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## **Meet the editor**

Prof. Celso Pereira is head-chief of Clinical Immunology at Medicine Faculty of Coimbra University. He is also a graduated specialist in Immuno-Allergy at Coimbra's University Hospital Centre, Portugal. He was the past-president of the Immuno-allergy Board of the Portuguese Medical Association. His main activities include clinical practice, education (pre and post graduate)

and clinical and laboratorial research. Prof. Pereira is member of national committee for vaccination and coordinator of some clinical guidelines approved and under application by Portuguese health authorities. He is also a member of a national committee for the diagnostic procedures in the field of Allergy and Clinical Immunology. His scientific interests include the participation and development of several research projects, but now his main interest is in the mechanisms of allergy and specific immunotherapy. Prof. Pereira edited two scientific books, and has more than 94 scientific publications including book chapters, and scientific papers. He has published over 300 abstracts related to the developed research projects and presented his work at scientific events. He was also honored with 34 scientific awards. Prof. Pereira was responsible for the first subcutaneous application of latex immunotherapy and for the first long term sublingual native *Pru p3* desensitization. During his Iberian Chapter Presidency of the Latin-American Allergy, Asthma and Immunology Society, he coordinated an international multicenter study focusing aeroallergen sensitization in the Iberian Peninsula. He is an associate editor of 5 scientific Journals related to Allergy and Clinical Immunology.

## Contents

#### **Preface XI**

## Chapter 8 **Rehabilitation of Allergic Diseases in Children — Climatothalassotherapy as a Power Tool in Achieving Optimal Disease Control 167** Silvije Šegulja, Milica Zvonarek-Valković, Daniel Buljević and Renata Volkmer-Starešina

## Preface

Chapter 8 **Rehabilitation of Allergic Diseases in Children —**

**in the Management Paradigm 179**

Rebecca Scherr and Mary Beth Hogan

**Disease Control 167**

Volkmer-Starešina

**VI** Contents

Chapter 10 **Eosinophilic Esophagitis 215**

**Climatothalassotherapy as a Power Tool in Achieving Optimal**

Silvije Šegulja, Milica Zvonarek-Valković, Daniel Buljević and Renata

José Manuel Lucas, Ana Moreno-Salvador and Luis García-Marcos

Chapter 9 **Specific Immunotherapy in Food Allergy — Towards a Change**

Increasing prevalence of allergic diseases has been observed all over the world, particularly in the last decades. Despite the progress in anti-inflammatory treatment and allergen specif‐ ic treatments or new immunemodulatory therapies, a significant proportion of patients still suffer clinically under controlled conditions and obvious interference on life quality.

The clinical expression of allergic diseases are heterogeneous, as well as the pathogenic mechanism, the inflammatory cells involved, the biological mediators, the genetic engage‐ ment and molecular pattern specific for each condition. So, it is not possible to develop in a sustainable way in this book all forms of allergic diseases. Therefore, in this book, we tried to include the crucial issues that are recent contributions to learning: less frequent topics, with very promising therapy approaches.

The quality and scientific meticulousness of the authors coming from several countries, con‐ tribute to the strength of this book, especially as it reflects local and regional realities that characterize the diversity of allergic expression. In fact, the etiology and clinical response of allergic patient is very different, depending on the location, habits, socio-economic condi‐ tions and even the specific interests of key lines of investigation developed, that the authors have incorporated into the chapters, but always guided by higher values of accuracy.

The chapters on treatment modalities and new strategies bring the latest concepts on the use of actual and very promising researches based on the better understanding of the mecha‐ nism of the allergic disease.

The effort and dedication of the authors in the time expended in the writing of the different chapters is fully demonstrated by the final quality of this project. To them goes my greatest of gratitude. The preparation of this edition for publication depends heavily on the excellent professional secretarial work of Ms Iva Simcic, Publishing Process Manager.

"Allergic diseases – New Insights" represents a shining example of a productive internation‐ al collaboration among colleagues, and the information inserted in this book should conduct to an improved health care for patients with allergic diseases which are the essence of our work.

> **Celso Pereira, PhD, MD** Clinical Immunology Medicine Faculty Coimbra University Coimbra - Portugal

## **Genetic Aspects of Respiratory Allergy**

Celso Pereira, Frederico S. Regateiro, Graça Loureiro, Beatriz Tavares and António Martinho

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59097

## **1. Introduction**

Genetic and environmental factors contribute to the pathogenesis of asthma [44]. A clear familial incidence and genetic susceptibility was recognized in early XXth century and subsequent studies established high levels heritability [14, 42, 49, 67]. Analysis of genetic association to asthma susceptibility and disease traits has long been used to try and identify pathways involved in asthma pathogenesis and potential therapeutic targets.

In this chapter we review the recent advances in the understanding of the genetic factors contributing to asthma development. We also discuss some of our recent results on the association between allergic asthma and human leukocyte antigen (HLA) and cytokine loci. We present gene and protein expression analysis for some of these relevant disease markers in the context of allergen challenging of asthmatic patients.

#### **1.1. Genetic studies**

Asthma is a complex disease with heterogeneous presentations in different populations [44]. These variable presentations have been grouped into "endotypes" [41]. The high phenotypic diversity led others to propose that asthma is not a single disease but instead that the term "asthma" has been used to define a spectrum of related, overlapping syndromes [41].

At present, no single gene was found to be determinant for asthma susceptibility or, indeed, disease endotypes. Multiple molecular mechanisms seem to be involved in the pathogenesis of each disease feature and many genetic variations have been associated with risk and/or phenotypes. While some genes seem to contribute to asthma susceptibility, others might specifically influence disease features such as age of onset, atopy, progression (severity) or response to treatment. On the other hand, asthma is a multifactorial disease. In addition to

genetic predisposition, there are well-established environmental or developmental factors that interact and influence gene expression [16, 52].

The knowledge in asthma genetics has evolved with the advances in biotechnology. Three major technical approaches have been used to search for associations between genes and disease (or phenotypes): genome-wide linkage studies, hypothesis-driven candidate gene association studies and genome-wide association studies (GWAS).

Genome-wide linkage studies using microsatellite markers performed in families identify chromosome segments associated with disease. Cloning of the "linked" alleles might then lead to the identification of genes and, potentially, to novel disease pathways, as it allows unbiased analysis of the genome. A downside of these linkage followed by positional candidate studies is the difficult study design and their poor resolution, as they often identify broad chromosome regions that might include hundreds of genes.

The sequencing of the whole human genome enabled the study of associations between disease/traits and single nucleotide polymorphisms (SNPs) in particular genes of interest. The candidate genes might be selected for their presumptive functional involvement in disease pathology or for their chromosomal position in a linkage region, or near a previous association signal (positional candidate). Association studies present some advantages over family linkage studies: use of independent case-controls and non-related populations, detect genes with modest effects, have a simpler study design and their results are easier to interpret. On the other hand, the candidate gene approaches require previous knowledge or assumptions about disease pathogenesis and, therefore, do not search for novel genes and pathways.

Linkage and candidate studies have contributed for pivotal discoveries in many monogenic diseases. However, genetic association studies provide limited information as only a small number of genes can be studied at a time. Furthermore, in complex diseases, such as asthma, the contribution of many genes is hard to encompass. Recently, the publication of the Human Genome Project in 2003 (www.genome.gov/) and the International HapMap Project (http:hap‐ map.ncbi.nlm.nih.gov) in 2005 (The International HapMap Consortium 2003) allowed the development of genome-wide association studies (GWAS), [26, 43]. GWAS analyse hundreds of thousands of genetic variants (such as SNPs) across the whole human genome and search for their association to a disease characteristic by comparing the frequency of a genetic variant among the afflicted population versus a control population. Powerful computational statistics are then required to analyse the data and find significant differences in frequency that support association and suggest involvement in disease pathogenesis. Case-control studies are useful to determine disease susceptibility. For particular disease characteristics, however, the associations might be better explored using case-only studies, comparing phenotypes, severity or response to treatment among subgroups of the diseased population.

GWAS brought significant improvement to previous genetic studies of disease because genetic variants are researched across the whole genome and a great number of patients and controls can be included. Importantly, the search is unbiased and does not require previous knowledge of the disease or the biological pathways involved, allowing the finding of unsuspected genes. This advantage has, sometimes, proved thorny, as some identified genes have little biological plausibility. One important limitation in GWAS is the low detection of uncommon risk variants, as genotyping platforms mostly include common gene variants.

genetic predisposition, there are well-established environmental or developmental factors that

The knowledge in asthma genetics has evolved with the advances in biotechnology. Three major technical approaches have been used to search for associations between genes and disease (or phenotypes): genome-wide linkage studies, hypothesis-driven candidate gene

Genome-wide linkage studies using microsatellite markers performed in families identify chromosome segments associated with disease. Cloning of the "linked" alleles might then lead to the identification of genes and, potentially, to novel disease pathways, as it allows unbiased analysis of the genome. A downside of these linkage followed by positional candidate studies is the difficult study design and their poor resolution, as they often identify broad chromosome

The sequencing of the whole human genome enabled the study of associations between disease/traits and single nucleotide polymorphisms (SNPs) in particular genes of interest. The candidate genes might be selected for their presumptive functional involvement in disease pathology or for their chromosomal position in a linkage region, or near a previous association signal (positional candidate). Association studies present some advantages over family linkage studies: use of independent case-controls and non-related populations, detect genes with modest effects, have a simpler study design and their results are easier to interpret. On the other hand, the candidate gene approaches require previous knowledge or assumptions about

Linkage and candidate studies have contributed for pivotal discoveries in many monogenic diseases. However, genetic association studies provide limited information as only a small number of genes can be studied at a time. Furthermore, in complex diseases, such as asthma, the contribution of many genes is hard to encompass. Recently, the publication of the Human Genome Project in 2003 (www.genome.gov/) and the International HapMap Project (http:hap‐ map.ncbi.nlm.nih.gov) in 2005 (The International HapMap Consortium 2003) allowed the development of genome-wide association studies (GWAS), [26, 43]. GWAS analyse hundreds of thousands of genetic variants (such as SNPs) across the whole human genome and search for their association to a disease characteristic by comparing the frequency of a genetic variant among the afflicted population versus a control population. Powerful computational statistics are then required to analyse the data and find significant differences in frequency that support association and suggest involvement in disease pathogenesis. Case-control studies are useful to determine disease susceptibility. For particular disease characteristics, however, the associations might be better explored using case-only studies, comparing phenotypes, severity

GWAS brought significant improvement to previous genetic studies of disease because genetic variants are researched across the whole genome and a great number of patients and controls can be included. Importantly, the search is unbiased and does not require previous knowledge of the disease or the biological pathways involved, allowing the finding of unsuspected genes.

disease pathogenesis and, therefore, do not search for novel genes and pathways.

or response to treatment among subgroups of the diseased population.

interact and influence gene expression [16, 52].

