Omalizumab response correlates with reduced IFN-γ-, IL-10- and IL-31-secreting cells in chronic spontaneous urticaria.

BACKGROUND: The anti-IgE antibody omalizumab has been approved for the treatment of chronic spontaneous urticaria (CSU) in patients insufficiently responding to antihistamines. However, its mode of action in CSU is not clearly understood. OBJECTIVES: The aim of this study was to get a better insight in the immune mechanisms involved in clinical improvement of CSU patients treated with omalizumab. METHODS: Chronic spontaneous urticaria patients (n = 15) were followed for 5 months after initiation of omalizumab treatment. Clinical symptoms were assessed by UCT and CU-Q2 oL. Cell-bound IgE was quantified on both FcεRI- and FcεRII-expressing cell populations in peripheral blood. In addition, IgE and IgG as well as their receptors were measured on basophils, and basophil activation was assessed with different concentrations of anti-FcεRI and fMLP. Furthermore, the frequencies of different T-cell subsets secreting IL-5, IL-10, IL-31 or IFN-γ were analysed by ELISpot assay. RESULTS: Seven patients showed a full, five a partial and three no clinical response to omalizumab. Cell-bound IgE was reduced on FcεRI-bearing cells, but not on FcεRII-expressing cells. Likewise, the expression of FcεRI declined. Basophil activation increased upon FcεRI-stimulation while their sensitivity was not affected. Both basophil and T-cell frequencies remained unchanged. However, when comparing the individual response to omalizumab treatment with distinct T-cell subsets, a significant correlation was found between improved UCT and decreased frequencies of IL-10-, IL-31- and IFN-γ-secreting T cells. CONCLUSIONS: We here show that besides addressing IgE-dependent immune mechanisms, omalizumab treatment of CSU patients has effects on distinct T-cell subsets, which correlate with clinical improvement.

[Personalized medicine in allergology]. / Personalisierte Medizin in der Allergologie.

BACKGROUND: Personalized medicine offers new perspectives for diagnostic measurements and medical treatment, but also puts greater demands on the physician. OBJECTIVES: Developments, potentials and potential pitfalls of personalized medicine in allergology. METHODS: Overview, evaluation and discussion of the current state of science on the basis of selected examples. RESULTS: Allergic diseases like allergic rhinitis, atopic eczema or anaphylaxis can be classified into various clinical phenotypes, which are based on different immunological endotypes. These can be captured and categorized by a wide variety of omics technologies. The identification of endotype specific biomarkers holds promising opportunities of more precise diagnostics, the implementation of novel targeted therapies or the development of optimized preventive strategies. However, individualized analysis and assessment of the significance of the measurements represent special challenges. CONCLUSIONS: Findings of the complex omics technologies need to be evaluated by comprehensive prospective studies in order to validate their clinical relevance and suitability for personalized medicine in allergology.

Development of a skin-friendly microemulsion for dermal allergen-specific immunotherapy.

Due to their role in immune responses, the skin dendritic cells (i.e. epidermal Langerhans cells and dermal dendritic cells) have become of great interest to researchers in the past decades. A dermal administration of allergens could target these professional antigen-presenting cells directly and build up immunotolerance. Additionally, many of the adverse side effects, which are seen in the current state of the art specific immunotherapy routes, could be avoided. Therefore, in this study a penetration enhancing microemulsion was developed and its physicochemical properties were determined under several storage conditions. The influence of different preservatives on the microemulsion stability was observed. We examined epidermal penetration of Alexa Fluor-647 labelled bee-venom phospholipase A2 (Api m 1) using the Franz diffusion cell set up and confocal laser-scanning microscopy. First results of an in-vivo Api m 1-allergic mouse model indicated the protective efficacy of dermal AIT with our newly developed microemulsion. Summarily, the developed microemulsion is a suitable, stable drug delivery system for the topical administration of proteogenic allergens into the epidermis and is able to reach dendritic cells in the skin.

Blocking antibodies induced by allergen-specific immunotherapy ameliorate allergic airway disease in a human/mouse chimeric model.

