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.

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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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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Prognostic impact of eosinophils in mastocytosis: analysis of 2350 patients collected in the ECNM Registry.

Systemic mastocytosis (SM) is frequently associated with eosinophilia. To examine its prevalence and clinical impact in all WHO classification-based subcategories, we analyzed eosinophil counts in 2350 mastocytosis patients using the dataset of the European Competence Network on Mastocytosis. Ninety percent of patients had normal eosinophil counts, 6.8% mild eosinophilia (0.5-1.5 × 109/l), and 3.1% hypereosinophilia (HE; >1.5 × 109/l). Eosinophilia/HE were mainly present in patients with advanced SM (17%/19%), and only rarely recorded in patients with indolent and smoldering SM (5%/1%), and some patients with cutaneous mastocytosis. The eosinophil count correlated with organomegaly, dysmyelopoiesis, and the WHO classification, but not with mediator-related symptoms or allergy. Eosinophilia at diagnosis had a strong prognostic impact (p < 0.0001) on overall survival (OS) and progression-free survival (PFS), with a 10-year OS of 19% for patients with HE, 70% for those with mild eosinophilia, and 88% for patients with normal eosinophil counts. In 89% of patients with follow-up data (n = 1430, censored at start of cytoreductive therapy), eosinophils remained stable. In those with changing eosinophil counts (increase/decrease or mixed pattern), OS and PFS were inferior compared with patients with stable eosinophil counts. In conclusion, eosinophilia and HE are more prevalent in advanced SM and are predictors of a worse outcome.

IL-10 signaling in dendritic cells is required for tolerance induction in a murine model of allergic airway inflammation.

Allergen specific tolerance induction efficiently ameliorates subsequent allergen induced inflammatory responses. The underlying regulatory mechanisms have been attributed mainly to interleukin (IL)-10 produced by diverse hematopoietic cells, while targets of IL-10 in allergen specific tolerance induction have not yet been well defined. Here, we investigate potential cellular targets of IL-10 in allergen specific tolerance induction using mice with a cell type specific inactivation of the IL-10 receptor gene. Allergic airway inflammation was effectively prevented by tolerance induction in mice with IL-10 receptor (IL-10R) deficiency in T or B cells. Similarly, IL-10R on monocytes/macrophages and/or neutrophils was not required for tolerance induction. In contrast, tolerance induction was impaired in mice that lack IL-10R on dendritic cells: those mice developed an allergic response characterized by a pronounced neutrophilic lung infiltration, which was not ameliorated by tolerogenic treatment. In conclusion, our results show that allergen specific tolerance can be effectively induced without a direct impact of IL-10 on cells of the adaptive immune system, and highlight dendritic cells, but not macrophages nor neutrophils, as the main target of IL-10 during tolerance induction.

Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages.

The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors.IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors.

Human IgE is efficiently produced in glycosylated and biologically active form in lepidopteran cells.

TH2-biased immunity to parasites and allergens is often associated with increased levels of antigen-specific and high affinity IgE. The role in reacting against minute amounts of target structures and to provoke severe anaphylactic reactions renders IgE a mechanistically outstanding isotype. IgE represents the least abundant serum antibody isotype and exhibits a variety of peculiarities including structure, extensive glycosylation and effector functions. Despite large progress in antibody technologies, however, the recombinant access to isotypes beyond IgG such as IgE still is scarce. The capacity of expression systems has to meet the complex structural conformations and the extensive posttranslational modifications that are indispensable for biological activity. In order to provide alternatives to mammalian expression systems with often low yield and a more complex glycosylation pattern we established the recombinant production of the highly complex IgE isotype in insect cells. Recombinant IgE (rIgE) was efficiently assembled and secreted into the supernatant in yields of >30 mg/L. Purification from serum free medium using different downstream processing methods provided large amounts of rIgE. This exhibited a highly specific interaction with its antigen, therapeutic anti-IgE and its high affinity receptor, the FcεRI. Lectins and glyco-proteomic analyses proved the presence of prototypic insect type N-glycans on the epsilon heavy chain. Mediator release assays demonstrated a biological activity of the rIgE comparable to IgE derived from mammalian cells. In summary the expression in insect cells provides rIgE with variant glycosylation pattern, but retained characteristics and biological activity. Therefore our data contribute to the understanding of functional and structural aspects and potential use of the IgE isotype.

Neutrophils are required for both the sensitization and elicitation phase of contact hypersensitivity.

Allergic contact dermatitis and its animal model, contact hypersensitivity (CHS), are T cell-mediated inflammatory skin diseases induced by contact allergens. Though numerous cellular and molecular players are known, the mechanism of chemical-induced sensitization remains poorly understood. Here, we identify neutrophils as crucial players in the sensitization phase of CHS. Genetic deficiency of neutrophils caused by myeloid-specific deletion of Mcl-1 or antibody-mediated depletion of neutrophils before sensitization abrogated the CHS response. Neutrophil deficiency reduced contact allergen-induced cytokine production, gelatinase release, and reactive oxygen species production in naive mice. Mast cell deficiency inhibited neutrophil accumulation at the site of sensitization. In turn, neutrophils were required for contact allergen-induced release of further neutrophil-attracting chemokines, migration of DCs to the draining lymph nodes, and priming of allergen-specific T cells. Lymph node cells from mice sensitized in the absence of neutrophils failed to transfer sensitization to naive recipients. Furthermore, no CHS response could be induced when neutrophils were depleted before elicitation or when normally sensitized lymph node cells were transferred to neutrophil-deficient recipients, indicating an additional role for neutrophils in the elicitation phase. Collectively, our data identify neutrophils to be critically involved in both the sensitization and elicitation phase of CHS.