2 Allergic Diseases - New Insights

regions that might include hundreds of genes.

association studies and genome-wide association studies (GWAS).

GWAS have now been used in many diseases with important results [26, 78]. The National Center for Biotechnology Information (part of the National Library of Medicine, USA) compiles data from GWAS performed in many diseases and conditions. Data can be accessed at the Database of Genotype and Phenotype at http://www.ncbi.nlm.nih.gov/gap and at the National Human Genome Research Institute, National Institutes of Health, USA: http:// www.genome.gov/gwastudies/.

#### **1.2. Linkage and candidate studies on the susceptibility and phenotypes of asthma**

The first genome-wide linkage screening for asthma susceptibility loci was published in 1989 [9] and, in 2002, *ADAM33*, located in chromosome 20, was the first gene associated with asthma by positional cloning [75]. This association has then been replicated numerous times in different populations [27]. Ironically, the function of the encoded metallopeptidase in asthma pathogenesis is still unclear [72]. Only 9 genes were identified by positional candidate studies in linked regions [4, 12, 52]: *ADAM33*, *DPP10*, *PHF11*, *NPSR1*, *HLA-G*, *CYFIP2*, *IRAK3*, *COL6A5* and *OPN3* ⁄ *CHML*.

Taking also into consideration the vast literature on candidate gene associations, more than 200 genes have been proposed as asthma related genes through human and murine studies [77]. In 2006, previous to the introduction of GWAS in asthma research, Ober and Hoff‐ jan extensively reviewed the literature and identified a "top10" of genes more frequently associated to asthma [51]: *ADAM33, IL4, IL4RA, IL13, CD14, ADRB2, TNF, HLA-DRB1, HLA-DQB1* and *FCER1B*. Several of these genes are positioned on region 5q31-33, an early recognized and replicated association in linkage studies. This region includes genes for Th2 mediators [22, 51] thought to be involved in asthma development (e.g. IL4 or IL13), and also other genes that might contribute for asthma pathogenesis, such as environment interaction genes (e.g. *CD14*) and drug-response genes (e.g. *ADRB2*). *ADRB2* encodes the β2 adrenergic receptor expressed on bronchial smooth muscle cells and influences both the bronchial tonus and the response to adrenergic medications (e.g. albuterol). Further to asthma susceptibility, it has been associated also with several phenotypes, such as severi‐ ty [8] or disease persistence into adulthood [24]. HLA genomic region (6p21) also has been repeatedly associated with asthma susceptibility, and in particular the *HLADRB1, HLA-DQB1* and *TNF* genes [45, 58, 60].

#### **1.3. Genome-wide association studies in asthma**

In 2007 the first GWAS on asthma susceptibility was published [46]. This study strongly associated gene *ORMDL3* in 17q21 with susceptibility to childhood asthma. This region includes also the gene *GSDBM*. Initially identified in populations of European ancestry [46, 61], many studies have now replicated the association of genes located in 17q21 with child‐ hood-onset asthma and several asthma phenotypes [3, 4] also in Latin American [19, 69], African American or African Caribbean [69, 19] European American [69], and Chinese [35] populations. *ORMDL3* gene encodes a transmembrane protein localized in the endoplasmic reticulum and may be involved in the endoplasmic reticulum response to stress and inflam‐ mation, increasing the unfolded-protein response [6]. The functional connections of *ORMDL3* and *GSDBM* to asthma development are not fully understood. Another GWAS on asthmatic children confirmed the relevance of 17q21 and found a novel association with the gene *DENND1B* (1q31) in children of European and African ancestry [62]. *DENND1B* is expressed by natural killer cells and dendritic cells and predicted to interact with TNF-alpha receptor [62].

So far, 43 GWAS were published on childhood and adult asthma using different populations – search at www.genome.gov, as of August 2014 – and a great deal of information is available about novel genes associations as well as confirmations of relevant genes previously described. This includes studies on asthma susceptibility as well as GWAS on several of asthma pheno‐ types, e.g., IgE [30], forced vital capacity [40], or response to glucocorticoid treatment [63]. Two important meta-analyses reviewed the published data.

In 2010, the **GABRIEL consortium** published a large-scale GWAS meta-analysis including European ancestry-matched 10365 asthmatic patients and 16110 controls [47]. Asthma was associated with *IL1RL1/IL18R1, HLA-DQB1, IL33, SMAD3, IL2R, GSDMB* and *GSDMA* genes, and using with a lower threshold to *SLC22A5*, IL13 and *RORA*. A stronger association was found between these genes and childhood-onset asthma than adult onset asthma, except for *HLA-DQ* which associates more with adult-onset. *ORMDL3* and *GSDMB* were associated only to childhood-onset asthma. No significant association was found for severe or occupational asthma. Notably, genes previously associated to asthma in other studies, such as *PDE4D, CHI3L1, DPP10, GPR154 [NPSR1], ADAM33, PHF11, OPN3, IRAK3, PCDH1, HLA-G,* and *DENND1B*, were not found to be significantly associated with asthma in this study. High IgE serum levels were associated with previously described *FCER1A, IL13, STAT6* and *IL4R/ IL21R* genes and to a new locus within MHC II – *HLA-DRB1*. Only *IL13* showed some associ‐ ation with asthma, whereas the others genes associated with serum high IgE levels were not associated with asthma. This is somewhat unexpected and suggests that asthma development is not related to the levels of IgE. Only *HLA-DR* was found to associate with high serum IgE levels [47].

The **EVE Consortium** meta-analysis searched for susceptibility genes across ethnic groups in North America: European American, African American or African Caribbean, and Latino ancestries [69]. Notably, four associations with asthma risk were found in all three ethnic groups: 17q21 region, *IL1RL1*, *TSLP* and *IL33*. Remarkably, some of these were associated in the two large published meta-analyses, GABRIEL and EVE [47, 69]. A novel asthma suscept‐ ibility locus was described by the EVE consortium at *PYHIN1* specifically in individuals of African ancestry [69].

It seems now clear that some genes are replicated across different populations whereas others seem to be ancestry specific. In another study, 6q14.1 was found to be related to asthma susceptibility in European and African patients [70].

#### **1.4. Biological significance of genetic studies**

African American or African Caribbean [69, 19] European American [69], and Chinese [35] populations. *ORMDL3* gene encodes a transmembrane protein localized in the endoplasmic reticulum and may be involved in the endoplasmic reticulum response to stress and inflam‐ mation, increasing the unfolded-protein response [6]. The functional connections of *ORMDL3* and *GSDBM* to asthma development are not fully understood. Another GWAS on asthmatic children confirmed the relevance of 17q21 and found a novel association with the gene *DENND1B* (1q31) in children of European and African ancestry [62]. *DENND1B* is expressed by natural killer cells and dendritic cells and predicted to interact with TNF-alpha receptor [62].

So far, 43 GWAS were published on childhood and adult asthma using different populations – search at www.genome.gov, as of August 2014 – and a great deal of information is available about novel genes associations as well as confirmations of relevant genes previously described. This includes studies on asthma susceptibility as well as GWAS on several of asthma pheno‐ types, e.g., IgE [30], forced vital capacity [40], or response to glucocorticoid treatment [63]. Two

In 2010, the **GABRIEL consortium** published a large-scale GWAS meta-analysis including European ancestry-matched 10365 asthmatic patients and 16110 controls [47]. Asthma was associated with *IL1RL1/IL18R1, HLA-DQB1, IL33, SMAD3, IL2R, GSDMB* and *GSDMA* genes, and using with a lower threshold to *SLC22A5*, IL13 and *RORA*. A stronger association was found between these genes and childhood-onset asthma than adult onset asthma, except for *HLA-DQ* which associates more with adult-onset. *ORMDL3* and *GSDMB* were associated only to childhood-onset asthma. No significant association was found for severe or occupational asthma. Notably, genes previously associated to asthma in other studies, such as *PDE4D, CHI3L1, DPP10, GPR154 [NPSR1], ADAM33, PHF11, OPN3, IRAK3, PCDH1, HLA-G,* and *DENND1B*, were not found to be significantly associated with asthma in this study. High IgE serum levels were associated with previously described *FCER1A, IL13, STAT6* and *IL4R/ IL21R* genes and to a new locus within MHC II – *HLA-DRB1*. Only *IL13* showed some associ‐ ation with asthma, whereas the others genes associated with serum high IgE levels were not associated with asthma. This is somewhat unexpected and suggests that asthma development is not related to the levels of IgE. Only *HLA-DR* was found to associate with high serum IgE

The **EVE Consortium** meta-analysis searched for susceptibility genes across ethnic groups in North America: European American, African American or African Caribbean, and Latino ancestries [69]. Notably, four associations with asthma risk were found in all three ethnic groups: 17q21 region, *IL1RL1*, *TSLP* and *IL33*. Remarkably, some of these were associated in the two large published meta-analyses, GABRIEL and EVE [47, 69]. A novel asthma suscept‐ ibility locus was described by the EVE consortium at *PYHIN1* specifically in individuals of

It seems now clear that some genes are replicated across different populations whereas others seem to be ancestry specific. In another study, 6q14.1 was found to be related to asthma

important meta-analyses reviewed the published data.

levels [47].

