Modern healthcare requires a proactive and individualized response to diseases, combining precision diagnosis and personalized treatment. Accordingly, the approach to patients with allergic diseases encompasses novel developments in the area of personalized medicine, disease phenotyping and endotyping and the development and application of reliable biomarkers. A detailed clinical history and physical examination followed by the detection of IgE immunoreactivity against specific allergens still represents the state of the art. However, nowadays, further emphasis focuses on the optimization of diagnostic and therapeutic standards and a large number of studies have been investigating the biomarkers of allergic diseases, including asthma, atopic dermatitis, allergic rhinitis, food allergy, urticaria and anaphylaxis. Various biomarkers have been developed by omics technologies, some of which lead to a better classification of the distinct phenotypes or endotypes. The introduction of biologicals to clinical practice increases the need for biomarkers for patient selection, prediction of outcomes and monitoring, to allow for an adequate choice of the duration of these costly and long-lasting therapies. Escalating healthcare costs together with questions on the efficacy of the current management of allergic diseases requires further development of a biomarker-driven approach. Here, we review biomarkers in diagnosis and treatment of asthma, atopic dermatitis, allergic rhinitis, viral infections, chronic rhinosinusitis, food allergy, drug hypersensitivity and allergen-immunotherapy with a special emphasis on specific IgE, microbiome and epithelial barrier. In addition, EAACI guidelines on biologicals are discussed within the perspective of biomarkers.
To the Editor: Asthma is a complex and heterogeneous chronic airway inflammatory disease with the involvement of environmental factors through epigenetic mechanisms.1 Accordingly, repeated injury, repair and regeneration of the airway epithelium following exposure to environmental factors and inflammation results in histological changes and functional abnormalities in the airway mucosal epithelium, which are associated with the pathophysiology of asthma.2Epigenetics is defined by heritable changes in gene expression without changes in the DNA sequence.3 Regulation of gene expression is mediated by different mechanisms such as DNA methylation, histone modifications and RNA-associated silencing by small non-coding RNAs. CpG sites are dinucleotides consisting of guanine and cytosine concentrated in clusters referred to CpG islands found at important regulatory sites, such as promoter and enhancer regions.4 Their de novo methylation occurs in response to various cellular stressors and signals by DNA methyltransferases (DNMT3a and 3b), which add a methyl group to position 5 of cytosine residues at the CpG site. During DNA replication both of the separated strands of DNA carry one methylated cytosine to be used as a template for duplication. Daughter DNA duplex strands will thus be hemi-methylated, which is recognized by a different DNA methyltransferase isoform (DNMT1).5 Because DNA methylation is a reversible process, the DNMTs are considered as a therapeutic target. Several DNMT inhibitors have been identified recently, among the non-nucleoside inhibitors, 4-aminoquoline-based inhibitors, such as SGI-1027 showed potent inhibitory activity. SGI-1027 occupies the binding site of DNMTs resulting in the prevention of access of target DNA to the substrate binding pocket.6We have demonstrated in previous studies from our laboratory that human primary bronchial epithelial cells (HBEC) isolated from patients with asthma showed lower barrier integrity compared to controls.7 To investigate the level of global methylation in HBEC, we investigated control and asthma samples for the long interspersed nuclear element-1 (LINE-1) methylation levels (Figure 1A). HBEC from asthma patients showed a tendency for higher global methylation levels, together with higher expression of 5-methylcytosine (5-mc) in immunofluorescence staining (Figure 1B). Next, we performed methylation profiling (Illumina Infinium EPIC array) to investigate genes methylated in ALI cultures of HBEC. Interestingly, in a highly methylated group of top 100 genes, we found many genes associated with cell growth, ion transport, and cytoskeletal remodeling (Figure S1). We kept our attention on the methylated epigenetic and tight junction (TJ) genes and further focused on TJs, especially zonula occludens and claudins which showed higher methylation in contrast to occludin, which was not methylated (Figure S2). As higher methylation levels were observed in HBEC of asthmatic origin, we inhibited the DNA methyltransferase enzyme with a specific inhibitor, SGI-1027, to demonstrate the role of CpG methylation on epithelial barrier integrity. ALI cultures were treated with the DNA methyltransferase inhibitor for 72 hours. Significantly decreased expression of 5-mc was observed after 48 hours of DNA-methyltransferase inhibition, demonstrating that the methylation of 5-methylcytosine (5-mc) in bronchial epithelium was reversed (Figure 2A). This prompted us to investigate the changes triggered by the inhibitor in epithelial cells. Further experiments showed increased transepithelial electric resistance (TER) in