Efficiency of dendritic cell vaccination against B16 melanoma depends on the immunization route.

Dendritic cells (DC) presenting tumor antigens are crucial to induce potent T cell-mediated anti-tumor immune responses. Therefore DC-based cancer vaccines have been established for therapy, however clinical outcomes are often poor and need improvement. Using a mouse model of B16 melanoma, we found that the route of preventive DC vaccination critically determined tumor control. While repeated DC vaccination did not show an impact of the route of DC application on the prevention of tumor growth, a single DC vaccination revealed that both the imprinting of skin homing receptors and an enhanced proliferation state of effector T cells was seen only upon intracutaneous but not intravenous or intraperitoneal immunization. Tumor growth was prevented only by intracutaneous DC vaccination. Our results indicate that under suboptimal conditions the route of DC vaccination crucially determines the efficiency of tumor defense. DC-based strategies for immunotherapy of cancer should take into account the immunization route in order to optimize tissue targeting of tumor antigen specific T cells.

A potential role for P2X7R in allergic airway inflammation in mice and humans.

P2X7R deficiency is associated with a less severe outcome in acute and chronic inflammatory disorders. Recently, we demonstrated that extracellular adenosine triphosphate is involved in the pathogenesis of asthma by modulating the function of dendritic cells (DCs). However, the role of the purinergic receptor subtype P2X7 is unknown. To elucidate the role of P2X7R in allergic airway inflammation (AAI) in vitro and in vivo, P2X7R expression was measured in lung tissue and immune cells of mice or in humans with allergic asthma. By using a specific P2X7R-antagonist and P2X7R-deficient animals, the role of this receptor in acute and chronic experimental asthma was explored. P2X7R was found to be up-regulated during acute and chronic asthmatic airway inflammation in mice and humans. In vivo experiments revealed the functional relevance of this finding because selective P2X7R inhibition or P2X7R deficiency was associated with reduced features of acute and chronic asthma in the ovalbumin-alum or HDM model of AAI. Experiments with bone marrow chimeras emphasized that P2X7R expression on hematopoietic cells is responsible for the proasthmatic effects of P2X7R signaling. In the DC-driven model of AAI, P2X7R-deficient DCs showed a reduced capacity to induce Th2 immunity in vivo. Up-regulation of P2X7R on BAL macrophages and blood eosinophils could be observed in patients with chronic asthma. Our data suggest that targeting P2X7R on hematopoietic cells (e.g., DCs or eosinophils) might be a new therapeutic option for the treatment of asthma.

Lack of the purinergic receptor P2X(7) results in resistance to contact hypersensitivity.

Sensitization to contact allergens requires activation of the innate immune system by endogenous danger signals. However, the mechanisms through which contact allergens activate innate signaling pathways are incompletely understood. In this study, we demonstrate that mice lacking the adenosine triphosphate (ATP) receptor P2X(7) are resistant to contact hypersensitivity (CHS). P2X(7)-deficient dendritic cells fail to induce sensitization to contact allergens and do not release IL-1ß in response to lipopolysaccharide (LPS) and ATP. These defects are restored by pretreatment with LPS and alum in an NLRP3- and ASC-dependent manner. Whereas pretreatment of wild-type mice with P2X(7) antagonists, the ATP-degrading enzyme apyrase or IL-1 receptor antagonist, prevents CHS, IL-1ß injection restores CHS in P2X(7)-deficient mice. Thus, P2X(7) is a crucial receptor for extracellular ATP released in skin in response to contact allergens. The lack of P2X(7) triggering prevents IL-1ß release, which is an essential step in the sensitization process. Interference with P2X(7) signaling may be a promising strategy for the prevention of allergic contact dermatitis.

Differential impact of diesel particle composition on pro-allergic dendritic cell function.

Diesel exhaust particles (DEP) were described as potent adjuvant in the induction and maintenance of allergic diseases, suggesting that they might play a role in the increase of allergic diseases in the industrialized countries. However, the cellular basis by which these particles enhance allergic immune responses is still a matter of debate. Thus, we exposed immature murine bone marrow-derived dendritic cells (BMDC) to different particles or particle-associated organic compounds in the absence or presence of the maturation stimuli lipopolysaccharide (LPS) and analyzed the cellular maturation, viability, and cytokine production. Furthermore, we monitored the functionality of particle-exposed BMDC to suppress B cell isotype switching to immunoglobulin (Ig) E. Only highly polluted DEP (standard reference material 1650a [SRM1650a]) but not particle-associated organic compounds or less polluted DEP from modern diesel engines were able to modulate the dendritic cell phenotype. SRM1650a particles significantly suppressed LPS-induced IL-12p70 production in murine BMDC, whereas cell-surface marker expression was not altered. Furthermore, SRM1650a-exposed immature BMDC lost the ability to suppress IgE isotype switch in B cells. This study revealed that highly polluted DEP not only interfere with dendritic cell maturation but also additionally with dendritic cell function, thus suggesting a role in T(h)2 immune deviation.