African ancestry [69].

4 Allergic Diseases - New Insights

susceptibility in European and African patients [70].

It is reassuring that many of the genes identified were related to immunological physiopa‐ thology, e.g. several cytokines and antigen presentation genes. Particularly, genes coding for cytokines and pathways associated with Th2 cells (e.g. *IL13*, *IL2R*) and regulatory cells (e.g. SMAD, TGF-beta) were among them. This agrees with the vast literature showing roles for these cells in asthma, atopy and immune regulation. The role of epithelial cells in asthma pathogenesis is underlined by the associations with *TSLP, IL33* and *IL1RL1* (that encodes for the receptor of IL33, expressed on mast cells, Th2 cells, T-regulatory cells, and macro‐ phages) [47, 69].

Some genes, however, do not immediately relate to immunological pathways. This was the case for *RAD50* [38]. In this study, Li and colleagues found that SNPs on *HLA-DQB1, HLA-DQA2* and *RAD50* region were associated with severe or difficult-to-treat asthma. *RAD 50* is a DNA repair gene and a connection with asthma pathogenesis is not obvious. Furthermore, the associated SNPs were found in an intron. Notably, previous research on Th2 cytokine expression in mice showed the presence of a locus control region (LCR) in this location that is an important regulator of the genes encoding the Th2 cytokines interleukins 4, 5 and 13 [33, 34]. Genetic ablation of this LCR did not affect the expression of a linked *RAD50* gene, but reduced long-range intrachromosomal interactions with the promoters of the Th2 cytokine genes. This example shows that some associations require further understanding of genetic or functional interactions of the genes. Nevertheless, some of the associated genes remain with no known immunological or genetic connection to asthma and more information is needed to understand their role in pathogenesis.

#### **1.5. Limitations of GWAS results in asthma**

The results obtained so far demonstrate the polygenic and multifactorial nature of asthma. Low levels of replication have been found between GWAS and individual study heritability estimates for the associated genes were found to be low [52] and, therefore, with little prog‐ nostic utility. These setbacks might be, at least in part, due to methodological or sampling differences between GWAS. A fundamental question is that some variants might increase risk only in specific individuals and not in others, for instance in the case of some genotypeenvironmental exposure interactions. Moreover, GWAS underestimate the role of rare genetic variants [65, 70] that might have an overall larger effect than common variants on the propor‐ tion of genetic risk for common diseases [15]. Epigenetic mechanisms [13] and epistatic effects might further difficult gene expression analysis [81].

The challenges with GWAS are discussed in further detail elsewhere [77]. Several methods have been proposed to improve the power of GWAS (De 2014). Utilizing a combination of approaches, in which GWAS-type studies are performed using selected SNPs of candidate genes, a strategy was devised and named "prioritized subset analysis" [37]. This strategy combines the strength of GWAS computational statistics with previous knowledge from the literature, allowing for power improvement [37]. Novel mathematical models have been proposed for data analysis in asthma [74].

In the future, the study of complex diseases might incorporate data from whole-genome sequencing [71] or exome sequencing to identify both common and rarer genetic variants in complex diseases [64]. These techniques and analysis are becoming more affordable and feasible on large numbers of individuals and therefore sufficient power will be possible for detecting associations with asthma. A recent study, for example, analysed the role of copy number variants asthma using whole genome sequencing [5].

Genetic approaches require replication and gene expression analysis. Ultimately, *in vitro/ex vivo/in vivo* studies are required to confirm the biological significance and to understand the precise functions of the associated genes in asthma pathogenesis. In this chapter, we analyse the expression of some candidate genes in the context of the allergen-specific response in allergy respiratory patients.

## **2. Experimental study**

#### **2.1. Cytokine single nucleotide polymorphism and HLA expression**

#### *2.1.1. Material and methods*

#### *<IP_ADDRESS>. Subjects*

85 voluntary adult patients with respiratory allergy to *Dermatophagoides pteronyssinus* (Dp) under observation in outpatient Immunoallergy were enrolled for study (Group A). This group included patients of both sexes (female= 55; male=30; average age: 30.09±9.22 years), wherein 36 of them had the diagnosis of allergic rhinitis and the others 49 patients had simultaneously allergic asthma and rhinitis diagnosis, according criteria from ARIA and GINA guidelines [2, 20]. At the moment of the study all the patients were under clinical stability, and never were submitted to specific immunotherapy in the past.

Pregnancy, systemic inflammatory illness or other chronic disease, any exacerbation of allergic disease or a respiratory tract infection in the previous month to the study, as well as nasal polyposis or previous nasal surgery in the past were considered exclusion criteria.

#### *<IP_ADDRESS>. Methods*

DNA was extracted from polymorphonuclear blood cells (PMBC) using MagAttract DNA Blood Mini M48 Kit on the BioRobot *M48*. After extraction, the quality and quantification of DNA was evaluated and PCR -RSSO Cytokine SNP Typing kit from LIFECODES was used to investigate single cytokine polymorphism (SNP's) from 14 cytokines using bead array xMAPTM technology from LuminexTM *.*

The *HLA* genomic typing to class I (*locus* A and B) and class II (*locus* DR) were obtained from peripheral blood samples. The study of cytokine single nucleotide polymorphisms (SNP's) were also performed in the promoter region of IL-1 (α,β,R,RA), IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ, TGF-β and TNF-α.

#### *<IP_ADDRESS>. Statistical analysis*

In the future, the study of complex diseases might incorporate data from whole-genome sequencing [71] or exome sequencing to identify both common and rarer genetic variants in complex diseases [64]. These techniques and analysis are becoming more affordable and feasible on large numbers of individuals and therefore sufficient power will be possible for detecting associations with asthma. A recent study, for example, analysed the role of copy

Genetic approaches require replication and gene expression analysis. Ultimately, *in vitro/ex vivo/in vivo* studies are required to confirm the biological significance and to understand the precise functions of the associated genes in asthma pathogenesis. In this chapter, we analyse the expression of some candidate genes in the context of the allergen-specific response in

85 voluntary adult patients with respiratory allergy to *Dermatophagoides pteronyssinus* (Dp) under observation in outpatient Immunoallergy were enrolled for study (Group A). This group included patients of both sexes (female= 55; male=30; average age: 30.09±9.22 years), wherein 36 of them had the diagnosis of allergic rhinitis and the others 49 patients had simultaneously allergic asthma and rhinitis diagnosis, according criteria from ARIA and GINA guidelines [2, 20]. At the moment of the study all the patients were under clinical stability, and never were

Pregnancy, systemic inflammatory illness or other chronic disease, any exacerbation of allergic disease or a respiratory tract infection in the previous month to the study, as well as nasal

DNA was extracted from polymorphonuclear blood cells (PMBC) using MagAttract DNA Blood Mini M48 Kit on the BioRobot *M48*. After extraction, the quality and quantification of DNA was evaluated and PCR -RSSO Cytokine SNP Typing kit from LIFECODES was used to investigate single cytokine polymorphism (SNP's) from 14 cytokines using bead array

The *HLA* genomic typing to class I (*locus* A and B) and class II (*locus* DR) were obtained from peripheral blood samples. The study of cytokine single nucleotide polymorphisms (SNP's) were also performed in the promoter region of IL-1 (α,β,R,RA), IL-2, IL-4, IL-6, IL-10, IL-12,

polyposis or previous nasal surgery in the past were considered exclusion criteria.

number variants asthma using whole genome sequencing [5].

**2.1. Cytokine single nucleotide polymorphism and HLA expression**

submitted to specific immunotherapy in the past.

xMAPTM technology from LuminexTM *.*

IFN-γ, TGF-β and TNF-α.

allergy respiratory patients.

6 Allergic Diseases - New Insights

**2. Experimental study**

*2.1.1. Material and methods*

*<IP_ADDRESS>. Subjects*

*<IP_ADDRESS>. Methods*

Statistical analysis was performed using SPSS® Statistics 17.0 software. Were analyzed the frequency distribution for the different qualitative variables. For quantitative variables Average and Standard Deviation (SD) were calculated, according the two patients groups (A and C).

Differences between the two groups were analysed by means of *χ* <sup>2</sup> tests and Mann–Whitney test (two independent samples) for qualitative and quantitative variables respectively. A statistical significant difference was assumed with p < 0.05.

#### *<IP_ADDRESS>. Results*

In allergic group (A), it was observed a higher genotype frequency of *A\*01* and *A\*68* alleles, while *A\*02* presented a lower frequency (Figure 1), compared to non-allergic healthy group (C). Concerning locus B (Figure 2), the most frequent allele was B\*51, while B\*35 was less frequent in the allergic population. With respect to HLA class II (Figure 3), the alleles DRB1\*03 and DRB1\*11 were more frequent in group A, while for DRB1\*04 and DRB1\*13 it was the opposite. None of these differences was statistically significant. The *A\*01 B\*08 DRB1\*03* haplotype was the most frequent in both groups. Nevertheless in allergic patients the *A\*02 B\*51 DRB1\*11* haplotype was the second most frequent, but the fifth one in healthy individuals (5.8% *vs* 1.4%, p=0.059).

**Figure 1.** HLA class I, locus A. Frequency distribution of allergic patients (blue) compared to control group (red), not significant

**Figure 2.** HLA class I, locus B. Frequency distribution of allergic patients (blue) compared to control group (red), not significant

**Figure 3.** HLA class II, locus DR. Frequency distribution of allergic patients (blue) compared to control group (red), not significant

Results of phenotypic frequency of cytokine single nucleotide polymorphisms (SNP's) in the promoter region for subjects in the study the results are expressed in Figures 4 and 5.