bronchial epithelial cells, in ALI from asthmatic donors after 48 hours of DNMT inhibition (Figure 2B). The link between barrier integrity and TER results were confirmed by the significantly decreased paracellular passage of FITC-labelled 4kD dextran after inhibition of DNMTs (Figure 2C). The reconstitution of TER in asthmatic ALI was associated with decreased protein DNMT1 expression and increased ZO-1 and claudin-18 proteins (Figure 2D). We also observed increased claudin-4, but not occludin expression upon DNMT inhibition (Figure S3). Increased expression of ZO-1 with an intact and honeycomb-like structure in the immunofluorescence staining of bronchial epithelial cells confirmed the effect on protein expression of bronchial epithelial barrier in asthma donors (Figure S4).Defective epithelial barrier has been established in asthma in addition to several chronic inflammatory diseases.8 Direct targeting of the epithelial barrier leakiness for the treatments represents an important target, however so far there is no treatment possibility targeting epigenetic mechanisms. The present study demonstrates an increased global methylation level in HBEC from asthmatic individuals. CpG methylation of specific genes is essential for the defect of epithelial barrier integrity, which is reversed upon DNMT inhibition. The inversion of CpG methylation, restores leakiness in the epithelium in asthma by increasing TER, decreasing paracellular flux and improves the structure of bronchial epithelial cells by increasing the expression of TJ proteins. The better understanding of the importance of epigenetic memory in chronic tissue inflammatory diseases together with the availability of treatment modalities targeting epigenetic mechanisms and transition of these molecules into the clinical studies may lead to curative treatment of allergic and autoimmune inflammatory diseases.9Paulina Wawrzyniak1, PhD,Krzysztof Krawczyk1,3, MSc,Swati Acharya5, PhD,Ge Tan1,7, PhD,Marcin Wawrzyniak1, PhD,Emmanuel Karouzakis4, PhD,Anita Dreher, Sci. Tech.,Bogdan Jakiela2, MD, PhD,Can Altunbulakli1, PhD,Marek Sanak2, MD, PhD,Liam O‘Mahony1,6, PD, PhD,Kari Nadeau5, MD, PhD,Cezmi A. Akdis1, MD1Swiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Davos, Switzerland, Christine Kühne-Center for Allergy Research and Education (CK-CARE)2Department of Medicine, Jagiellonian University Medical College, Krakow, Poland3Faculty of Biology and Environmental Protection, Department of Cellular Immunology, Lodz, Poland4Department of Rheumatology, University Hospital of Zurich5Departament of Medicine, Stanford University, United States6 Department of Medicine and School of Microbiology, APC Microbiome Ireland, University College Cork, Cork, Ireland.7 Functional Genomics Center Zurich, ETH Zurich/University of ZurichCorresponding author:Paulina WawrzyniakSwiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Davos, SwitzerlandObere Strasse 22,7270 Davos, SwitzerlandTel: +41 81 410 08 48Fax: +41 81 410 08 email@example.comConflict of interest:The authors declare that they have no conflicts of interest.Founding sources:Supported by Swiss National Science Foundation grants 310030_156823, and 320030_176190.Word count: 765Keywords: asthma, tight junction, CpG methylation, DNA methyltransferases,
Large scale, complex biobanking of biofluids for immunology research and testingRohit K. Gupta1, Vanitha Sampath2, Kari C. Nadeau2, Holden T. Maecker3,1 Biospecimen Resource Program, Office of Research, University of California, San Francisco, CA, USA.2 Sean N. Parker Center for Allergy and Asthma Research at Stanford University and Division of Pulmonary and Critical Care Medicine, Department of Medicine, Stanford University, Stanford, CA, USA.3 Institute for Immunity, Transplantation, and Infection, Stanford University, Stanford, CA, USA.Corresponding Author: Prof. Holden Maecker, Department of Microbiology and Immunology, Fairchild Science Building, room D039, 299 Campus Drive, Stanford, CA 94305-5124, USA, Email: firstname.lastname@example.org, Phone: (650) 723-1671Funding: Sean N. Parker Center for Allergy and Asthma Research at Stanford University and Sunshine Foundation.Conflict of Interest: Dr. Nadeau reports grants from National Institute of Allergy and Infectious Diseases (NIAID), Food Allergy Research & Education (FARE), End Allergies Together (EAT), Allergenis, and Ukko Pharma; Grant awardee at NIAID, National Institute of Environmental Health Sciences (NIEHS), National Heart, Lung, and Blood Institute (NHLBI), and the Environmental Protection Agency (EPA); is involved in Clinical trials with Regeneron, Genentech, AImmune Therapeutics, DBV Technologies, AnaptysBio, Adare Pharmaceuticals, and Stallergenes-Greer; Research Sponsorship by Novartis, Sanofi, Astellas, Nestle; Data and Safety Monitoring Board member at Novartis and NHLBI; Cofounded Before Brands, Alladapt, ForTra, and Iggenix; Chief Intellectual Office at FARE, Director of the World Allergy Organization (WAO) Center of Excellence at Stanford, Personal fees from Regeneron, Astrazeneca, ImmuneWorks, and Cour Pharmaceuticals; Consultant and Advisory Board Member at European Academy of Allergy and Clinical Immunology (EAACI) Research and Outreach Committee, Ukko, Before Brands, Alladapt, IgGenix, Probio, Vedanta, Centecor, Seed, Novartis, NHBLI, EPA, National Scientific Committee of Immune