Atopic Keratoconjunctivitis: Pathophysiology, Clinic, and Potential New Therapeutic Concepts. / Atopische Keratokonjunktivitis: Pathophysiologie, Klinik und potenzielle neue Therapiekonzepte.

Atopic dermatitis (AD) is a chronic recurrent inflammatory skin disease with a bipolar age distribution in childhood, adolescence and middle adulthood. Up to 50% of AD patients show ocular involvement, which can be potentially sight threatening. Clinically, the majority of cases present with atopic blepharo(kerato)conjunctivitis or atopic keratoconjunctivitis (AKC); other clinical variants from this group of inflammatory ocular surface diseases are keratoconjunctivitis vernalis in childhood and adolescence and allergic conjunctivitis. In addition to the aforementioned blepharitis, keratitis and conjunctivitis, AD is also associated with eyelid involvement with subsequent eyelid malposition, limbal insufficiency with the development of pseudopterygia, (chronic) cicatrizing conjunctivitis with symblephara formation and fornix shortening, as well as ocular surface malignancies such as conjunctival intraepithelial neoplasia (CIN) and squamous cell carcinoma. In addition, an association with AD or AKC has been described for keratoconus. Whereas the therapy of AD in dermatology has made revolutionary advances in recent years through the use of biologicals, the primary use of these biologicals in ophthalmological complications is still very hesitant. Treatment here is often provided using topical steroids and calcineurin inhibitors. The following article summarises recent developments in basic and clinical dermatological research and discusses them in the context of current concepts for ophthalmological therapy.

Efficacy and safety of on-demand versus daily rupatadine in chronic spontaneous urticaria: A randomized trial.

BACKGROUND: Non-sedating H1 -antihistamines (nsAH) are the most commonly used treatment for chronic spontaneous urticaria (CSU). Many patients use them as on-demand (OD) therapy rather than a maintenance treatment. Here, we compared OD versus daily maintenance treatment with the nsAH rupatadine, assessed the efficacy of rupatadine updosing, and investigated potential long-term disease-modifying effects. METHODS: This multicenter, randomized study consisted of 2 weeks of screening, 8 weeks of double-blind treatment, and 6 weeks of treatment-free follow-up (OD allowed). Adult patients were randomized to 10 mg rupatadine OD or 10 mg rupatadine daily. At Week 4, if patients did not have a complete response, they switched from 10 to 20 mg rupatadine daily or underwent sham updosing (patients on 10 mg rupatadine OD). The primary aim was to compare CSU disease activity at the end of follow-up between daily versus OD. Additionally, we assessed the efficacy of rupatadine updosing. Major outcomes were disease activity, CSU-related quality of life (QoL), and disease control. RESULTS: At Week 4, disease activity and QoL significantly improved in daily versus OD-treated patients. Updosing of rupatadine did not improve the mean disease activity, but the number of complete responders increased during updosing from 5% to 22%. At the end of follow-up, the disease activity of patients treated OD versus daily was not significantly different. CONCLUSIONS: Daily rupatadine treatment significantly improved CSU disease activity and QoL during treatment versus OD treatment but not after discontinuation of rupatadine, indicating the benefits of a daily maintenance nsAH schedule.

Diagnosis and treatment of Hymenoptera venom allergy: S2k Guideline of the German Society of Allergology and Clinical Immunology (DGAKI) in collaboration with the Arbeitsgemeinschaft für Berufs- und Umweltdermatologie e.V. (ABD), the Medical Association of German Allergologists (AeDA), the German Society of Dermatology (DDG), the German Society of Oto-Rhino-Laryngology, Head and Neck Surgery (DGHNOKC), the German Society of Pediatrics and Adolescent Medicine (DGKJ), the Society for Pediatric Allergy and Environmental Medicine (GPA), German Respiratory Society (DGP), and the Austrian Society for Allergy and Immunology (ÖGAI).