**Figure 4.** Cytokine single nucleotide polymorphisms (SNP's) : IFNg, IL-10, IL-6, TNFa, and IL-12b Frequency distribu‐ tion of allergic patients (blue) compared to control group (red)

**Figure 2.** HLA class I, locus B. Frequency distribution of allergic patients (blue) compared to control group (red), not

**Figure 3.** HLA class II, locus DR. Frequency distribution of allergic patients (blue) compared to control group (red), not

significant

8 Allergic Diseases - New Insights

significant

**Figure 5.** CCL4 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

It was found a higher phenotypic frequency for the following cytokine SNP's: TNFα-238GG (p<0.0001), TGFβ+869CT (p<0.0001) and IL4-1098TT (p=0.003). On the other hand, there were lower frequencies for TNFα-238AG (p<0.0001), TGFβ+915CC (p=0.024) and IL4-1098GG (p=0.005). In the allergic group it was not found any homozygous individual for TGFβ+869TT (p<0.0001), but conversely There was an increase of heterozygous individual compared with the control group (p=0.004).

No differences were observed between patients with concomitant asthma complaints and those with only rhinitis symptoms.

### **2.2. Gene expression and allergy challenge test**

#### *2.2.1. Subjects*

From the previous mentioned 85 voluntary adult patients studied, with respiratory allergy to *Dermatophagoides pteronyssinus* (Dp) we selected a sample of 42 patients. These patients were enrolled to being submitted to an specific allergic challenge test, so H1-antihistamines and corticosteroids, either nasal or oral, were stopped 2 weeks and 4 weeks prior to the challenge test, respectively.

The specific allergic challenge test was performed using a standardized lyophilized *Dp* extract by nasal and ocular in 21 patients for each methodological procedure [21]. All patients were submitted to a previous respiratory function test (pletismography using *Master screen Body Jaeger*®) to assure the *standard* safe criteria (FEV1 ≥ 80% and FEV1/FVC ≥ 80).

Symptoms as dyspnoea, thoracic oppression, wheezing or cough were asked to being men‐ tioned to all patients after challenge test.

The local ethics committee approved the study and all the participants gave written informed consent before entry.

#### *2.2.2. Specific nasal and conjunctival challenge tests*

All the patients performed skin prick tests with an aqueous extract of *Dp* with 5 mg/ml concentration (23 mg/ml of *Der p 1*, BialAristegui, Bilbao, Spain), with the dilutions of 1/1, 1/10, 1/100 and 1/1000 and negative and positive controls, according to standardized procedures [21, 54] (Dreborg 1993). The concentration used for specific provocation was the minimum that induced a prick test wheal at least equal to that induced by histamine.

All the specific challenge tests were performed after a period of adapting of room temperature for at least 30 minutes, and at the same period of the morning.

Nasal provocation test (NPT) were carry out by the administration of 160μl, corresponding a 2 consecutive puffs, of aerosolized spraying of *Dp* extract administered to the unilateral inferior nasal turbinate of the less congested nostril, and asking patients to perform apnea during the application of the allergen. Conjunctival provocation test (CPT) comprised on the unilateral administration of 50μl, one drop, of allergen extract in the inferior and external quadrant of the bulbar conjunctiva.

Both ocular and nasal symptoms were recorded after challenge test and registered filled at the 1st and the 5th minutes, according specific clinical score system [21, 39].

#### *2.2.3. Clinical score scaling*

It was found a higher phenotypic frequency for the following cytokine SNP's: TNFα-238GG (p<0.0001), TGFβ+869CT (p<0.0001) and IL4-1098TT (p=0.003). On the other hand, there were lower frequencies for TNFα-238AG (p<0.0001), TGFβ+915CC (p=0.024) and IL4-1098GG (p=0.005). In the allergic group it was not found any homozygous individual for TGFβ+869TT (p<0.0001), but conversely There was an increase of heterozygous individual compared with

No differences were observed between patients with concomitant asthma complaints and

From the previous mentioned 85 voluntary adult patients studied, with respiratory allergy to *Dermatophagoides pteronyssinus* (Dp) we selected a sample of 42 patients. These patients were enrolled to being submitted to an specific allergic challenge test, so H1-antihistamines and corticosteroids, either nasal or oral, were stopped 2 weeks and 4 weeks prior to the challenge

The specific allergic challenge test was performed using a standardized lyophilized *Dp* extract by nasal and ocular in 21 patients for each methodological procedure [21]. All patients were submitted to a previous respiratory function test (pletismography using *Master screen Body*

Symptoms as dyspnoea, thoracic oppression, wheezing or cough were asked to being men‐

The local ethics committee approved the study and all the participants gave written informed

All the patients performed skin prick tests with an aqueous extract of *Dp* with 5 mg/ml concentration (23 mg/ml of *Der p 1*, BialAristegui, Bilbao, Spain), with the dilutions of 1/1, 1/10, 1/100 and 1/1000 and negative and positive controls, according to standardized procedures [21, 54] (Dreborg 1993). The concentration used for specific provocation was the minimum that

All the specific challenge tests were performed after a period of adapting of room temperature

Nasal provocation test (NPT) were carry out by the administration of 160μl, corresponding a 2 consecutive puffs, of aerosolized spraying of *Dp* extract administered to the unilateral inferior nasal turbinate of the less congested nostril, and asking patients to perform apnea during the application of the allergen. Conjunctival provocation test (CPT) comprised on the unilateral

*Jaeger*®) to assure the *standard* safe criteria (FEV1 ≥ 80% and FEV1/FVC ≥ 80).

induced a prick test wheal at least equal to that induced by histamine.

for at least 30 minutes, and at the same period of the morning.

the control group (p=0.004).

10 Allergic Diseases - New Insights

*2.2.1. Subjects*

test, respectively.

consent before entry.

those with only rhinitis symptoms.

**2.2. Gene expression and allergy challenge test**

tioned to all patients after challenge test.

*2.2.2. Specific nasal and conjunctival challenge tests*

For evaluation the clinical responses an adaptation of the previously used a nasal clinical score, NCS, [39] and ocular clinical score, OCS, [48] were applied at the 1st and the 5th minutes [21, 54] as well as the total clinical score (TCS), representing the sum of NCS (range: 0-15) and OCS (range: 0-13) ranging from 0 to 28 points.

Although the late record symptoms occurred 5 minutes later after the challenge test all patients remained under strictly clinical observation for at least 4 additional hours. Clinical evaluation scores was interrupted after the 5th minute, but all the patients maintained medical observa‐ tion during the next 4 hours.

#### *2.2.4. Patient groups*

Blood samples were collect to PAXgene blood RNA tubes to all patients before the allergic challenge test (NPT or CPT) and at 60 or 240 minutes, according to the following subgroups.

#### *2.2.5. Methods*

Peripheral blood was collected in a PAXGene Blood RNA tube (Qiagen) and total RNA extraction performed with the PAXgene Blood RNA kit (Qiagen) according to the supplier's instructions in a QIAcube BioRobot [59]. Total RNA quantification and RNA integrity evaluation was analyzed using a 6000 Nano Chip kit, in an Agilent 2100 bioanalyzer (Agilent Technologies) and 2100 expert software.

RNA was reverse transcribed with SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen), according to the manufacturer's instructions. Relative quantification of gene expression by real-time PCR was performed in the LightCycler 480 II (Roche Diagnostics).

Real-time PCR reactions were carried out using QuantiTect SYBR Green PCR Master Mix and QuantiTect Primer Assay (Qiagen) and 20 ng of cDNA sample, in a total volume of 10 μl. All samples were run in duplicate.

Real-time PCR results were analyzed with the LightCycler software (Roche Diagnostics). GeNorm Reference Gene Selection kit (PrimerDesign Ltd) in conjunction with the geNorm software (PrimerDesign Ltd.) were used to select the reference genes to normalize data. The normalized expression levels of the genes of interest were calculated by using the delta-Ct method.

#### *2.2.6. Statistical analysis*

Statistical analyses were performed using SPSS® Statistics 17.0 software. To determine the statistical significance of the differences observed, the non-parametric Mann–Whitney U-test and Wilcoxon paired-sample test were performed. A statistical significant difference was assumed with p < 0.05.

#### *2.2.7. Results*

All the patient groups demonstrated clinical positive response to a single dose of *Dp* extract [21], the symptoms were progressively decreasing, without need of therapeutic intervention. There were neither bronchial symptoms nor systemic reactions in any of the provocation tests.

It was observed in most of the patients, even at 60 minutes after challenge, differences in gene expression for cytokines, chemokines and nuclear transcription factors related to allergen exposure.

No differences were observed between patients with only nasal complaints compared to those that had associated clinical asthma.

We did not observe a similar pattern between the two routes of specific allergen challenge and in the two different periods of time in analysis.

Results of some chemokine gene expression after specific allergen challenge test are presented in figures 6 to 8.

The cytokine gene response to allergens did not show a homogeneous profile in the subgroups of patients studied, but it was evident that individually, there were changes in mRNA expression in many patients, as seen in Figures 9 and 12.

So we can distinguish two different groups of patients: one with very high values (also with high values of total serum IgE but without correlation with the clinical severity- data not shown) and the other with low expression of gene levels (Figures 13 and 14). However, this difference was not found for the cytokine receptor gene, IL-4R, Figure 15.

Figures 16-18 show the results of specific response to allergens in genes related to nuclear transcription factors on the four subgroups of patients.

ures 5 to 8.

p R

e did not ob periods of time Results of som

bserve a simila e in analysis. me chemokine g

ar pattern betw

ween the two

routes of spec

allergen challe

enge test are pre

esented in figu

n after specific

gene expression

F

F c

F

Figure 7. CXCL2

2 gene expression

n in the patients s

Figure 6. CXCL1

Figure 6. CXCL1 challenge (indivi

gene expression

 gene expression idual response v

n in the patients s

n in the patients s values , average a

Real-time PCR reactions were carried out using QuantiTect SYBR Green PCR Master Mix and QuantiTect Primer Assay (Qiagen) and 20 ng of cDNA sample, in a total volume of 10 μl. All

12 Allergic Diseases - New Insights W

Real-time PCR results were analyzed with the LightCycler software (Roche Diagnostics). GeNorm Reference Gene Selection kit (PrimerDesign Ltd) in conjunction with the geNorm software (PrimerDesign Ltd.) were used to select the reference genes to normalize data. The normalized expression levels of the genes of interest were calculated by using the delta-Ct

Statistical analyses were performed using SPSS® Statistics 17.0 software. To determine the statistical significance of the differences observed, the non-parametric Mann–Whitney U-test and Wilcoxon paired-sample test were performed. A statistical significant difference was

All the patient groups demonstrated clinical positive response to a single dose of *Dp* extract [21], the symptoms were progressively decreasing, without need of therapeutic intervention. There were neither bronchial symptoms nor systemic reactions in any of the provocation tests.

