Paving the way of systems biology and precision medicine in allergic diseases: the MeDALL success story: Mechanisms of the Development of ALLergy; EU FP7-CP-IP; Project No: 261357; 2010-2015.

MeDALL (Mechanisms of the Development of ALLergy; EU FP7-CP-IP; Project No: 261357; 2010-2015) has proposed an innovative approach to develop early indicators for the prediction, diagnosis, prevention and targets for therapy. MeDALL has linked epidemiological, clinical and basic research using a stepwise, large-scale and integrative approach: MeDALL data of precisely phenotyped children followed in 14 birth cohorts spread across Europe were combined with systems biology (omics, IgE measurement using microarrays) and environmental data. Multimorbidity in the same child is more common than expected by chance alone, suggesting that these diseases share causal mechanisms irrespective of IgE sensitization. IgE sensitization should be considered differently in monosensitized and polysensitized individuals. Allergic multimorbidities and IgE polysensitization are often associated with the persistence or severity of allergic diseases. Environmental exposures are relevant for the development of allergy-related diseases. To complement the population-based studies in children, MeDALL included mechanistic experimental animal studies and in vitro studies in humans. The integration of multimorbidities and polysensitization has resulted in a new classification framework of allergic diseases that could help to improve the understanding of genetic and epigenetic mechanisms of allergy as well as to better manage allergic diseases. Ethics and gender were considered. MeDALL has deployed translational activities within the EU agenda.

Are allergic multimorbidities and IgE polysensitization associated with the persistence or re-occurrence of foetal type 2 signalling? The MeDALL hypothesis.

Allergic diseases [asthma, rhinitis and atopic dermatitis (AD)] are complex. They are associated with allergen-specific IgE and nonallergic mechanisms that may coexist in the same patient. In addition, these diseases tend to cluster and patients present concomitant or consecutive diseases (multimorbidity). IgE sensitization should be considered as a quantitative trait. Important clinical and immunological differences exist between mono- and polysensitized subjects. Multimorbidities of allergic diseases share common causal mechanisms that are only partly IgE-mediated. Persistence of allergic diseases over time is associated with multimorbidity and/or IgE polysensitization. The importance of the family history of allergy may decrease with age. This review puts forward the hypothesis that allergic multimorbidities and IgE polysensitization are associated and related to the persistence or re-occurrence of foetal type 2 signalling. Asthma, rhinitis and AD are manifestations of a common systemic immune imbalance (mesodermal origin) with specific patterns of remodelling (ectodermal or endodermal origin). This study proposes a new classification of IgE-mediated allergic diseases that allows the definition of novel phenotypes to (i) better understand genetic and epigenetic mechanisms, (ii) better stratify allergic preschool children for prognosis and (iii) propose novel strategies of treatment and prevention.

Severe chronic allergic (and related) diseases: a uniform approach--a MeDALL--GA2LEN--ARIA position paper.

Concepts of disease severity, activity, control and responsiveness to treatment are linked but different. Severity refers to the loss of function of the organs induced by the disease process or to the occurrence of severe acute exacerbations. Severity may vary over time and needs regular follow-up. Control is the degree to which therapy goals are currently met. These concepts have evolved over time for asthma in guidelines, task forces or consensus meetings. The aim of this paper is to generalize the approach of the uniform definition of severe asthma presented to WHO for chronic allergic and associated diseases (rhinitis, chronic rhinosinusitis, chronic urticaria and atopic dermatitis) in order to have a uniform definition of severity, control and risk, usable in most situations. It is based on the appropriate diagnosis, availability and accessibility of treatments, treatment responsiveness and associated factors such as comorbidities and risk factors. This uniform definition will allow a better definition of the phenotypes of severe allergic (and related) diseases for clinical practice, research (including epidemiology), public health purposes, education and the discovery of novel therapies.

MeDALL (Mechanisms of the Development of ALLergy): an integrated approach from phenotypes to systems medicine.

The origin of the epidemic of IgE-associated (allergic) diseases is unclear. MeDALL (Mechanisms of the Development of ALLergy), an FP7 European Union project (No. 264357), aims to generate novel knowledge on the mechanisms of initiation of allergy and to propose early diagnosis, prevention, and targets for therapy. A novel phenotype definition and an integrative translational approach are needed to understand how a network of molecular and environmental factors can lead to complex allergic diseases. A novel, stepwise, large-scale, and integrative approach will be led by a network of complementary experts in allergy, epidemiology, allergen biochemistry, immunology, molecular biology, epigenetics, functional genomics, bioinformatics, computational and systems biology. The following steps are proposed: (i) Identification of 'classical' and 'novel' phenotypes in existing birth cohorts; (ii) Building discovery of the relevant mechanisms in IgE-associated allergic diseases in existing longitudinal birth cohorts and Karelian children; (iii) Validation and redefinition of classical and novel phenotypes of IgE-associated allergic diseases; and (iv) Translational integration of systems biology outcomes into health care, including societal aspects. MeDALL will lead to: (i) A better understanding of allergic phenotypes, thus expanding current knowledge of the genomic and environmental determinants of allergic diseases in an integrative way; (ii) Novel diagnostic tools for the early diagnosis of allergy, targets for the development of novel treatment modalities, and prevention of allergic diseases; (iii) Improving the health of European citizens as well as increasing the competitiveness and boosting the innovative capacity of Europe, while addressing global health issues and ethical issues.

