Here we assessed the expression of TRAIL on both mRNA and protein level in cord blood derived MC (CBMC) and HMC-1. The TRAIL release either spontaneous or induced by LPS, IFN-gamma and IgE-dependent activation, was evaluated as well. The protein location was restricted to the intracellular compartment in CBMC, but not in HMC-1. The intracellular TRAIL was not localized inside the granules. The treatment with IFN-gamma and LPS up-regulated intracellular TRAIL expression in CBMC, but did not induce its release. These in vitro data show that human MC can produce and express intracellular TRAIL whose location could not be altered by different stimuli.

General considerations on rapid desensitization for drug hypersensitivity - a consensus statement

Drug hypersensitivity reactions can occur with most drugs, are unpredictable, may affect any organ or system, and range widely in clinical severity from mild pruritus to anaphylaxis. In most cases, the suspected drug is avoided in the future. However, for certain patients, the particular drug may be essential for optimal therapy. Under these circumstances, desensitization may be performed. Drug desensitization is defined as the induction of a temporary state of tolerance of a compound responsible for a hypersensitivity reaction. It is performed by administering increasing doses of the medication concerned over a short period of time (from several hours to a few days) until the total cumulative therapeutic dose is achieved and tolerated. It is a high-risk procedure used only in patients in whom alternatives are less effective or not available after a positive risk/benefit analysis. Desensitization protocols have been developed and are used in patients with allergic reactions to antibiotics (mainly penicillin), insulins, sulfonamides, chemotherapeutic and biologic agents, and many other drugs. Desensitization is mainly performed in IgE-mediated reactions, but also in reactions where drug-specific IgE have not been demonstrated. Desensitization induces a temporary tolerant state, which can only be maintained by continuous administration of the medication. Thus, for treatments like chemotherapy, which have an average interval of 4 weeks between cycles, the procedure must be repeated for every new course. In this paper, some background information on rapid desensitization procedures is provided. We define the drugs and drug reactions indicated for such procedures, describe the possible mechanism of action, and discuss the indications and contraindications. The data should serve as background information for a database (accessible via the EAACI-homepage) with standardized protocols for rapid desensitization for antibiotics, chemotherapeutic agents, monoclonal antibodies/fusion proteins, and other drugs.

Allergen cross reactions: a problem greater than ever thought?

Cross reactions are an often observed phenomenon in patients with allergy. Sensitization against some allergens may cause reactions against other seemingly unrelated allergens. Today, cross reactions are being investigated on a per-case basis, analyzing blood serum specific IgE (sIgE) levels and clinical features of patients suffering from cross reactions. In this study, we evaluated the level of sIgE compared to patients' total IgE assuming epitope specificity is a consequence of sequence similarity.

Individual IL-3 priming is crucial for consistent in vitro activation of donor basophils in patients with chronic urticaria

The in vivo autologous serum skin test (ASST) is the diagnostic gold standard to detect autoantibodies against FcεRI or IgE itself, as well as other autoreactive serum components, in patients with chronic spontaneous urticaria (CU). Coincubation of patient sera with donor basophils and measuring their degranulation in vitro could be a safe alternative but has shown inconsistent results.

Anti-idiotypic Fab Fragments Image a Conserved N-terminal Epitope Patch of Grass Pollen Allergen Phl p 1

BACKGROUND AND AIMS: Naturally occurring anti-idiotypic antibodies structurally mimic the original antibody epitope. Anti-idiotypes, therefore, are interesting tools for the portrayal of conformational B-cell epitopes of allergens. In this study we used this strategy particularly for major timothy grass pollen (Phleum pratense) allergen Phl p 1. METHODS AND RESULTS: We used a combinatorial phage display library constructed from the peripheral IgG repertoire of a grass pollen allergic patient which was supposed to contain anti-idiotypic Fab specificities. Using purified anti-Phl p 1 IgG for biopanning, several Fab displaying phage clones could be isolated. 100 amplified colonies were screened for their binding capacity to anti-Phl p 1-specific antibodies, finally resulting in four distinct Fab clones according to sequence analysis. Interestingly, heavy chains of all clones derived from the same germ line sequence and showed high homology in their CDRs. Projecting their sequence information on the surface of the natural allergen Phl p 1 (PDB ID: 1N10) indicated matches on the N-terminal domain of the homo-dimeric allergen, including the bridging region between the two monomers. The resulting epitope patches were formed by spatially distant sections of the primary allergen sequence. CONCLUSION: In this study we report that anti-idiotypic specificities towards anti-Phl p 1 IgG, selected from a Fab library of a grass pollen allergic patient, mimic a conformational epitope patch being distinct from a previously reported IgE epitope area.

Educational case series: Mechanisms of drug allergy

Once administered, a drug can activate the immune system by various mechanisms and lead to a large range of clinical manifestations closely related to the type of immune reaction elicited. Administration of the drug can classically result in an immunoglobulin E (IgE)-type sensitization, but can also result in more complex activation of the immune system potentially resulting in severe syndromes, such as the drug-induced hypersensitivity syndrome (DIHS). Although there has been a major increase in our knowledge over the last years, the exact mechanisms of drug allergy are not well understood for most clinical manifestations. A complex interaction between individual characteristics, environmental factors, and the drug itself is usually responsible for adverse reactions to drugs. In this educational review series, we described three cases of drug allergy: first, a child with a typical IgE-mediated drug allergy, second, a child with a non-immediate reaction to penicillin, and in the third patient, we will discuss the drug-induced hypersensitivity syndrome, which is rare but potentially fatal. These cases are correlated to the immune mechanism potentially involved.