It was observed in most of the patients, even at 60 minutes after challenge, differences in gene expression for cytokines, chemokines and nuclear transcription factors related to allergen

No differences were observed between patients with only nasal complaints compared to those

We did not observe a similar pattern between the two routes of specific allergen challenge and

Results of some chemokine gene expression after specific allergen challenge test are presented

The cytokine gene response to allergens did not show a homogeneous profile in the subgroups of patients studied, but it was evident that individually, there were changes in mRNA

So we can distinguish two different groups of patients: one with very high values (also with high values of total serum IgE but without correlation with the clinical severity- data not shown) and the other with low expression of gene levels (Figures 13 and 14). However, this

Figures 16-18 show the results of specific response to allergens in genes related to nuclear

samples were run in duplicate.

method.

*2.2.6. Statistical analysis*

assumed with p < 0.05.

that had associated clinical asthma.

in the two different periods of time in analysis.

expression in many patients, as seen in Figures 9 and 12.

transcription factors on the four subgroups of patients.

difference was not found for the cytokine receptor gene, IL-4R, Figure 15.

*2.2.7. Results*

exposure.

in figures 6 to 8.

c challenge (indivi idual response v values , average a and SD). **Figure 6.** CXCL1 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

T and NPT, befo

T and NPT, befo

ore challenge (T0)

ore challenge (T0)

and at 60 and 2

) and at 60 and 2

240 minutes after

fferent

240 minutes after

r

submitted to CPT

submitted to CPT and SD).

c challenge (indivi idual response v values , average a and SD). **Figure 7.** CXCL2 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

T and NPT, befo

ore challenge (T0)

) and at 60 and 2

240 minutes after

submitted to CPT

F c

F c

F

Figure 8. CXCL5

5 gene expression

n in the patients s

y, there were ch

to allergens did y, there were ch

hat individually

gene response t hat individually

was evident th

The cytokine g was evident th

w

T w

W p R

We did not ob periods of time Results of som

bserve a simila e in analysis. me chemokine g

ar pattern betw

ween the two

routes of spec

cific allergen c

challenge and

in the two dif

fferent

fferent

ures 5 to 8.

240 minutes after

240 minutes after

esented in figu

) and at 60 and 2

) and at 60 and 2

enge test are pre

ore challenge (T0)

ore challenge (T0)

allergen challe

T and NPT, befo

T and NPT, befo

n after specific

submitted to CPT and SD).

submitted to CPT and SD).

gene expression

n in the patients s values , average a

n in the patients s values , average a

 gene expression idual response v

2 gene expression idual response v

Figure 6. CXCL1 challenge (indivi

Figure 7. CXCL2 challenge (indivi

c T challenge (indivi The cytokine g idual response v gene response t values , average a to allergens did and SD). d not show a h omogeneous p profile in the su ubgroups of pa atients studied, rrr but it **Figure 8.** CXCL5 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD). F c Figure 8. CXCL5 challenge (indivi 5 gene expression idual response v n in the patients s values , average a submitted to CPT and SD). T and NPT, befo ore challenge (T0) ) and at 60 and 2 240 minutes after rrr but it

T and NPT, befo

ore challenge (T0)

) and at 60 and 2

240 minutes after

Figures 9 and 1

atients studied, Figures 9 and 1

12.

12.

nts, as seen in F

ubgroups of pa nts, as seen in F

in many patien

profile in the su in many patien

NA expression

omogeneous p NA expression

submitted to CPT

hanges in mRN

d not show a h hanges in mRN

**Figure 9.** IL-10 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

W p R

We did not ob periods of time Results of som

bserve a simila e in analysis. me chemokine g

ar pattern betw

ween the two

routes of spec

cific allergen c

challenge and

in the two dif

fferent

rrr but it

fferent

ures 5 to 8.

240 minutes after

240 minutes after

in the two dif

ures 5 to 8.

240 minutes after

esented in figu

) and at 60 and 2

) and at 60 and 2

challenge and

esented in figu

) and at 60 and 2

enge test are pre

ore challenge (T0)

ore challenge (T0)

cific allergen c

enge test are pre

ore challenge (T0)

allergen challe

T and NPT, befo

T and NPT, befo

routes of spec

allergen challe

T and NPT, befo

n after specific

submitted to CPT and SD).

submitted to CPT and SD).

ween the two

n after specific

submitted to CPT and SD).

gene expression

n in the patients s values , average a

n in the patients s values , average a

ar pattern betw

gene expression

n in the patients s values , average a

n in the patients s values , average a

 gene expression idual response v

2 gene expression idual response v

bserve a simila e in analysis. me chemokine g

> gene expression idual response v

Figure 6. CXCL1 challenge (indivi

Figure 7. CXCL2 challenge (indivi

F c

F c

14 Allergic Diseases - New Insights

W p R

We did not ob periods of time Results of som

Figure 6. CXCL1 challenge (indivi

Figure 7.  CXCL5 challenge (indivi The cytokine g was evident th

Figure 8. CXCL5 challenge (indivi The cytokine g was evident th

5 gene expression idual response v gene response t hat individually

5 gene expression idual response v gene response t hat individually

2 gene expression idual response v

minutes after challenge (individual response values, average and SD).

minutes after challenge (individual response values, average and SD).

n in the patients s values , average a to allergens did y, there were ch

n in the patients s values , average a to allergens did y, there were ch

submitted to CPT and SD). d not show a h hanges in mRN

submitted to CPT and SD). d not show a h hanges in mRN

submitted to CPT and SD).

**Figure 8.** CXCL5 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240

**Figure 9.** IL-10 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240

T and NPT, befo

T and NPT, befo

T and NPT, befo

ore challenge (T0)

ore challenge (T0)

ore challenge (T0)

) and at 60 and 2

) and at 60 and 2

) and at 60 and 2

240 minutes after

240 minutes after

240 minutes after

atients studied, Figures 9 and 1

atients studied, Figures 9 and 1

12.

12.

rrr but it

ubgroups of pa nts, as seen in F

ubgroups of pa nts, as seen in F

profile in the su in many patien

profile in the su in many patien

omogeneous p NA expression

omogeneous p NA expression

F c igure 10. IL-1β hallenge (indivi gene expression dual response v in the patients s alues , average a ubmitted to CPT nd SD). and NPT, before challenge (T0) and at 60 and 240 minutes after **Figure 10.** IL-1β gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

F c igure 11. IL-1R2 hallenge (indivi gene expression dual response v in the patients alues , average a submitted to CP nd SD). T and NPT, before challenge (T0) and at 60 and 240 minutes after **Figure 11.** IL-1R2 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

F

igure 12. IL-17R

RA gene expressi

ion in the patient

with the clinica r, this differenc

with the clinica r, this differenc

ut correlation w d 14). However

ut correlation w d 14). However

IgE but withou (Figures 13 and

IgE but withou (Figures 13 and

I

I

F

Figure 13. IL-4 (l

low levels) gene

expression in the

c S challenge (indivi So we can dist idual response v tinguish two d values , average a ifferent groups and SD). s of patients: o one with very h high values (al so with high v values of total s serum **Figure 12.** IL-17RA gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD). <sup>S</sup> o we can dist tinguish two d ifferent groups s of patients: o one with very h high values (al so with high v values of total s serum

CPT and NPT, be

efore challenge (T

T0) and at 60 and

d 240 minutes aft

ter

levels

levels

ession of gene gure 15.

ession of gene gure 15.

with low expre gene, IL-4R, Fig

with low expre gene, IL-4R, Fig

and the other w kine receptor g

and the other w kine receptor g

ta not shown) a nd for the cytok

ta not shown) a nd for the cytok

ts submitted to C

al severity- dat ce was not foun

al severity- dat ce was not foun

a after challenge (i individual respo onse values , aver rage and SD). **Figure 13.** IL-4 (low levels) gene expression in the patients submitted to CPT and NPT before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

tted to CPT and

NPT before chal

llenge (T0) and a

t 60 and 240 min

utes

e patients submit

t 60 and 240 min

values of total s ession of gene gure 15.

serum levels

nutes

S I

So we can dist IgE but withou (Figures 13 and

tinguish two d ut correlation w d 14). However

ifferent groups with the clinica r, this differenc

s of patients: o al severity- dat ce was not foun

one with very h ta not shown) a nd for the cytok

high values (al and the other w kine receptor g

so with high v with low expre gene, IL-4R, Fig

llenge (T0) and a

NPT before chal

tted to CPT and

e patients submit rage and SD).

expression in the onse values , aver

low levels) gene individual respo

Figure 13.  IL-4R

gene expression

n in the patients s

s of patients.

four subgroup

f

Figure 14. IL-4 (l

Figure 14. IL-4 (l after challenge (i

lhigh levels) gene

lhigh levels) gene individual respo

e expression in th

e expression in th onse values , aver

a after challenge (i individual respo onse values , aver rage and SD). **Figure 14.** IL-4 (lhigh levels) gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

mitted to CPT and

mitted to CPT and

d NPT, before ch

d NPT, before ch

hallenge (T0) and

hallenge (T0) and

d at 60 and 240 m

d at 60 and 240 m

minutes

minutes

nutes

serum levels

he patients subm

he patients subm rage and SD).

F c S I

16 Allergic Diseases - New Insights

I

F a

Figure 13. IL-4 (l after challenge (i

low levels) gene individual respo

and 240 minutes after challenge (individual response values, average and SD).

expression in the onse values , aver

e patients submit rage and SD).