BACKGROUND: Allergen-specific immunotherapy (AIT) induces specific blocking antibodies (Ab), which are claimed to prevent IgE-mediated reactions to allergens. Additionally, AIT modulates cellular responses to allergens, for example, by desensitizing effector cells, inducing regulatory T and B lymphocytes and immune deviation. It is still enigmatic which of these mechanisms mediate(s) clinical tolerance. We sought to address the role of AIT-induced blocking Ab separately from cellular responses in a chimeric human/mouse model of respiratory allergy. METHODS: Nonobese diabetic severe combined immunodeficient γc-/- (NSG) mice received intraperitoneally allergen-reactive PBMC from birch pollen-allergic patients together with birch pollen extract and human IL-4. Engraftment was assessed by flow cytometry. Airway hyperresponsiveness (AHR) and bronchial inflammation were analyzed after intranasal challenges with allergen or PBS. Sera collected from patients before and during AIT with birch pollen were added to the allergen prior to intranasal challenge. The IgE-blocking activity of post-AIT sera was assessed in vitro. RESULTS: Human cells were detected in cell suspensions of murine lungs and spleens indicating successful humanization. Humanized mice displayed a more pronounced AHR and bronchial inflammation when challenged with allergen compared to negative controls. Post-AIT sera exerted IgE-blocking activity. In contrast to pre-AIT sera, the presence of heterologous and autologous post-AIT sera significantly reduced the allergic airway inflammation and matched their IgE-blocking activity determined in vitro. CONCLUSION: Our data demonstrate that post-AIT sera with IgE-blocking activity ameliorate allergic airway inflammation in a human/mouse chimeric model of respiratory allergy independently of AIT-induced cellular changes.

Immunological differences between insect venom-allergic patients with and without immunotherapy and asymptomatically sensitized subjects.

BACKGROUND: Currently available tests are unable to distinguish between asymptomatic sensitization and clinically relevant Hymenoptera venom allergy. A reliable serological marker to monitor venom immunotherapy (VIT) does also not exist. Our aim was to find reliable serological markers to predict tolerance to bee and vespid stings. METHODS: We included 77 asymptomatically sensitized subjects, 85 allergic patients with acute systemic sting reactions, and 61 allergic patients currently treated with VIT. Levels of sIgE and sIgG4 to bee and vespid venom, rApi m 1, and rVes v 5 were measured immediately after allergic sting reactions or before sting challenges and 4 weeks later. All sting challenges were tolerated. The inhibitory activity was determined using BAT inhibition and ELIFAB assay. RESULTS: Median sIgG4 levels were 96-fold higher in VIT patients (P < .001) while sIgE/sIgG4 ratios were consistently lower (P < .001). The ELIFAB assay was paralleled by low sIgE/sIgG4 ratios in VIT patients, showing markedly higher allergen-blocking capacity (P < .001). An almost complete inhibition of the basophil response was seen in all patients treated with vespid venom, but not in those treated with bee venom. Four weeks after the sting, sIgE and sIgG4 levels were increased in allergic and asymptomatically sensitized patients, but not in VIT patients. CONCLUSION: Immunological responses after stings varied in bee and vespid venom-allergic patients. In patients under VIT, sIgE and sIgG4 remained completely stable after sting challenges. Monitoring VIT efficacy was only possible in vespid venom allergy, and the sIgG4 threshold for rVes v 5 had the highest sensitivity to confirm tolerance. The BAT inhibition test was the most reliable tool to confirm tolerance on an individual basis.

Functional and phenotypic analysis of basophils allows determining distinct subtypes in patients with chronic urticaria.