Hymenoptera venom (HV) is injected into the skin during a sting by Hymenoptera such as bees or wasps. Some components of HV are potential allergens and can cause large local and/or systemic allergic reactions (SAR) in sensitized individuals. During their lifetime, ~ 3% of the general population will develop SAR following a Hymenoptera sting. This guideline presents the diagnostic and therapeutic approach to SAR following Hymenoptera stings. Symptomatic therapy is usually required after a severe local reaction, but specific diagnosis or allergen immunotherapy (AIT) with HV (VIT) is not necessary. When taking a patient's medical history after SAR, clinicians should discuss possible risk factors for more frequent stings and more severe anaphylactic reactions. The most important risk factors for more severe SAR are mast cell disease and, especially in children, uncontrolled asthma. Therefore, if the SAR extends beyond the skin (according to the Ring and Messmer classification: grade > I), the baseline serum tryptase concentration shall be measured and the skin shall be examined for possible mastocytosis. The medical history should also include questions specific to asthma symptoms. To demonstrate sensitization to HV, allergists shall determine concentrations of specific IgE antibodies (sIgE) to bee and/or vespid venoms, their constituents and other venoms as appropriate. If the results are negative less than 2 weeks after the sting, the tests shall be repeated (at least 4 - 6 weeks after the sting). If only sIgE to the total venom extracts have been determined, if there is double sensitization, or if the results are implausible, allergists shall determine sIgE to the different venom components. Skin testing may be omitted if in-vitro methods have provided a definitive diagnosis. If neither laboratory diagnosis nor skin testing has led to conclusive results, additional cellular testing can be performed. Therapy for HV allergy includes prophylaxis of reexposure, patient self treatment measures (including use of rescue medication) in the event of re-stings, and VIT. Following a grade I SAR and in the absence of other risk factors for repeated sting exposure or more severe anaphylaxis, it is not necessary to prescribe an adrenaline auto-injector (AAI) or to administer VIT. Under certain conditions, VIT can be administered even in the presence of previous grade I anaphylaxis, e.g., if there are additional risk factors or if quality of life would be reduced without VIT. Physicians should be aware of the contraindications to VIT, although they can be overridden in justified individual cases after weighing benefits and risks. The use of ß-blockers and ACE inhibitors is not a contraindication to VIT. Patients should be informed about possible interactions. For VIT, the venom extract shall be used that, according to the patient's history and the results of the allergy diagnostics, was the trigger of the disease. If, in the case of double sensitization and an unclear history regarding the trigger, it is not possible to determine the culprit venom even with additional diagnostic procedures, VIT shall be performed with both venom extracts. The standard maintenance dose of VIT is 100 µg HV. In adult patients with bee venom allergy and an increased risk of sting exposure or particularly severe anaphylaxis, a maintenance dose of 200 µg can be considered from the start of VIT. Administration of a non-sedating H1-blocking antihistamine can be considered to reduce side effects. The maintenance dose should be given at 4-weekly intervals during the first year and, following the manufacturer's instructions, every 5 - 6 weeks from the second year, depending on the preparation used; if a depot preparation is used, the interval can be extended to 8 weeks from the third year onwards. If significant recurrent systemic reactions occur during VIT, clinicians shall identify and as possible eliminate co-factors that promote these reactions. If this is not possible or if there are no such co-factors, if prophylactic administration of an H1-blocking antihistamine is not effective, and if a higher dose of VIT has not led to tolerability of VIT, physicians should should consider additional treatment with an anti IgE antibody such as omalizumab as off lable use. For practical reasons, only a small number of patients are able to undergo sting challenge tests to check the success of the therapy, which requires in-hospital monitoring and emergency standby. To perform such a provocation test, patients must have tolerated VIT at the planned maintenance dose. In the event of treatment failure while on treatment with an ACE inhibitor, physicians should consider discontinuing the ACE inhibitor. In the absence of tolerance induction, physicians shall increase the maintenance dose (200 µg to a maximum of 400 µg in adults, maximum of 200 µg HV in children). If increasing the maintenance dose does not provide adequate protection and there are risk factors for a severe anaphylactic reaction, physicians should consider a co-medication based on an anti-IgE antibody (omalizumab; off-label use) during the insect flight season. In patients without specific risk factors, VIT can be discontinued after 3 - 5 years if maintenance therapy has been tolerated without recurrent anaphylactic events. Prolonged or permanent VIT can be considered in patients with mastocytosis, a history of cardiovascular or respiratory arrest due to Hymenoptera sting (severity grade IV), or other specific constellations associated with an increased individual risk of recurrent and/or severe SAR (e.g., hereditary α-tryptasemia). In cases of strongly increased, unavoidable insect exposure, adults may receive VIT until the end of intense contact. The prescription of an AAI can be omitted in patients with a history of SAR grade I and II when the maintenance dose of VIT has been reached and tolerated, provided that there are no additional risk factors. The same holds true once the VIT has been terminated after the regular treatment period. Patients with a history of SAR grade ≥ III reaction, or grade II reaction combined with additional factors that increase the risk of non response or repeated severe sting reactions, should carry an emergency kit, including an AAI, during VIT and after regular termination of the VIT.

