Mechanisms of Allergen-Antibody Interaction of Cockroach Allergen Bla g 2 with Monoclonal Antibodies That Inhibit IgE Antibody Binding

BACKGROUND: Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2. METHODOLOGY/PRINCIPAL FINDINGS: Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab' fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-π interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients. CONCLUSIONS/SIGNIFICANCE: Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates

Impaired innate interferon induction in severe therapy resistant atopic asthmatic children

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

In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients

Budesonide and Formoterol Reduce Early Innate Anti-Viral Immune Responses In Vitro

Asthma is a chronic inflammatory airways disease in which respiratory viral infections frequently trigger exacerbations. Current treatment of asthma with combinations of inhaled corticosteroids and long acting beta2 agonists improves asthma control and reduces exacerbations but what impact this might have on innate anti-viral immunity is unclear. We investigated the in vitro effects of asthma drugs on innate anti-viral immunity. Peripheral blood mononuclear cells (PBMC) from healthy and asthmatic donors were cultured for 24 hours with the Toll-like receptor 7 agonist, imiquimod, or rhinovirus 16 (RV16) in the presence of budesonide and/or formoterol. Production of proinflammatory cytokines and expression of anti-viral intracellular signalling molecules were measured by ELISA and RT-PCR respectively. In PBMC from healthy donors, budesonide alone inhibited IP-10 and IL-6 production induced by imiquimod in a concentration-dependent manner and the degree of inhibition was amplified when budesonide and formoterol were used in combination. Formoterol alone had little effect on these parameters, except at high concentrations (10−6 M) when IL-6 production increased. In RV16 stimulated PBMC, the combination of budesonide and formoterol inhibited IFNα and IP-10 production in asthmatic as well as healthy donors. Combination of budesonide and formoterol also inhibited RV16-stimulated expression of the type I IFN induced genes myxovirus protein A and 2′, 5′ oligoadenylate synthetise. Notably, RV16 stimulated lower levels of type Myxovirus A and oligoadenylate synthase in PBMC of asthmatics than control donors. These in vitro studies demonstrate that combinations of drugs commonly used in asthma therapy inhibit both early pro-inflammatory cytokines and key aspects of the type I IFN pathway. These findings suggest that budesonide and formoterol curtail excessive inflammation induced by rhinovirus infections in patients with asthma, but whether this inhibits viral clearance in vivo remains to be determined

Viruses exacerbating chronic pulmonary disease: the role of immune modulation

Chronic pulmonary diseases are a major cause of morbidity and mortality and their impact is expected to increase in the future. Respiratory viruses are the most common cause of acute respiratory infections and it is increasingly recognized that respiratory viruses are a major cause of acute exacerbations of chronic pulmonary diseases such as asthma, chronic obstructive pulmonary disease and cystic fibrosis. There is now increasing evidence that the host response to virus infection is dysregulated in these diseases and a better understanding of the mechanisms of abnormal immune responses has the potential to lead to the development of new therapies for virus-induced exacerbations. The aim of this article is to review the current knowledge regarding the role of viruses and immune modulation in chronic pulmonary diseases and discuss avenues for future research and therapeutic implications

Der Modellstudiengang Humanmedizin in Oldenburg - European Medical School Oldenburg-Groningen

In the summer of 2012 the School of Medicine and Health Sciences at the Carl von Ossietzky University of Oldenburg became the first new medical faculty to be founded in Germany in more than 20 years. The faculty was established within the framework of the European Medical School Oldenburg Groningen, a cooperation project between the University of Oldenburg and the University of Groningen. In addition to the University of Groningen and its faculty of medical sciences (Universitair Medisch Centrum Groningen - UMCG), four hospitals in Oldenburg are involved in the programme as cooperation partners, as well as a network of general practitioner practices that provide training and academic teaching hospitals across northwest Germany. The programme itself is a model medical degree programme with a modular structure, a highly integrative approach and an early and consistent focus on practical skills and patient-centredness. In addition to the early introduction to outpatient care in the first years of study, longitudinal pathways and a strong focus on research with early integration of scientific activities into medical studies are the defining characteristics of this programme. The two faculties in Oldenburg and Groningen coordinated their respective curriculums during the founding phase and recognise each other's study modules as equivalent to their own. This has created the preconditions for students from Oldenburg to obtain Dutch qualifications (Bachelor of Human Life Sciences and/or Master of Science in Medicine) in addition to the German "Staatsexamen" (the state examination in medicine) under certain circumstances. Irrespective of whether they intend to obtain these qualifications, all students from Oldenburg must spend at least a year studying at the partner university in Groningen. In exchange, up to 40 students from Groningen have the option to complete part of their studies in Oldenburg.Im Sommer 2012 wurde mit der Fakultät für Medizin und Gesundheitswissenschaften an der Carl von Ossietzky Universität Oldenburg zum ersten Mal seit über 20 Jahren wieder eine medizinische Fakultät in Deutschland gegründet. Diese Fakultät entstand im Rahmen der sog. European Medical School Oldenburg Groningen, einem Kooperationsprojekt mit der Rijksuniversiteit Groningen. Neben der Rijksuniversiteit Groningen mit ihrer universitätsmedizinischen Einrichtung des UMCG (Universitair Medisch Centrum Groningen) sind in Oldenburg vier Krankenhäuser als Kooperationspartner beteiligt. Dazu kommen allgemeinärztliche Hospitationspraxen und akademische Lehrkrankenhäuser im gesamten Nordwesten.Der Studiengang selbst ist ein modularisierter Modellstudiengang mit stark ausgeprägter Integration und früher und konsequenter Praxis- und Patientenorientierung. Neben der Einführung der ambulanten Medizin in die ersten Studienjahre sind longitudinale Pfade und insbesondere der Forschungsbezug mit früher Einbindung der Wissenschaftlichkeit in das Studium Kennzeichen dieses Studiengangs.Die beiden Fakultäten in Oldenburg und Groningen haben in der Gründungsphase ihre Curricula aufeinander abgestimmt und erkennen gegenseitig Studienmodule als gleichwertig an. Dadurch wurden Voraussetzungen geschaffen, dass Studierende aus Oldenburg unter bestimmten Voraussetzungen neben dem deutschen Staatsexamen auch niederländische Abschlüsse (Bachelor of Human Life Sciences und/oder Master of Science in Geneeskunde) erwerben konnten. Unabhängig von diesen Abschlüssen müssen alle Studierenden aus Oldenburg mindestens ein Jahr lang an der Partneruniversität in Groningen studieren. Gleichzeitig können bis zu 40 Studierende aus Groningen einen Teil ihres Studiums in Oldenburg verbringen

6 Jahre European Medical School Oldenburg-Groningen: offen für neue Wege im Medizinstudium

T. Green's Flying Coaster - FC3 - photographed building up 29 September 1980