**Figure 13.** IL-4 (low levels) gene expression in the patients submitted to CPT and NPT before challenge (T0) and at 60

tted to CPT and

NPT before chal

llenge (T0) and a

t 60 and 240 min

utes

igure 12. IL-17R challenge (indivi So we can dist IgE but withou (Figures 13 and

o we can dist IgE but withou (Figures 13 and

RA gene expressi idual response v tinguish two d ut correlation w d 14). However

tinguish two d ut correlation w d 14). However

minutes after challenge (individual response values, average and SD). <sup>S</sup>

ion in the patient values , average a ifferent groups with the clinica r, this differenc

ifferent groups with the clinica r, this differenc

ts submitted to C and SD). s of patients: o al severity- dat ce was not foun

s of patients: o al severity- dat ce was not foun

**Figure 12.** IL-17RA gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240

CPT and NPT, be

efore challenge (T

T0) and at 60 and

d 240 minutes aft

ter

serum levels

serum levels

values of total s ession of gene gure 15.

values of total s ession of gene gure 15.

so with high v with low expre gene, IL-4R, Fig

so with high v with low expre gene, IL-4R, Fig

high values (al and the other w kine receptor g

high values (al and the other w kine receptor g

one with very h ta not shown) a nd for the cytok

one with very h ta not shown) a nd for the cytok

c F challenge (indivi Figures 16-18 s idual response v show the resul values , average a lts of specific r and SD). response to all lergens in gene es related to n nuclear transcri iption factors o on the **Figure 15.** IL-4R gene expression in the patients submitted to CPT and NPT before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

T and NPT before

e challenge (T0)

and at 60 and 24

40 minutes after

submitted to CPT

F f

F

igure 17. EGR1

gene expression

in the patients s

S I

So we can dist IgE but withou (Figures 13 and

tinguish two d ut correlation w d 14). However

ifferent groups with the clinica r, this differenc

s of patients: o al severity- dat ce was not foun

one with very h ta not shown) a nd for the cytok

high values (al and the other w kine receptor g

so with high v with low expre gene, IL-4R, Fig

values of total s ession of gene gure 15.

serum levels

nutes

minutes

on the

t 60 and 240 min

d at 60 and 240 m

40 minutes after

iption factors o

llenge (T0) and a

hallenge (T0) and

and at 60 and 24

nuclear transcri

NPT before chal

d NPT, before ch

e challenge (T0)

es related to n

tted to CPT and

mitted to CPT and

T and NPT before

lergens in gene

e patients submit rage and SD).

he patients subm rage and SD).

submitted to CPT and SD). response to all

expression in the onse values , aver

e expression in th onse values , aver

n in the patients s values , average a lts of specific r

low levels) gene individual respo

lhigh levels) gene individual respo

show the resul s of patients.

Figure 13. IL-4 (l after challenge (i

Figure 14.  JAK2 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

c hallenge (indivi dual response v alues , average a nd SD). **Figure 17.** EGR1 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

and NPT, before challenge (T0) and at 60 and 240 minutes after

ubmitted to CPT

S I

So we can dist IgE but withou (Figures 13 and

tinguish two d ut correlation w d 14). However

ifferent groups with the clinica r, this differenc

s of patients: o al severity- dat ce was not foun

one with very h ta not shown) a nd for the cytok

high values (al and the other w kine receptor g

so with high v with low expre gene, IL-4R, Fig

values of total s ession of gene gure 15.

serum levels

nutes

minutes

on the

t 60 and 240 min

d at 60 and 240 m

40 minutes after

iption factors o

llenge (T0) and a

hallenge (T0) and

and at 60 and 24

nuclear transcri

NPT before chal

d NPT, before ch

e challenge (T0)

es related to n

tted to CPT and

mitted to CPT and

T and NPT before

lergens in gene

e patients submit rage and SD).

he patients subm rage and SD).

submitted to CPT and SD). response to all

**Figure 16.** JAK2 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240

expression in the onse values , aver

e expression in th onse values , aver

n in the patients s values , average a lts of specific r

low levels) gene individual respo

lhigh levels) gene individual respo

gene expression idual response v show the resul s of patients.

minutes after challenge (individual response values, average and SD).

Figure 13. IL-4 (l after challenge (i

Figure 14. IL-4 (l after challenge (i

Figure 15.  EGR1 hallenge (indivi

gene expression dual response v

minutes after challenge (individual response values, average and SD).

 in the patients s alues , average a

ubmitted to CPT nd SD).

**Figure 17.** EGR1 gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240

and NPT, before challenge (T0) and at 60 and 240 minutes after

c O challenge (indivi Overall, there idual response v seems that do values , average a oes not exist a and SD). specific profil le of genes in response to cy ytokines, chem mokines and nu uclear **Figure 18.** JUNB gene expression in the patients submitted to CPT and NPT, before challenge (T0) and at 60 and 240 minutes after challenge (individual response values, average and SD).

T and NPT, befor

re challenge (T0)

) and at 60 and 24

40 minutes after

wed marked cha

anges,

y patients show

ge (T60), many

e after challeng

submitted to CPT

t period of tim

F

Figure 18. JUNB

gene expression

n in the patients s

er, even in first

minutes).

actors. Howeve

minutes; B: 240 m

transcription fa

in study (A: 60 m

t

i

s F since the begin Figure 19. Globa nning of the alle al nuclear transcr ergic reaction ( ription factor gen (Figures 19 to 2 ne responses in th 21). he patients subm mitted to CPT and d NPT, according g to the periods o of time Overall, there seems that does not exist a specific profile of genes in response to cytokines, chemokines and nuclear transcription factors. However, even in first period of time after challenge (T60), many patients showed marked changes, since the beginning of the allergic reaction (Figures 19 to 21).

Fig. 19. Global nuclear transcription factor gene responses in the patients submitted to CPT and NPT, according to the periods of time in study (A: 60 minutes; B: 240 **Figure 19.** minutes). Global nuclear transcription factor gene responses in the patients submitted to CPT and NPT, according to the periods of time in study (A: 60 minutes; B: 240 minutes).

**Figure 20.** Global cytokine gene responses in the patients submitted to CPT and NPT, according to the periods of time in study (A: 60 minutes; B: 240 minutes).

Fig. 20. Global cytokine gene responses in the patients submitted to CPT and NPT, according to the periods of time in study (A: 60 minutes; B: 240 minutes).

Fig. 21. Global chemokine gene responses in the patients submitted to CPT and NPT, according to the periods of time in study (A: <sup>60</sup> minutes; B: <sup>240</sup> minutes). **Figure 21.** Global chemokine gene responses in the patients submitted to CPT and NPT, according to the periods of time in study (A: 60 minutes; B: 240 minutes).

#### *2.2.8. Discussion*

Allergic diseases and particularly respiratory allergy are complex disorders often present in the same family or closely related subjects. Genetic factors undoubtedly contribute to disease susceptibility but the expression of the disease can be modulated by environmental exposures and the interactions between the two. In this context it is emphasized the role of infection, allergens, pollutants, oxidative stress and cytokine dysregulation [50]. In the case of allergy and asthma in particular because they constitute a complex and multifactorial disease, each of the individual factors by itself can determine distinct phenotypes that result in the polarization of distinct cellular pathways, resulting into the final clinical expression.

The last decade has been marked by the publication of a lot of genome-wide association studies (GWAS) in asthma or allergy phenotypes. GWAS have reported novel and interesting genes but have also confirmed the role of some functionally relevant genes previously described. However, heritability of allergic diseases has not been elucidated completely so far [1].

Considering that respiratory allergy, like epigenetic mechanisms, is heritable, nevertheless exists a strongly familial condition (36% to 79% heritability) with a non-Mendelian pattern of inheritance and polymorphisms in more than 100 genes, but these associations have infre‐ quently been replicated, scientifically supported, and so the genetic has explained only a small portion of the cause of this disease [80, 81].

Fig. 20. Global cytokine gene responses in the patients submitted to CPT and NPT, according to the periods of time in study (A: 60 minutes; B: 240 minutes). **Figure 20.** Global cytokine gene responses in the patients submitted to CPT and NPT, according to the periods of time

Fig. 21. Global chemokine gene responses in the patients submitted to CPT and NPT, according to the periods of time in study (A: <sup>60</sup> minutes; B: <sup>240</sup> minutes). **Figure 21.** Global chemokine gene responses in the patients submitted to CPT and NPT, according to the periods of

Allergic diseases and particularly respiratory allergy are complex disorders often present in the same family or closely related subjects. Genetic factors undoubtedly contribute to disease

in study (A: 60 minutes; B: 240 minutes).

20 Allergic Diseases - New Insights

time in study (A: 60 minutes; B: 240 minutes).

*2.2.8. Discussion*

In the selection of the sample of our patients we were intended to be homogenous, in particular to the type of allergen sensitivity, ethnicity and area of residence and similarly of environ‐ mental surrounding.

Although we found some more frequent haplotypes in allergic patients we could not identify significant differences compared to healthy subjects. However, the second most frequent haplotype was *A\*02 B\*51 DRB1\*11*, corresponding to the fifth most frequent in control group.

Ours results are according to others studies, namely in Greek allergic children, that also failed to demonstrate a positive correlation between *HLA* haplotypes and allergy to *Dermatopha‐ goides* species [53]. So, as previously reported by a European study, it seems reasonable to assume that specific sensitization is influenced by genetic variants leading to its initiation, as well as to its enhancement [32].

*HLA* super-locus is a genomic region in the chromosomal position 6p21 [31]. It is highly promising for future research particularly since it carries on the correlation of *HLA-DRB1* in allergic asthma, *HLA-DQB1* in occupational asthma and *HLA-DPB1* in aspirin-sensitive asthma. However, it is difficult to study the role of class II genes *in vivo* because of the tremendous heterogeneity of human population, the strong linkage disequilibrium among different class II genes, and also the highly complexity of MHC.