BACKGROUND: Chronic urticaria (CU) is a frequent skin disease characterized by relapsing appearance of pruritic hives. While clinical symptoms are due to the release of histamine by cutaneous mast cells, the underlying pathophysiology is still unknown. However, previous studies indicate that basophils might be of relevance. Besides, the occurrence of autoantibodies against IgE or its receptor, FcεRI, and the therapeutic efficacy of anti-IgE antibodies imply that IgE-mediated mechanisms also play an important role in CU. METHODS: Reactivity of CU patients' peripheral blood basophils (n=60) to specific anti-FcεRI and IgE-independent fMLP stimulation was determined by basophil activation test in comparison with patients suffering from IgE-mediated allergic rhinitis (n=10) and healthy controls (n=10). In addition, immunoglobulin receptor (FcεRI, FcγRII) expression and surface bound antibodies (IgE, IgG) were quantified on basophils. Furthermore, the autoreactive capacity of CU sera was evaluated and urticaria-related symptoms were assessed by both UCT and CU-Q2 oL. RESULTS: Stimulating CU patients' basophils via FcεRI, we identified three distinct immunologic phenotypes. One subgroup of patients' basophils reacted to FcεRI stimulation, whereas the others had anti-FcεRI nonreactive basophils. Among the latter, a subgroup with pronounced basopenia was identified. Of note, this group was characterized by augmented serum-induced basophil activation, increased levels of autoantibodies against thyroid peroxidase, and also exhibited the strongest disease impact on their quality of life. CONCLUSIONS: Patients with CU can be categorized into three immunologic subgroups based on their basophil reactivity and frequency. These phenotypes are associated with different clinical characteristics, pointing to basophils as important players in CU pathophysiology.

[Diagnostics of drug hypersensitivity reactions]. / Diagnostik von Arzneimittelüberempfindlichkeiten.

Drug hypersensitivity reactions comprise approximately 25% of all adverse drug reactions and can be classified into allergic and pseudoallergic drug reactions. Immediate type anaphylactic and delayed type rash reactions of various clinical patterns can be distinguished, depending on the pathogenesis and clinical symptoms. The diagnostic work-up encompasses a thorough but also focused evaluation of the medical history, skin tests and when indicated challenge tests. Furthermore, in vitro tests, such as basophil activation tests and T cell assays not only add valuable additional information but can also yield decisive results for the diagnosis, especially in cases of severe drug reactions or reactions which cannot be further clarified by provocation tests. The aim of these measurements is not only the proof of drug intolerance and the detection of the causal drug but also the disclosure of the type of adverse reaction and the identification of potential, tolerated alternative drugs. This information is very important for the counseling of the patient and for prevention of new drug hypersensitivity reactions in the future.

IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen.

BACKGROUND: Allergen-specific immunotherapy (AIT) with birch pollen generates Bet v 1-specific immunoglobulin (Ig)G4 which blocks IgE-mediated hypersensitivity mechanisms. Whether IgG4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG4 antibodies are developed is under debate. OBJECTIVE: We sought to analyze the epitope specificities of IgE and IgG4 antibodies from sera of patients who received AIT. METHODS: 15 sera of patients (13/15 received AIT) with Bet v 1a-specific IgE and IgG4 were analyzed. The structural arrangements of recombinant (r)Bet v 1a and rBet v 1a_11x , modified in five potential epitopes, were analyzed by circular dichroism and nuclear magnetic resonance spectroscopy. IgE binding to Bet v 1 was assessed by ELISA and mediator release assays. Competitive binding of monoclonal antibodies specific for Bet v 1a and serum IgE/IgG4 to rBet v 1a and serum antibody binding to a non-allergenic Bet v 1-type model protein presenting an individual epitope for IgE was analyzed in ELISA and western blot. RESULTS: rBet v 1a_11x had a Bet v 1a - similar secondary and tertiary structure. Monomeric dispersion of rBet v 1a_11x was concentration and buffer-dependent. Up to 1500-fold increase in the EC50 for IgE-mediated mediator release induced by rBet v 1a_11x was determined. The reduction of IgE and IgG4 binding to rBet v 1a_11x was comparable in 67% (10/15) of sera. Bet v 1a-specific monoclonal antibodies inhibited binding of serum IgE and IgG4 to 66.1% and 64.9%, respectively. Serum IgE and IgG4 bound specifically to an individual epitope presented by our model protein in 33% (5/15) of sera. CONCLUSION AND CLINICAL RELEVANCE: Patients receiving AIT develop Bet v 1a-specific IgG4 which competes with IgE for partly identical or largely overlapping epitopes. The similarities of epitopes for IgE and IgG4 might stimulate the development of epitope-specific diagnostics and therapeutics.