Specific gene expression signature associated with development of autoimmune type-I diabetes using whole-blood microarray analysis.

Understanding the pathogenesis of type-I diabetes (T1D) is hindered in humans by the long autoimmune process occurring before clinical onset and by the difficulty to study the pancreas directly. Alternatively, exploring body fluids and particularly peripheral blood can provide some insights. Indeed, circulating cells can function as 'sentinels', with subtle changes in gene expression occurring in association with disease. Therefore, we investigated the gene expression profiles of circulating blood cells using Affymetrix microarrays. Whole-blood samples from 20 first-degree relatives of T1D children with autoimmune diabetes-related antibodies, 19 children immediately after the onset of clinical T1D and 20 age- and sex-matched healthy controls were collected in PAXgene tubes. A global gene expression analysis with MDS approach allowed the discrimination of pre-diabetic subjects, diabetic patients and healthy controls. Univariate statistical analysis highlighted 107 distinct genes differently expressed between these three groups. Two major gene expression profiles were characterized, including type-I IFN-regulated genes and genes associated with biosynthesis and oxidative phosphorylation. Our results showed the presence of early functional modifications associated with T1D, which could help to understand the disease and suggest possible avenues for therapeutic interventions.

Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.

The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3-expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.

Towards a European health research and innovation cloud (HRIC).

The European Union (EU) initiative on the Digital Transformation of Health and Care (Digicare) aims to provide the conditions necessary for building a secure, flexible, and decentralized digital health infrastructure. Creating a European Health Research and Innovation Cloud (HRIC) within this environment should enable data sharing and analysis for health research across the EU, in compliance with data protection legislation while preserving the full trust of the participants. Such a HRIC should learn from and build on existing data infrastructures, integrate best practices, and focus on the concrete needs of the community in terms of technologies, governance, management, regulation, and ethics requirements. Here, we describe the vision and expected benefits of digital data sharing in health research activities and present a roadmap that fosters the opportunities while answering the challenges of implementing a HRIC. For this, we put forward five specific recommendations and action points to ensure that a European HRIC: i) is built on established standards and guidelines, providing cloud technologies through an open and decentralized infrastructure; ii) is developed and certified to the highest standards of interoperability and data security that can be trusted by all stakeholders; iii) is supported by a robust ethical and legal framework that is compliant with the EU General Data Protection Regulation (GDPR); iv) establishes a proper environment for the training of new generations of data and medical scientists; and v) stimulates research and innovation in transnational collaborations through public and private initiatives and partnerships funded by the EU through Horizon 2020 and Horizon Europe.

Sputum proteomic signature of gastro-oesophageal reflux in patients with severe asthma.

Gastro-oesophageal reflux disease (GORD) has long been associated with poor asthma control without an established cause-effect relationship. 610 asthmatics (421 severe/88 mild-moderate) and 101 healthy controls were assessed clinically and a subset of 154 severe asthmatics underwent proteomic analysis of induced sputum using untargeted mass spectrometry, LC-IMS-MSE. Univariate and multiple logistic regression analyses (MLR) were conducted to identify proteins associated with GORD in this cohort. When compared to mild/moderate asthmatics and healthy individuals, respectively, GORD was three- and ten-fold more prevalent in severe asthmatics and was associated with increased asthma symptoms and oral corticosteroid use, poorer quality of life, depression/anxiety, obesity and symptoms of sino-nasal disease. Comparison of sputum proteomes in severe asthmatics with and without active GORD showed five differentially abundant proteins with described roles in anti-microbial defences, systemic inflammation and epithelial integrity. Three of these were associated with active GORD by multiple linear regression analysis: Ig lambda variable 1-47 (p = 0·017) and plasma protease C1 inhibitor (p = 0·043), both in lower concentrations, and lipocalin-1 (p = 0·034) in higher concentrations in active GORD. This study provides evidence which suggests that reflux can cause subtle perturbation of proteins detectable in the airways lining fluid and that severe asthmatics with GORD may represent a distinct phenotype of asthma.

The major histocompatibility complex in the chicken.

The chicken B complex is the first non-mammalian MHC characterized at the molecular level. It differs from the human HLA and murine H-2 complexes in the small size of the class I (B-F) and class II (B-L) genes and their close proximity. This proximity accounts for the absence of recombination between B-F and B-L genes and leaves no space for class III genes. Moreover the B-F and B-L genes are tightly linked to unrelated genes absent from mammalian MHCs, such as the polymorphic B-G genes and a member of the G protein beta subunit family. This linkage could form the basis for resistance to viral-induced tumors associated with some B complex haplotypes.