Tolerance Network (ITN) and NIH Programs; US patents for basophil testing, multifood immunotherapy and prevention, monoclonal antibody from plasmoblasts, and device for diagnostics. HM, RG, and VS indicate no conflict of interest.Author Contributions : All authors wrote and edited the final manuscript.Word Count: 978To the Editor:Biobanks have evolved from simple localized storage of samples in individual labs and clinics to large industrialized repositories with sophisticated sample life cycle infrastructure. By enabling collaborations between researchers working on different aspects of a disease, biobanks can bridge the gap between clinical care and research, accelerating medical care towards precision medicine. The concomitant advances in trans-omic technologies, big data analytics, and biorepositories make possible a coordinated, robust systems biology approach. Biobanks can be envisioned as a central hub responsible for compliant custodianship of specimens and associated clinical and biological data. Operationally, biobanks should strive to provide universal consent, standardized processing, cold-chain management, and quality control checks. Here, we discuss biobanks with respect to optimal utilization of biofluid derivatives, such as cells, supernatants, and genomic material, for immunology research and testing.A number of parameters need to be considered for specimen optimization and standardization based on sample type and downstream assays to be performed. Choices begin with the blood collection tubes to be used. For DNA and RNA analysis, EDTA anticoagulated blood is most common, as heparin can inhibit downstream polymerase reactions. However, at least one source suggests that citrate may provide higher quality RNA and DNA than other stabilizers.1For immunoassays, either serum or plasma can be effectively used, but there are subtle differences for some cytokine analytes.2 As such, a minimal requirement should be to use the same matrix (serum or plasma) and same anticoagulant if using plasma, for all samples to be compared in a study. For metabolome and lipidome studies, a report by Yin et al suggests EDTA plasma as the preferred matrix, since clotting in serum tubes activates additional processes, including the release of metabolites and enzymes from activated platelets.3For cellular assays such as flow cytometry, CyTOF, or single-cell RNAseq, viably cryopreserved peripheral blood mononuclear cells (PBMCs) or other cells of interest are key. Protocols for cryopreservation are readily available, but careful attention to both freezing and thawing protocols is particularly important to maintain viability and recovery. Traditionally, heparinized blood is used for Ficoll isolation of PBMCs, but other anticoagulants are generally equivalent for functionality of cryopreserved PBMC. Concomitant use of the whole blood for other purposes (e.g., stimulation or DNA isolation) may dictate the optimal anticoagulant—for example, EDTA would inhibit T cell receptor-based stimulation, but would be compatible with molecular assays). There are also variations to traditional Ficoll protocols, using Cell Preparation Tubes (CPTs) or SepMate tubes.4 These should be considered, as they can save time and labor and overcome some hurdles for standardization of the Ficoll procedure. The main drawbacks are a slight reduction in yield or increase in erythrocyte contamination. Importantly, training and protocol adherence are still important to prevent, for example, breakage of CPT from improper centrifuge holders, inadequate PBMC separation from improper spin speed, or loss of separation if CPT are shipped in very cold temperatures. Another variable to be considered is time to processing5, which is of course highly related to whether samples are shipped prior to processing (see Figure 1). This is particularly relevant to functional cellular assays. An alternative to overnight shipping and PBMC cryopreservation for functional assays is to perform on-site stimulation and stabilization of whole blood (e.g., Smart Tube Inc., http://smarttubeinc.com); however, proper monitoring of cold-chain storage is critical to ensure frozen specimens are not compromised. For example, when using the Smart Tube system, biobanks must maintain the frozen samples at -80°C, as micro-fluctuations in temperature can cause the specimens to coagulate, rendering them unusable. In any case, there are a number of potential variables that can be detrimental to downstream analysis and even reproducibility; biobanks should strive to harmonize collection, processing, and storage of samples related to biofluids.Research institutes often have multiple laboratories, each of which may be supporting various collections of human specimens. Unfortunately, most labs have employed their own data solutions to track and search for specimens, which has led to fragmented processes and inconsistent ontologies. Utilization of biospecimens that have been collected for scientific purposes continues to be problematic and may be more effective when paired with informatics tools that enable researchers to track, annotate, and interrogate.6 Biobanks should have a sample management system (SMS) which permits labs to accurately register, label (Figure 2), and track biospecimen inventory related to study participants7; in addition, the software should be configurable