Guideline for allergological diagnosis of drug hypersensitivity reactions: S2k Guideline of the German Society for Allergology and Clinical Immunology (DGAKI) in cooperation with the German Dermatological Society (DDG), the Association of German Allergologists (ÄDA), the German Society for Pediatric Allergology (GPA), the German Contact Dermatitis Research Group (DKG), the German Society for Pneumology (DGP), the German Society of Otorhinolaryngology, Head and Neck Surgery, the Austrian Society of Allergology and Immunology (ÖGAI), the Austrian Society of Dermatology and Venereology (ÖGDV), the German Academy of Allergology and Environmental Medicine (DAAU), and the German Documentation Center for Severe Skin Reactions (dZh).

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EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH 2 responses.

BACKGROUND: Immune responses to N-glycan structures from allergens and parasites are often associated with pronounced, high affinity IgE reactivities. Cross-reactive carbohydrate determinants (CCDs) are constituted by modified N-glycan core structures and represent the most frequently recognized epitopes in allergic immune responses. Although recently accepted as potentially allergenic epitopes, the biological and clinical relevance as well as structural and functional characteristics of CCD-specific antibodies remain elusive. METHODS: In order to gain structural insights into the recognition of CCDs, two specific antibody fragments were isolated from a leporid immune repertoire library and converted into human/leporid IgE and IgG formats. The antibody formats were assessed by ELISA and surface plasmon resonance, structural and functional analyses were performed by X-ray crystallography, mediator release, and ELIFAB assays. RESULTS: The recombinant IgE exhibited highly specific interactions with different types of CCDs on numerous CCD-carrying glycoproteins. Crystal structures of two CCD-specific antibodies, one of which in complex with a CCD-derived disaccharide emphasize that mechanisms of core glycan epitope recognition are as specific as those governing protein epitope recognition. The rIgE triggered immediate cellular responses via FcεRI cross-linking and mediated facilitated antigen presentation by binding of IgE/antigen complexes to CD23, a process that also could be blocked by IgG of allergic patients. CONCLUSIONS: Our study provides evidence for the relevance of N-glycan recognition in TH 2 responses and corroborates that IgE and IgG antibodies to ubiquitous carbohydrate epitopes can be equivalent to those directed against proteinaceous epitopes with implications for diagnostic and immunotherapeutic concepts.

Stimulation of naïve B cells with a fusion protein consisting of FlaA and Bet v 1 induces regulatory B cells ex vivo.

BACKGROUND: The experimental fusion protein rFlaA:Betv1 was shown to efficiently suppress allergen-specific sensitization in mice. However, the detailed mechanism of rFlaA:Betv1-mediated immune modulation is not fully understood. In this study, we investigated the effect of rFlaA:Betv1 on naïve murine B cells. METHODS: Immune modulating capacity of rFlaA:Betv1 was screened in IL-10 reporter mice. B cells were isolated from spleens of naïve C57Bl/6, TLR5-/- , or MyD88-/- mice, stimulated with rFlaA:Betv1 and controls, and monitored for the expression of the regulatory B cell markers CD1d, CD24, CD38, and surface IgM by flow cytometry. Secreted cytokines, antibodies, and reactivity of the induced antibodies were investigated by ELISA and intracellular flow cytometry. Suppressive capacity of rFlaA:Betv1-stimulated B cells was tested in mDC:CD4+ T cell:B cell triple cultures. RESULTS: Upon in vivo application of rFlaA:Betv1 into IL-10-GFP reporter mice, CD19+ B cells were shown to produce anti-inflammatory IL-10, suggesting B cells to contribute to the immune-modulatory properties of rFlaA:Betv1. rFlaA:Betv1-induced IL-10 secretion was confirmed in human B cells isolated from buffy coats. In vitro stimulation of naïve murine B cells with rFlaA:Betv1 resulted in an mTOR- and MyD88-dependent production of IL-10 and rFlaA:Betv1 induced Bet v 1-reactive IgG production, which was not observed for IgA. rFlaA:Betv1-stimulated B cells formed a CD19+ CD24+ CD1d+ IgM+ CD38+ Breg subpopulation capable of suppressing Bet v 1-induced TH2 cytokine secretion in vitro. CONCLUSION: rFlaA:Betv1 can act as a thymus-independent B cell antigen, stimulating the mTOR- and MyD88-dependent differentiation of B cells displaying a regulatory phenotype, IL-10 secretion, antigen-binding antibody production, and a suppressive capacity in vitro.

