Research needs in allergy: an EAACI position paper, in collaboration with EFA

Abstract In less than half a century, allergy, originally perceived as a rare disease, has become a major public health threat, today affecting the lives of more than 60 million people in Europe, and probably close to one billion worldwide, thereby heavily impacting the budgets of public health systems. More disturbingly, its prevalence and impact are on the rise, a development that has been associated with environmental and lifestyle changes accompanying the continuous process of urbanization and globalization. Therefore, there is an urgent need to prioritize and concert research efforts in the field of allergy, in order to achieve sustainable results on prevention, diagnosis and treatment of this most prevalent chronic disease of the 21 st century. The European Academy of Allergy and Clinical Immunology (EAACI) is the leading professional organization in the field of allergy, promoting excellence in clinical care, education, training and basic and translational research, all with the ultimate goal of improving the health of allergic patients. The European Federation of Allergy and Airways Diseases Patients' Associations (EFA) is a non-profit network of allergy, asthma and Chronic Obstructive Pulmonary Disorder (COPD) patients' organizations. In support of their missions, the present EAACI Position Paper, in collaboration with EFA, highlights the most important research needs in the field of allergy to serve as key recommendations for future research funding at the national and European levels. Although allergies may involve almost every organ of the body and an array of diverse external factors act as triggers, there are several common themes that need to be prioritized in research efforts. As in many other chronic diseases, effective prevention, curative treatment and accurate, rapid diagnosis represent major unmet needs. Detailed phenotyping/endotyping stands out as widely required in order to arrange or re-categorize clinical syndromes into more coherent, uniform and treatment-responsive groups. Research efforts to unveil the basic pathophysiologic pathways and mechanisms, thus leading to the comprehension and resolution of the pathophysiologic complexity of allergies will allow for the design of novel patient-oriented diagnostic and treatment protocols. Several allergic diseases require well-controlled epidemiological description and surveillance, using disease registries, pharmacoeconomic evaluation, as well as large biobanks. Additionally, there is a need for extensive studies to bring promising new biotechnological innovations, such as biological agents, vaccines of modified allergen molecules and engineered components for allergy diagnosis, closer to clinical practice. Finally, particular attention should be paid to the difficult-to-manage, precarious and costly severe disease forms and/or exacerbations. Nonetheless, currently arising treatments, mainly in the fields of immunotherapy and biologicals, hold great promise for targeted and causal management of allergic conditions. Active involvement of all stakeholders, including Patient Organizations and policy makers are necessary to achieve the aims emphasized herein

Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC)-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds

Use of nasal filters for allergen exposure measurements in veterinary practices

Objectives: In this study, we applied novel nasal filters to assess animal allergen exposure of veterinary staff during their normal daily routine. Methods: Rhinix nasal filters were worn during work by 94 employees at different veterinary practices and 18 employees at a research institute, who acted as controls representing an animal-free environment. Contact with animals and the activities performed were documented by the study participants using a short questionnaire. Major allergens of cats (Fel d 1), dogs (Can f 1), and domestic mites (DM) were measured using fluorescence enzyme immunoassays. Results: At the practices, Can f 1 was detected in 98%, Fel d 1 in 82%, and DM allergens in 39% of the samples. Allergens were also detected in some control samples (6% for Can f 1, 39% for Fel d 1, and 17% for DM) but in very low concentrations. There was a highly significant difference between allergen levels in veterinary workers who treated at least one cat or dog during the sampling period and those who did not (2.66 vs. 0.70 ng/filter for Can f 1 and 1.01 vs. 0.20 ng/filter for Fel d 1). The amount of sampled Fel d 1 increased significantly with increasing duration of contact with cats. This effect was not observed for Can f 1. Conclusions: The majority of veterinary workers are exposed to dog and cat allergens, even without direct contact with these animals. Rhinix nasal filters may be considered a simple and easily applicable method for monitoring personal allergen exposure

Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status

Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific alpha MHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77-90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo

Upper and lower respiratory airway complaints among female veterinary staff

$\bf Objective$ Working with animals is characterized by exposure to particulate, biological or chemical matter, and respiratory complaints are common. The aim of our cross-sectional study was to assess the prevalence of respiratory symptoms and diagnoses among veterinary staff. $\bf Methods$ Participants working in veterinary practices were examined and a detailed questionnaire was used to collect data. IgE tests to common and animal allergens were performed to specify sensitization. Associations with respiratory outcomes were analysed using logistic regression models while controlling for potential confounders. $\bf Results$ Atopy was seen in 31% of the 109 female participants. Symptoms of rhinoconjunctivitis were the most frequent complaints ($\it n$ = 92; 84%). In 18% the diagnosis was confirmed by physicians. Symptoms of upper and lower airways were highly correlated and an asthma diagnosis was confirmed in 11% of participants. Modelling revealed that sensitization against cats/dogs was a significant risk factor for respiratory symptoms of upper [odds ratio (OR) 4.61; 95% confidence interval (CI) 1.13–18.81] and lower airways (OR 5.14; 95% CI 1.25–21.13), physician-confirmed rhinoconjunctivitis (OR 13.43; 95% CI 1.69–106.5) and asthma (OR 9.02; 95% CI 1.16–70.39) in assistant staff of small-animal practices. $\bf Conclusions$ In several cases, rhinoconjunctivitis worsened after entering the profession. Atopy and specific sensitization to cats/dogs were risk factors for health impairments. Thus, to implement preventive measures, veterinary practice staff should be educated that upper respiratory tract symptoms are not harmless and should be diagnosed and treated early

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

<div><p>Rationale</p><p>Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.</p><p>Methods and Results</p><p>In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.</p><p>Conclusion</p><p>Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.</p></div

Functional subtype characterization of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.

<p><b>(A)</b> Top graph, typical ventricular-like action potential (AP) of a cell from the EL group. Bottom graph, typical atrial-like AP from a CMs of the EH group. <b>(B)</b> Distribution of the cells in the two sorted groups. <b>(C)</b> Statistical analysis of AP parameters: left, action potential duration at 90% of repolarization (ADP90); mid, maximum rate of rise of the AP (max dV/dt); right, maximum diastolic polarization (MDP). Data are expressed as mean ± SEM. *** p ≤ 0.001 EL vs. EH. <b>(D)</b> Representative voltage ramps recordings from an E15.5 ERBB-2⁺/ITGA6^low CM (left) and an E15.5 ERBB-2⁺/ITGA6^high CM (right) show functional expression of inward and outward current components. Abbreviation: EL = E15.5 ERBB-2⁺/ITGA6^low, EH = E15.5 ERBB-2⁺/ITGA6^high, PL = P2 ITGA6^low, PH = P2 ITGA6^high.</p

Gene expression analysis of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.

<p><b>(A)</b> Normalized signal intensities of CM-specific marker genes: general CM-specific <i>Tnnt2</i> and <i>Nkx2-5</i>, ventricle-specific <i>Hey2</i> and <i>Irx4</i>, atrium-specific <i>Nr2f2</i> and <i>Fgf12</i>. Data are expressed as mean ± SD, n = 4. Statistical analysis: ANOVA, Benjamini-Hochberg correction for multiple testing p ≤ 0.05, Tukey post-hoc test *** p ≤ 0.001, ns = not significant. <b>(B)</b> Heat-map shows median-centered log2-transformed signal intensities of selected genes. The color code indicates expression relative to the gene-wise median of all samples. Abbreviation: EL = E15.5 ERBB-2⁺/ITGA6^low, EH = E15.5 ERBB-2⁺/ITGA6^high, PL = P2 ITGA6^low, PH = P2 ITGA6^high.</p

List of fold-change values of selected genes with general or subtype-specific expression in mouse cardiomyocytes.

<p>Positive fold-change values indicate a higher abundance in ITGA6^high as compared to ITGA6^low-sorted cells, negative values demonstrate a higher abundance in ITGA6^low-sorted cells in comparison to ITGA6^high. Differential gene expression was assumed for fold-change values ≥ 3.0 or ≤ -3.0.</p><p>List of fold-change values of selected genes with general or subtype-specific expression in mouse cardiomyocytes.</p

Differential expression of ITGA5 and ITGA6 on atrial and ventricular cardiomyocytes.

<p><b>(A)</b> E13.5 whole hearts and mechanically separated atrial and ventricular tissue were co-labeled for ITGA6 or ITGA5 and α-actinin. Histograms, ITGA6 or ITGA5 expression gated on α-actinin+ cells. <b>(B)</b> E13.5 whole-heart preparations co-stained with antibodies against ITGA6 or ITGA5 and MLC-2a or MLC-2v (labeled with AlexaFluor^® 488 goat anti-mouse IgG). Analysis gates set according to the secondary antibody control. Rectangles indicate ITGA6 low (green) and high (red) expressing myocytes. <b>(C)</b> Co-labeling of E11.5 –P2 mouse hearts for ITGA6 and α-actinin.</p