to align with lab workflows, while maintaining best practices for biobanking and ensuring governance can be maintained by the individual laboratory or institute. Further, for bioinventory tracking, it is critical to connect de-identified clinical attributes from electronic health records to biological assays following analysis of specimens in a central ecosystem; this enables researchers to rapidly search and request specimens for further analysis.8 To date, although many solutions have been developed to support virtual sample catalogs, most require extensive software engineering support in order to be deployed and require data to be migrated to a central database; robust and innovative solutions for identifying unused biospecimens in the life sciences are still desired.Long thought of as freezer farms, a biobank’s primary role has always been to provide proper cold-chain storage and logistics related to biospecimens. While much literature exists on optimal storage conditions and management,9 biobanks have evolved to now facilitate research in the life sciences that extend from the physical management of the sample life cycle to supporting standardized processing, assay optimization, and modernized data infrastructure. As compliant use of biospecimens continues to be a major component being addressed through community engagement, biobanks are poised to play an important role in medical research with increasing demand for high quality biospecimens. However, a number of questions and challenges exist regarding standardization, classification, management, sustainability, as well as ethical considerations including ownership and informed consent. Ultimately, improving how biospecimens are utilized for downstream analysis can accelerate our understanding of biological mechanisms and fuel a better tomorrow.Rohit K. Gupta1Vanitha Sampath2Kari C. Nadeau2Holden T. Maecker31 Biospecimen Resource Program, Office of Research, University of California, San Francisco, CA, USA.2 Sean N. Parker Center for Allergy and Asthma Research at Stanford University and Division of Pulmonary and Critical Care Medicine, Department of Medicine, Stanford University, Stanford, CA, USA.3 Institute for Immunity, Transplantation, and Infection, Stanford University, Stanford, CA, USA.
Atopic diseases have increased in prevalence over the last few decades and the rapid increases suggest that the predominant driving forces behind these increases are environmental factors rather than genetic alterations. A number of environmental factors have been implicated in the increased prevalence of allergic diseases. Predominant among them are increased exposure to pollutants and decreased exposure to microbes and parasitic infections. The hygiene hypothesis suggests that increased hygiene and lack of exposure to microbes and parasitic infections at an early age prevents the necessary stimulus to train the developing immune system to develop tolerogenic responses. Lifestyle factors, such as increased time spent indoors, use of antibiotics, and consumption of processed foods and decreased exposure to farm animals and pets, limit exposure to environmental allergens, infectious parasitic worms, and microbes. The lack of exposure to these factors is thought to prevent proper education and training of the immune system. Other factors that are also associated with increased risk of allergic diseases are Caesarian birth, birth order, tobacco smoke exposure and psychosomatic factors. Here, we review current knowledge on the environmental factors that have been shown to affect the development of allergic diseases and the recent developments in the field.
In December 2019, China reported the first cases of the coronavirus disease 2019 (COVID-19). This disease, caused by the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), has developed into a pandemic. To date it has resulted in ~5.6 million confirmed cases and caused 353,334 related deaths worldwide. Unequivocally, the COVID-19 pandemic is the gravest health and socio-economic crisis of our time. In this context, numerous questions have emerged in demand of basic scientific information and evidence-based medical advice on SARS-CoV-2 and COVID-19. Although the majority of the patients show a very mild, self-limiting viral respiratory disease, many clinical manifestations in severe patients are unique to COVID-19, such as severe lymphopenia and eosinopenia, extensive pneumonia, a “cytokine storm” leading to acute respiratory distress syndrome, endothelitis, thrombo-embolic complications and multiorgan failure. The epidemiologic features of COVID-19 are distinctive and have changed throughout the pandemic. Vaccine and drug development studies and clinical trials are rapidly growing at an unprecedented speed. However, basic and clinical research on COVID-19-related topics should be based on more coordinated high-quality studies. This paper answers pressing questions, formulated by young clinicians and scientists, on SARS-CoV-2, COVID-19 and allergy, focusing on the following topics: virology, immunology, diagnosis, management of patients with allergic disease and asthma, treatment, clinical trials, drug discovery, vaccine development and epidemiology. Over 140 questions were answered by experts in the field providing a comprehensive and practical overview of COVID-19 and allergic disease.