Potential and limitations of epitope mapping and molecular targeting in Hymenoptera venom allergy.

Hymenoptera venom (HV) allergy can lead to life threatening conditions by specific IgE (sIgE)-mediated anaphylactic reactions. The knowledge about major allergens from venom of different clinically relevant species increased in the last decades, allowing the development of component-resolved diagnostics in which sIgE to single allergens is analysed. Despite these advances, the precise regions of the allergens that bind to IgE are only known for few HV allergens. The detailed characterization of IgE epitopes may provide valuable information to improve immunodiagnostic tests and to develop new therapeutic strategies using allergen-derived peptides or other targeted approaches. Epitope-resolved analysis is challenging, since the identification of conformational epitopes present in many allergens demands complex technologies for molecular analyses. Furthermore, functional analysis of the epitopes interaction with their respective ligands is needed to distinguish epitopes that can activate the allergic immune response, from those that are recognized by irrelevant antibodies or T cell receptors from non-effector cells. In this review, we focus on the use of mapping and molecular targeting approaches for characterization of the epitopes of the major venom allergens of clinically relevant Hymenoptera species. The screening of the most relevant allergen peptides by epitope mapping could be helpful for the development of molecules that target major and immunodominant epitopes blocking the allergen induced cellular reactions as novel approach for the treatment of HV allergy.

Arsenic Trioxide Decreases Lymphangiogenesis by Inducing Apoptotic Pathways and Inhibition of Important Endothelial Cell Receptors.

Tumor-induced lymphangiogenesis is strongly associated with the formation of tumor metastasis. Therefore, the regulation of lymphangiogenesis offers a promising target in cancer therapy. Arsenic trioxide (ATO) is highly effective in the treatment of patients with acute promyelocytic leukemia (APL). As ATO mediates anti-angiogenic effects on endothelial and tumor cells, we aimed to explore the impact of ATO on lymphangiogenesis in human lymphatic endothelial cells (LEC). The BrdU assay and flow cytometry analysis were used to evaluate the influence of ATO on the proliferation and cell cycle distribution of LECs. The lymphatic suppression effects of ATO were investigated in vitro using the lymphatic tube formation assay. The effects of ATO on apoptosis, mitochondrial membrane potential and endothelial cell receptors were investigated by Western blotting, ELISA, flow cytometry and qRT-PCR. The treatment of LECs with ATO attenuated cell proliferation, blocked tube formation and induced subG0/G1 arrest in LECs, thus suggesting enhanced apoptosis. Although subG0/G1 arrest was accompanied by the upregulation of p21 and p53, ATO treatment did not lead to visible cell cycle arrest in LECs. In addition, ATO caused apoptosis via the release of cytochrome c from mitochondria, activating caspases 3, 8 and 9; downregulating the anti-apoptotic proteins survivin, XIAP and cIAP-2; and upregulating the pro-apoptotic protein Fas. Furthermore, we observed that ATO inhibited the VEGF-induced proliferation of LECs, indicating that pro-survival VEGF/VEGFR signaling was affected by ATO treatment. Finally, we found that ATO inhibited the expression of the important endothelial cell receptors VEGFR-2, VEGFR-3, Tie-2 and Lyve-1. In conclusion, we demonstrate that ATO inhibits lymphangiogenesis by activating apoptotic pathways and inhibiting important endothelial cell receptors, which suggests that this drug should be further evaluated in the treatment of tumor-associated lymphangiogenesis.

Guideline on allergen immunotherapy in IgE-mediated allergic diseases: S2K Guideline of the German Society of Allergology and Clinical Immunology (DGAKI), Society of Pediatric Allergology and Environmental Medicine (GPA), Medical Association of German Allergologists (AeDA), Austrian Society of Allergology and Immunology (ÖGAI), Swiss Society for Allergology and Immunology (SSAI), German Dermatological Society (DDG), German Society of Oto-Rhino-Laryngology, Head and Neck Surgery (DGHNO-KHC), German Society of Pediatrics and Adolescent Medicine (DGKJ), Society of Pediatric Pulmonology (GPP), German Respiratory Society (DGP), German Professional Association of Otolaryngologists (BVHNO), German Association of Paediatric and Adolescent Care Specialists (BVKJ), Federal Association of Pneumologists, Sleep and Respiratory Physicians (BdP), Professional Association of German Dermatologists (BVDD).