Sequence analysis of the human major histocompatibility gene SX alpha.

The DP subregion of the human major histocompatibility complex contains two closely linked gene pairs, DP alpha, DP beta and SX alpha, SX beta. The exon-intron organization and the complete DNA sequence of the SX alpha gene are reported here. There are several mutations within the SX alpha gene which strongly suggest that it is a pseudogene. These include two frameshift mutations, one in the alpha 1 domain and the other in the cytoplasmic domain. A 5' splice site mutation at the end of the alpha 1 exon also exists. DNA sequence homology between DP alpha and SX alpha suggests that these genes arose through a gene duplication event.

Synthetic CD4 exocyclic peptides antagonize CD4 holoreceptor binding and T cell activation.

We have developed peptide analogs to analyze precise human CD4 substructures involved in MHC class II binding. Forms of the complementarity determining-like regions (CDRs) of the D1 domain of human CD4 were reproduced as synthetic aromatically modified exocyclic (AME) analogs and tested for their ability to block CD4-MHC II interactions and T cell activation. The exocyclic derived from CDR3 (residues 82-89) of human CD4, which specifically associated with CD4 on the T cell surface to create a heteromeric CD4 complex, blocked IL-2 production and antagonized the normal function of the CD4 receptor. The approach of creating novel synthetic antagonistic receptor complexes may represent a new receptor specific pharmaceutical approach to modulate biological function.

Bridging Systems Medicine and Patient Needs.

While there is widespread consensus on the need both to change the prevailing research and development (R&D) paradigm and provide the community with an efficient way to personalize medicine, ecosystem stakeholders grapple with divergent conceptions about which quantitative approach should be preferred. The primary purpose of this position paper is to contrast these approaches. The second objective is to introduce a framework to bridge simulation outputs and patient outcomes, thus empowering the implementation of systems medicine.

CD4-targeted immune intervention: a strategy for the therapy of AIDS and autoimmune disease.

The cell-surface antigen CD4 plays a pivotal role in the class II MHC-restricted response of specific T lymphocytes, and serves as the major receptor of human immunodeficiency virus (HIV). The recent elucidation of CD4 function in physiological and pathological conditions has improved prospects for CD4-targeted immune therapy by facilitating the design of therapeutic strategies aimed at blocking CD4 function, delivering immunosuppressive signals via this receptor molecule, or selectively depleting CD4+ cells.

Detailed transcript map of a 810-kb region at 11p14 involving identification of 10 novel human 3' exons.

A limited number of genes, including the human brain-derived neutrotrophic factor (BDNF) gene, have been identified in the human chromosome 11p14 region. Since this area is involved in a genetic disorder (WAGR syndrome) and because of interest in studying the regulation of the human BDNF gene, we have established a detailed transcript map of a 810-kb region clone in a yeast artificial chromosome (YAC), corresponding to a portion of this genomic locus. A set of nested deletion mutants has been generated to map genes at a mean resolution of 75kb. Four genic markers from available mapping databases have been mapped on the YAC. Ten potential novel human exons have been isolated by a 3' terminal exon trapping procedure directly applied to purified YAC DNA. Most of these exons display polyadenylation signals and they all yield positive signals in RT-PCR experiments, confirming their status of transcribed sequences. The BDNF gene is now co-localised with three other genes on a 120 kb DNA fragment.

Nucleotide sequence of a cloned cDNA corresponding to secreted mu chain of mouse immunoglobulin.

The cDNA complementary to a mouse immunoglobulin mu heavy chain mRNA has been cloned into the PstI site of the plasmid vector pBR322. A hybrid plasmid pmu 183.5 containing a 1850 bp insert has been selected by differential screening. The nucleotide sequence of the inset encodes the four constant domains, the terminal piece and the 3'-untranslated region.

Rat immunoglobulin delta heavy chain gene: nucleotide sequence derived from cloned cDNA.

Rat immunoglobulin delta heavy-chain mRNA has been isolated. RNA blot analysis revealed that this mRNA with a length of 1.8 kb encodes for the secreted form of IgD. The corresponding cDNA was cloned in plasmid pBR322 and its sequence was determined. The hybrid plasmid contains a 775-bp insert comprising a partial C delta 1 sequence and complete C delta H, C delta 3, C delta DC and 3' untranslated sequences. Rat and mouse IgD amino acid sequences show striking homology in C delta 3 and C delta DC regions.

Cloning and mapping of 5' exons from the gene encoding chicken beta nerve growth factor.

An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs.

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.

Mapping of 59 EST gene markers in 31 intervals spanning the human X chromosome.

The positioning of Expressed Sequence Tags (ESTs) constitutes an important step towards a functional map of the human genome, including candidate genes for human genetic disorders that have been localized by linkage analysis. We localized 59 ESTs on the human X chromosome, including 44 derived from infant brain and 15 from adult muscle cDNA libraries. Localizations by a somatic cell hybrid panel were refined for five cDNAs by mapping them in yeast artificial chromosome (YAC) contigs.