An increase in serum tryptase even below 11.4 ng/mL may indicate a mast cell-mediated hypersensitivity reaction: a prospective study in Hymenoptera venom allergic patients

During a systemic hypersensitivity reaction (SR), an increase in serum tryptase compared to the baseline value is an indicator of mast cell activation, most often due to an IgE-mediated mechanism.

CD4(+)CD25(+) T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-beta1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.

Skin-infiltrating T cells and cytokine expression in Icelandic horses affected with insect bite hypersensitivity: a possible role for regulatory T cells

Equine insect bite hypersensitivity (IBH) is a seasonally recurrent, pruritic skin disorder caused by an IgE-mediated reaction to salivary proteins of biting flies, predominantly of the genus Culicoides. The aim of this study was to define T cell subsets and cytokine profile in the skin of IBH-affected Icelandic horses with particular focus on the balance between T helper (Th) 1, Th2 and T regulatory (Treg) cells. Distribution and number of CD4+, CD8+ and Forkhead box P3 (FoxP3)+ T cells were characterized by immunohistochemical staining in lesional and non-lesional skin of moderately and severely IBH-affected horses (n=14) and in the skin of healthy control horses (n=10). Using real-time quantitative reverse transcription-polymerase chain reaction, mRNA expression levels of Th2 cytokines (Interleukin (IL)-4, IL-5, IL-13), Th1 cytokines (Interferon-gamma), regulatory cytokines (Transforming Growth Factor beta1, IL-10) and the Treg transcription factor FoxP3 were measured in skin and blood samples. Furthermore, Culicoides nubeculosus specific serum IgE levels were assessed. Lesions of IBH-affected horses contained significantly higher numbers of CD4+ cells than skin of healthy control horses. Furthermore, the total number of T cells (CD4+ and CD8+) was significantly increased in lesional compared to non-lesional skin and there was a tendency (p=0.07) for higher numbers of CD4+ cells in lesional compared to non-lesional skin. While the number of FoxP3+ T cells did not differ significantly between the groups, the ratio of Foxp3 to CD4+ cells was significantly lower in lesions of severely IBH-affected horses than in moderately affected or control horses. Interestingly, differences in FoxP3 expression were more striking at the mRNA level. FoxP3 mRNA levels were significantly reduced in lesional skin, compared both to non-lesional and to healthy skin and were also significantly lower in non-lesional compared to healthy skin. Expression levels of IL-13, but not IL-4 or IL-5, were significantly elevated in lesional and non-lesional skin of IBH-affected horses. IL-10 levels were lower in lesional compared to non-lesional skin (p=0.06) and also lower (p=0.06) in the blood of IBH-affected than of healthy horses. No significant changes were observed regarding blood expression levels of Th1 and Th2 cytokines or FoxP3. Finally, IBH-affected horses had significantly higher Culicoides nubeculosus specific serum IgE levels than control horses. The presented data suggest that an imbalance between Th2 and Treg cells is a characteristic feature in IBH. Treatment strategies for IBH should thus aim at restoring the balance between Th2 and 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.

Increased IL-4 and decreased regulatory cytokine production following relocation of Icelandic horses from a high to low endoparasite environment

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but IgE levels than horses in Switzerland. Nevertheless, horses in Iceland displayed a low proportion of IL-4-producing cells in PBMC cultures after polyclonal or parasite extracts stimulation. No IL-4-producing cells were found in PBMC from horses after stimulation by Culicoides extract. Addition of anti-IL-10 and anti-TGF-beta1 to PBMC cultures of horses in Iceland increased the proportion of IL-4-producing cells after polyclonal or parasite antigens stimulation but not stimulation with Culicoides extract. This paralleled the high levels of IL-10 and TGF-beta1 found in supernatants from PBMC cultures of horses in Iceland. Collectively, horses living in Iceland have a high parasite burden but low IL-4 production. This supports the hypothesis that heavy helminth infections have a suppressive effect on IL-4 production mediated by IL-10 and TGF-beta1.

Distinct reactivities of interleukin-4-specific antibodies with recombinant and native canine interleukin-4 in various assays

Interleukin 4 (IL-4) plays a central role in immune responses to parasites and allergens. IL-4 drives the differentiation of naive T cells into Th2 cells and regulates immunoglobulin class switching to IgE.Little is known about the role of IL-4 in canine allergies and parasite infections. Most of the information derives from measurement of IL-4 mRNA expression in dog tissues, but detection of IL-4 protein has been difficult so far, probably due to low sensitivity of available methods. Antibodies (Ab) specific for canine IL-4 are available from various sources, but these Ab have been produced against recombinant Escherichia coli-expressed canine IL-4 and there is only limited information on their reactivities with native canine IL-4. Therefore, in the present study, we tested six available canine IL-4-specific Ab for their reactivities with recombinant canine IL-4 expressed in E. coli (rec.IL-4) or in mammalian cells (mam.IL-4), and with supernatants from stimulated canine peripheral blood mononuclear cells (PBMCs) using several detection methods, including Western blotting, ELISA, cytokine bead assay, and intracellular IL-4 staining. Additionally, we tested a bovine IL-4-specific antibody that has been previously shown to cross-react with canine IL-4. All tested Ab except anti-bovine IL-4 reacted with rec.IL-4, and most of them reacted with mam.IL-4. However, only the cytokine bead assay was sensitive enough to allow the detection of IL-4 in supernatants of canine PBMCs.

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.