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Role of the Steroid Sulfate Uptake Transporter Soat (Slc10a6) in Adipose Tissue and 3T3-L1 Adipocytes.

In addition to the endocrine and paracrine systems, peripheral tissues such as gonads, skin, and adipose tissue are involved in the intracrine mechanisms responsible for the formation of sex steroids via the transformation of dehydroepiandrosterone and dehydroepiandrosterone sulfate (DHEA/DHEAS) into potent androgenic and estrogenic hormones. Numerous studies have examined the relationship between overweight, central obesity, and plasma levels of DHEA and DHEAS. The sodium-dependent organic anion transporter Soat (Slc10a6) is a plasma membrane uptake transporter for sulfated steroids. Significantly increased expression of Slc10a6 mRNA has been previously described in organs and tissues of lipopolysaccharide (LPS)-treated mice, including white adipose tissue. These findings suggest that Soat plays a role in the supply of steroids in peripheral target tissues. The present study aimed to investigate the expression of Soat in adipocytes and its role in adipogenesis. Soat expression was analyzed in mouse white intra-abdominal (WAT), subcutaneous (SAT), and brown (BAT) adipose tissue samples and in murine 3T3-L1 adipocytes. In addition, adipose tissue mass and size of the adipocytes were analyzed in wild-type and Slc10a6 -/- knockout mice. Soat expression was detected in mouse WAT, SAT, and BAT using immunofluorescence. The expression of Slc10a6 mRNA was significantly higher in 3T3-L1 adipocytes than that of preadipocytes and was significantly upregulated by exposure to lipopolysaccharide (LPS). Slc10a6 mRNA levels were also upregulated in the adipose tissue of LPS-treated mice. In Slc10a6 -/- knockout mice, adipocytes increased in size in the WAT and SAT of female mice and in the BAT of male mice, suggesting adipocyte hypertrophy. The serum levels of adiponectin, resistin, and leptin were comparable in wild-type and Slc10a6 -/- knockout mice. The treatment of 3T3-L1 adipocytes with DHEA significantly reduced lipid accumulation, while DHEAS did not have a significant effect. However, following LPS-induced Soat upregulation, DHEAS also significantly inhibited lipid accumulation in adipocytes. In conclusion, Soat-mediated import of DHEAS and other sulfated steroids could contribute to the complex pathways of sex steroid intracrinology in adipose tissues. Although in cell cultures the Soat-mediated uptake of DHEAS appears to reduce lipid accumulation, in Slc10a6 -/- knockout mice, the Soat deletion induced adipocyte hyperplasia through hitherto unknown mechanisms.

IRE1 and PERK signaling regulates inflammatory responses in a murine model of contact hypersensitivity.

BACKGROUND: Contact sensitizers may interfere with correct protein folding. Generation of un-/misfolded proteins can activate the IRE-1 or PERK signaling pathways initiating the unfolded protein response (UPR) and thereby determine inflammatory immune responses. We have analyzed the effect of sensitizers with different potencies on the induction of UPR activation/inhibition and the subsequent generation of a pro-inflammatory micromilieu in vitro as well as the effect of UPR modulation on the inflammatory response in the murine contact hypersensitivity (CHS) in vivo. METHODS: Semi-quantitative and quantitative PCR, fluorescence microscopy, ELISA, NF-κB activation and translocation assays, DC/keratinocyte co-culture assay, FACS, and in vivo CHS experiments were performed. RESULTS: Sensitizers and irritants activate IRE-1 and PERK in murine and human keratinocytes. Synergistic effects occur after combination of different weak sensitizers / addition of irritants. Moreover, tolerogenic dinitrothiocyanobenzene can be converted into a strong sensitizer by pre-activation of the UPR. Blocking UPR signaling results in decreased NF-κB activation and cytokine production in keratinocytes and in activation marker downregulation in a HaCaT/THP-1 co-culture. Interestingly, not only systemic but also topical application of UPR inhibitors abrogates CHS responses in vivo. CONCLUSION: These observations highlight an important role of the UPR in determination of the inflammatory response in vitro and in vivo further underlining the importance of tissue stress and damage responses in the development of ACD and provide mechanistically based concepts as a basis for the development of new therapeutic approaches to treat allergic contact dermatitis.