An interesting issue in this subject is the "epistasis", representing the action between 2-loci, in opposite side to Mendel´s classical law of heredity. Epistasis designation applies when two or more loci interact to create a new phenotype, and when an allele at one locus masks or modifies the effects of alleles at one or more other loci. The phenotypic effect of a specific gene mutation entirely depends on the overall genetic background affected by many other gene mutations. Furthermore, the combined effects of more than 2 loci on phenotypes are not simply additive, but are either synergistic or antagonistic [81].

A wide range of studies has demonstrated the enormous variability of clinical phenotypes in allergic disease and respiratory allergy, representing the tremendous variability and gene diversity.

An interesting study, focused on single nucleotide polymorphisms (SNPs) from 17q21 locus, demonstrated scattering different frequencies of SNPs between distinct ethnic populations [36]. Therefore, comparisons of results between different populations are often hazardous as explained earlier.

Regarding the results of SNPs in our patients with respiratory allergy we stress relevant differences concerning IL-4, TNF-α and TGF-β genes.

TNF-α and IL-4 are relevant cytokines interested in the allergic reaction and also promotes the development of Th2 cell response to aeroallergens [7]. In the population that we studied we found strong increment of TNFα-238GG and IL4-1098TT phenotypes compared to healthy individuals. Conversely a decrease in phenotypic expression was found to TNFα-238AG and IL4-1098GG.

TGF-β is a master regulator of the immune response and exerts important anti-inflammatory functions [68] and is deeply involved in allergy. In our allergic patients TGFβ+869CT pheno‐ type had a clearly increased frequency, and the genotype TGFβ+869TT was completely absent compared to healthy population.

So far, as our knowledge, this phenotype was not previously involved in allergy or asthma. A study in cystic fibrosis demonstrated that higher plasma levels of TGFβ1 were observed in patients carrying the TGFβ1 +869TT genotype, intermediate levels for +869CT genotype and low levels to +869CC genotype. Together with these results it was also observed that the +869CC genotype had a more severe phenotype, and the +869TT genotype had the higher rate of decline of lung function, compared to +869CT genotype [17].

The IgE-mediated allergic reaction dictates a premature systemic effect, which will develop simultaneously to immuno-inflammatory mechanisms at the local of allergen exposure. In previous studies we have demonstrated that in parallel to the immediate response that results from an IgE-dependent allergenic mechanism, the cellular response occurs early in the process, and the involvement of the central immune organs is crucial since the beginning of the reaction [54, 55].

In this study we provide evidence that mRNA gene expression take place earlier on peripheral T cells after allergen exposure, at least 60 minutes later. This fact occurs independently of the technique of allergen provocation, in spite of the differences on the mechanisms and cell traffic ways involved, because they are distinct mucosae. Obviously, this modification in the production of mRNA does not imply that all of it will be completed in an efficient manner, particularly since there are strictly necessary requirements in cell regulation and biological skill conditions that allows the synthesis of the protein or its biological mediator.

Naturally, this modification in the production of mRNA does not imply that all of it will be completed in an efficient manner, particularly since in cell regulation there are specific requirements and biological conditions allowing the synthesis of the corresponding protein or its biological mediator.

A wide range of studies has demonstrated the enormous variability of clinical phenotypes in allergic disease and respiratory allergy, representing the tremendous variability and gene

An interesting study, focused on single nucleotide polymorphisms (SNPs) from 17q21 locus, demonstrated scattering different frequencies of SNPs between distinct ethnic populations [36]. Therefore, comparisons of results between different populations are often hazardous as

Regarding the results of SNPs in our patients with respiratory allergy we stress relevant

TNF-α and IL-4 are relevant cytokines interested in the allergic reaction and also promotes the development of Th2 cell response to aeroallergens [7]. In the population that we studied we found strong increment of TNFα-238GG and IL4-1098TT phenotypes compared to healthy individuals. Conversely a decrease in phenotypic expression was found to TNFα-238AG and

TGF-β is a master regulator of the immune response and exerts important anti-inflammatory functions [68] and is deeply involved in allergy. In our allergic patients TGFβ+869CT pheno‐ type had a clearly increased frequency, and the genotype TGFβ+869TT was completely absent

So far, as our knowledge, this phenotype was not previously involved in allergy or asthma. A study in cystic fibrosis demonstrated that higher plasma levels of TGFβ1 were observed in patients carrying the TGFβ1 +869TT genotype, intermediate levels for +869CT genotype and low levels to +869CC genotype. Together with these results it was also observed that the +869CC genotype had a more severe phenotype, and the +869TT genotype had the higher rate

The IgE-mediated allergic reaction dictates a premature systemic effect, which will develop simultaneously to immuno-inflammatory mechanisms at the local of allergen exposure. In previous studies we have demonstrated that in parallel to the immediate response that results from an IgE-dependent allergenic mechanism, the cellular response occurs early in the process, and the involvement of the central immune organs is crucial since the beginning

In this study we provide evidence that mRNA gene expression take place earlier on peripheral T cells after allergen exposure, at least 60 minutes later. This fact occurs independently of the technique of allergen provocation, in spite of the differences on the mechanisms and cell traffic ways involved, because they are distinct mucosae. Obviously, this modification in the production of mRNA does not imply that all of it will be completed in an efficient manner, particularly since there are strictly necessary requirements in cell regulation and biological

Naturally, this modification in the production of mRNA does not imply that all of it will be completed in an efficient manner, particularly since in cell regulation there are specific

skill conditions that allows the synthesis of the protein or its biological mediator.

diversity.

explained earlier.

22 Allergic Diseases - New Insights

IL4-1098GG.

compared to healthy population.

of the reaction [54, 55].

differences concerning IL-4, TNF-α and TGF-β genes.

of decline of lung function, compared to +869CT genotype [17].

In this context, it is very important to analyse the individual results in the different subgroups, since the high individual variation does not allow an unequivocal evaluation of the average values of the sample. For this study we did not considered to be relevant the existence of a separate control group of allergic patients, since the time point T0 would be a better internal control [54]. It was also evident that the individual response of each patient is highly variable, most likely depending on the genetic endowment of each patient, although the clinical pattern and the allergic sensitization (Dp) was the same.

During the inflammatory mechanism a set of different cytokine and chemokine molecules are able to promote not only the appropriate regulation of leukocytes and other cells recruitment but also the efficient network that permitting an accurate intracellular signaling control [73].

The existence of 23 chemokine receptors and 48 chemokine ligands guarantees a tight control and fine-tuning of the immune system [83]. Conceptually, it is considered that these biological mediators require long periods of cell maturation and synthesis, as described in several papers using culture cells [25; 29].

However, our results support the involvement of genic cytokines and chemokines are earlier events, indeed consistent with the early cell trafficking between places of allergenic aggression and those dependent on the central and secondary immune system, as noted in previous work

However, our results support that the involvement of genic cytokines and chemokines are earlier events, indeed consistent with the quickly cell trafficking between the local site of allergenic aggression and those sites dependents on the central and secondary immune system, as noted in previous research from our group [55].

More than a common standard for the same respiratory allergic disease and for the same the most important allergic sensitization is the observation of the individual profile of each patient. Indeed, IgE-mediated allergy in spite of a pathogenic mechanism conductor has at clinical level, is extremely heterogeneous. The results shown in Figures 18 to 20 also showed a tremendous heterogeneity in gene expression after allergen provocation, between our patients.

The gene expression levels of IL-4 results are extremely interesting, because unquestionably two distinct profiles are observed (high and low levels), but without correspondence with those achieved for IL-4R. Also, the level of gene expression of IL-4 was correlated with total serum IgE levels, but without correlation with the clinical severity profile, as supported by clinical practice [18].

Unlike other diseases, allergy and asthma appear to be arguably multifactorial and multigenic disorders, and therefore it has not been possible identify till now one or a set of specific genes that may be genetic markers [57, 81], although recent paper identifies the involvement of *GSTO2 loci* in asthma, and *GSTA1* as risk factor for asthma and allergies in Italian patients [56], while Genome-wide association studies (GWAS) represent the most powerful approach for asthma, have identified several distinct others genotypes from IL-18R1, IL-33, *SMAD3, ORMDL3*, *HLA-DQ* and IL-2RB loci [31].

As mentioned before, allergy and asthma are chronic inflammatory and polygenic disorders, and so any isolated specific gene could claim be an asthma gene. Therefore, recently in order to best understanding the intricate heterogeneity of this disease the designation of phenotype and endotype have been used to stress the complexity of the mechanisms and clinical patterns [10, 31].

In the pathogenesis of chronic inflammatory diseases such as asthma several transcription (signal transducer and activator) factors are known to play a crucial role, acting as agonists or antagonists [79]. A transcription factor is formed by a sequence-specific DNA-binding factor. It is a protein that specific binds to specific DNA sequences, so it is able to controlling the rate of transcription of genetic information from DNA to messenger RNA.

These factors perform this purpose alone or with other proteins in a complex, by promoting (activator), or blocking (repressor) the recruitment of RNA polymerase. These enzymes permit the efficient genetic transcription from DNA to RNA to a specific gene.

A defining feature of transcription factors is that they contain one or more DNA-binding domains, which linked to specific sequences of DNA adjacent to the genes that they regulate.

For an efficient transcription are needed additional proteins (not classified as transcription factors) such as coactivators, chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases, having crucial roles in gene regulation, but lack DNA-binding domains.

So these factors are very important and therefore we also studied three of them.

*EGR1*, also knew as early growth response protein 1, have engaged in multi functions such as transcriptional regulator, mitogenesis, cell proliferation and differentiation regulation, apoptosis and tumor suppressor gene [28]. Previous report had highlighted the potential stimulatory and inhibitory effects on *EGR1* by *STAT6* (Th2 differentiation enhancer) and *STAT1* (Th1 differentiation enhancer), respectively.

Overall our patients, we have observed at 60 minutes later the allergen exposure an increase of expression of *EGR1*, favoring the allergic reaction, but at 240 minutes after provocation levels had decreased, possibly due to mechanisms of self-control of allergic reaction.