Decline of Ves v 5-specific blocking capacity in wasp venom-allergic patients after stopping allergen immunotherapy.

While allergen-specific immunotherapy (AIT) is very efficient in hymenoptera venom (HV)-allergic patients, long-term outcome after finishing AIT is not well investigated, especially regarding mechanisms that are suggested to contribute to allergen-specific tolerance. Here, we analyse the Ves v 5-inhibitory activity of sera from wasp venom-allergic patients using the novel cell-free enzyme-linked immunosorbent facilitated antigen binding (ELIFAB) assay. Compared to pre-AIT, sera from patients undergoing AIT displayed an increased ability to inhibit Ves v 5 binding by IgE antibodies. In contrast, this inhibitory activity was reduced in patients having finished AIT 5-12 years ago. Allergen-blocking capacity correlated with serum concentrations of Ves v 5-specific IgG4 which rose during AIT but almost reached pretreatment levels in patients who had stopped AIT more than 5 years ago. These data raise questions about how long allergen tolerance is maintained in AIT-treated HV-allergic patients and suggest that the ELIFAB assay might be an easy-to-use tool assessing long-term tolerance in patients treated with HV-AIT.

Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen.

BACKGROUND: IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP-SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross-reactivity of BP-SIT-induced Bet v 1-specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and IgE-blocking activity of Bet v 1-specific IgG4 antibodies emerging during conventional BP-SIT and whether IgG4-epitopes overlapped with IgE epitopes. METHODS: IgE and IgG4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen-allergic patients before and during BP-SIT. Inhibition of IgE binding was studied by IgE-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of IgE-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by IgE and IgG4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3D structure of the allergens using a computer-based algorithm. RESULTS: BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies. In parallel, allergen-specific IgE levels decreased significantly. Sera containing food allergen-reactive IgG4 antibodies inhibited IgE binding, basophil activation, and IgE-mediated food allergen-induced T-cell proliferation. Predicted IgE and IgG4 epitopes on all allergens showed high overlap. CONCLUSION: Our results indicate that BP-SIT may induce Bet v 1-specific IgG4 antibodies that cross-react with related food allergens and inhibit IgE binding by epitope competition.

Combined treatment with immunoadsorption and rituximab leads to fast and prolonged clinical remission in difficult-to-treat pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune bullous disorder which is characterized by blisters and erosions of the skin and mucous membranes. A frequently applied first-line therapy for PV consists of systemic corticosteroids (CS) combined with immunosuppressive agents. In refractory cases, novel therapeutic strategies such as immunoadsorption (IA) and the anti-CD20 antibody rituximab (Rtx) aim at directly interfering with pathogenic autoantibodies (auto-Abs). OBJECTIVES: To investigate the long-term efficacy of IA in combination with Rtx in patients with difficult-to-treat PV, we assessed the clinical response to treatment by monitoring the Autoimmune Bullous Skin Disorder Intensity Score, IgG auto-Abs against desmoglein 1 and 3 (Dsg1 and Dsg3) and the dose of systemic CS. METHODS: We retrospectively analysed clinical and serological parameters of 10 patients with difficult-to-treat PV who received IA at 4-week intervals, followed by Rtx either twice at 1000 mg or four times at 375mg m(-2) . During a 12-month follow-up period, CS were tapered according to the individual clinical status. RESULTS: Six months after the first IA treatment eight of 10 patients were in complete remission on therapy while one patient showed a partial response and one patient was unresponsive to the treatment. At 12 months, six of eight patients were in complete remission on therapy, one patient showed stable disease and one patient had relapsed. Overall, anti-Dsg3 IgG and anti-Dsg1 IgG auto-Abs correlated well with the clinical activity and systemic CS were tapered gradually. CONCLUSIONS: The present findings show that the combination of IA and Rtx induces rapid clinical remission and long-term control in difficult-to-treat pemphigus.