In opposite, the *JUNB* expression decreased at least after 60 minutes of the beginning of the allergic reaction, and going increasing later at 240 minutes. This factor is mainly a Th2 cell related transcription factor (Il-4), but also have role favoring other cytokines: IL-1, IL-2, and IL-10 [79]. So, we hypothesize that the earlier involvement could favouring the beginning of the allergic reaction, but also could be later a down-regulator factor.

*JAK2* belongs to a tyrosine kinase family, and its activation engages the transcription factor *STAT3* to initiate *JAK-STAT* pathway [82]. It signalizes the interferon receptors, and GM-CSF receptor family (IL3R, IL5R, GM-CSF-R). In our patients we did not observed. In our patients, we did not observe relevant performance for this transcription factor.

Allergic diseases are complex and multifaceted, and asthma in particular was widely recog‐ nized as condition in which both genes and the environment play critical roles.

Currently, GWAS try to identifying a number of candidate genes evocative of biological relevance, but have failed to report for more than a reduced amount of phenotypic variance – a situation that has come to be known as the problem of the missing or hidden heritability [13, 76].

As mentioned before, allergy and asthma are chronic inflammatory and polygenic disorders, and so any isolated specific gene could claim be an asthma gene. Therefore, recently in order to best understanding the intricate heterogeneity of this disease the designation of phenotype and endotype have been used to stress the complexity of the mechanisms and clinical patterns

In the pathogenesis of chronic inflammatory diseases such as asthma several transcription (signal transducer and activator) factors are known to play a crucial role, acting as agonists or antagonists [79]. A transcription factor is formed by a sequence-specific DNA-binding factor. It is a protein that specific binds to specific DNA sequences, so it is able to controlling the rate

These factors perform this purpose alone or with other proteins in a complex, by promoting (activator), or blocking (repressor) the recruitment of RNA polymerase. These enzymes permit

A defining feature of transcription factors is that they contain one or more DNA-binding domains, which linked to specific sequences of DNA adjacent to the genes that they regulate.

For an efficient transcription are needed additional proteins (not classified as transcription factors) such as coactivators, chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases, having crucial roles in gene regulation, but lack DNA-binding domains.

*EGR1*, also knew as early growth response protein 1, have engaged in multi functions such as transcriptional regulator, mitogenesis, cell proliferation and differentiation regulation, apoptosis and tumor suppressor gene [28]. Previous report had highlighted the potential stimulatory and inhibitory effects on *EGR1* by *STAT6* (Th2 differentiation enhancer) and

Overall our patients, we have observed at 60 minutes later the allergen exposure an increase of expression of *EGR1*, favoring the allergic reaction, but at 240 minutes after provocation levels

In opposite, the *JUNB* expression decreased at least after 60 minutes of the beginning of the allergic reaction, and going increasing later at 240 minutes. This factor is mainly a Th2 cell related transcription factor (Il-4), but also have role favoring other cytokines: IL-1, IL-2, and IL-10 [79]. So, we hypothesize that the earlier involvement could favouring the beginning of

*JAK2* belongs to a tyrosine kinase family, and its activation engages the transcription factor *STAT3* to initiate *JAK-STAT* pathway [82]. It signalizes the interferon receptors, and GM-CSF receptor family (IL3R, IL5R, GM-CSF-R). In our patients we did not observed. In our patients,

Allergic diseases are complex and multifaceted, and asthma in particular was widely recog‐

So these factors are very important and therefore we also studied three of them.

had decreased, possibly due to mechanisms of self-control of allergic reaction.

the allergic reaction, but also could be later a down-regulator factor.

we did not observe relevant performance for this transcription factor.

nized as condition in which both genes and the environment play critical roles.

*STAT1* (Th1 differentiation enhancer), respectively.

of transcription of genetic information from DNA to messenger RNA.

the efficient genetic transcription from DNA to RNA to a specific gene.

[10, 31].

24 Allergic Diseases - New Insights

In our patients we could verify that the allergen challenge determines a very early cellular response with gene and protein expression.

At present, the environmental influence with direct effects on epigenetic plan offers an explanation for this extreme individual variability, one clinical disease but different pheno‐ types and endotypes, not yet fully characterized.

So, in spite of the same genetic heritage, each individual and each patient, with an equal number of chromosomes and genes, have a specificity and uniqueness: all equals but all different from one to each other.

In Figure 22 we present the results of 4 allergic patients with asthma and rhinitis.

Fig. 22. Overall results of <sup>4</sup> patients with allergic asthma and rhinitis **Figure 22.** Overall results of 4 patients with allergic asthma and rhinitis

Gene expression of cytokines and chemokines seem to have a strictly correlation with the results obtained in the study of nuclear transcription factors, *ERG1* and *JUNB*, as discussed before.

The susceptibility to elicit an allergic response or in opposite side its own inhibition is under liability of a time-dependent dynamic response since the allergen exposure. It might be more appropriate to view genes as a frame surrounding a complex network of biological processes. The frame delimits and supports the network, but the connections within the network determine its functions.

Besides the identification of novel pathological mechanisms and therapeutics, the wealth of information leads to the vision of genes forecasting asthma risk and phenotypes, with genetic scores and eventual personalized medicine with more effective treatments. Gene-environment interactions might difficult the achievement of such knowledge. Thus, even after the completed sequence of the human genome, the scientific community maintains expectations that genetics may be a decisive tool in the allergic risk prediction and personalized tailor-made treatments. At present asthma epigenetics is still in its infancy and a promise to keep [13].

## **Author details**

Celso Pereira\* , Frederico S. Regateiro, Graça Loureiro, Beatriz Tavares and António Martinho

\*Address all correspondence to<EMAIL_ADDRESS>
Clinic Immunology, Medicine Faculty, Coimbra University. Immunoallergology Depart‐ ment, Coimbra University Center Hospital. Blood and Transplantation Center of Coimbra / Portuguese Institute of Blood and Transplantation., Portugal

## **References**

[5] Campbell CD, Mohajeri K, Malig M, et al. (2014) Whole-Genome Sequencing of Indi‐ viduals from a Founder Population Identifies Candidate Genes for Asthma. PLoS ONE, Vol. 9, nº 8, (August 2014), pp. e104396, ISSN 1932-6203.

The susceptibility to elicit an allergic response or in opposite side its own inhibition is under liability of a time-dependent dynamic response since the allergen exposure. It might be more appropriate to view genes as a frame surrounding a complex network of biological processes. The frame delimits and supports the network, but the connections within the network

Besides the identification of novel pathological mechanisms and therapeutics, the wealth of information leads to the vision of genes forecasting asthma risk and phenotypes, with genetic scores and eventual personalized medicine with more effective treatments. Gene-environment interactions might difficult the achievement of such knowledge. Thus, even after the completed sequence of the human genome, the scientific community maintains expectations that genetics may be a decisive tool in the allergic risk prediction and personalized tailor-made treatments.

At present asthma epigenetics is still in its infancy and a promise to keep [13].

\*Address all correspondence to<EMAIL_ADDRESS>
Portuguese Institute of Blood and Transplantation., Portugal

ber 2012), pp. 1049-62, ISSN 0091-6749.

1985-94, ISSN 1533-4406.

, Frederico S. Regateiro, Graça Loureiro, Beatriz Tavares and

Clinic Immunology, Medicine Faculty, Coimbra University. Immunoallergology Depart‐ ment, Coimbra University Center Hospital. Blood and Transplantation Center of Coimbra /

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**Author details**

António Martinho

Celso Pereira\*

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32 Allergic Diseases - New Insights

**Chapter 2**

## **Airborne Allergens**

Olga Ukhanova and Evgenia Bogomolova

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59300

## **1. Introduction**

A huge number of substances of various origin forming atmospheric aerosols constantly circulates in the atmospheric air (Table 1). One of the most important components of such aerosols are airborne allergens. Airborne allergens are substances of biological origin, which getting into the human body promote the induction of the immune response with the subse‐ quent development of an allergic disease. Allergic reaction is directly caused by proteins and glycoproteins forming the airborne allergen.

**Table 1.** The components of the atmospheric aerosols and their size [4].

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

More than 15 million people in Europe suffer from allergic rhinitis, conjunctivitis, bronchial asthma, atopic dermatitis and food allergy. Climatic and geographic features of the Russian Federation (its Northern and Southern parts, as well as European and Asian territories) contribute to a different sickness rate among the population [1, 2].

So far there are many questions concerning monitoring and a variety of the airborne allergens, and especially their influence on the human body. The study of the airborne allergens is of theoretical and practical interest for many specialists (allergists, laboratorians, ecologists, biologists). The biological study of the airborne allergens is connected with the problem of the origin, transport, protein structure and protein interaction with the macroorganism. Under the influence of the environment the airborne allergens may be a subject to significant changes and they may change their properties.

This chapter presents the main types of the airborne allergens: their origin, transport, proper‐ ties, monitoring, etc.

## **2. Classification of the airborne allergens**

The most common airborne allergens are pollen, fungal spores, house dust, house dust mites, animal allergens, insect allergens, industrial allergens, food and drug allergens.

**Table 2.** Classification of the airborne allergens.

The scientific line-molecular allergology-intensively develops in modern allergology for the last 10-15 years. The modern allergen nomenclature adopted and approved in 2013 by WAO is now becoming routine in practice of the allergist [3].

We will present the most well-known and popular airborne allergens in applied medicine which are used for the diagnosis and treatment of allergic diseases of reaginic type (IgEdependent allergic reactions or type I of immune response). Such diseases include allergic rhinitis, allergic conjunctivitis, atopic dermatitis, bronchial asthma, urticaria, etc.

The knowledge of the airborne allergens types is very useful in routine practice of a medical specialist for the purpose of individual selection of methods of diagnosis and treatment of patients with respiratory allergosis, as well as for the prevention of allergic diseases in genetically predisposed to atopy or already sensitized patients.

## **3. Pollen allergens**

More than 15 million people in Europe suffer from allergic rhinitis, conjunctivitis, bronchial asthma, atopic dermatitis and food allergy. Climatic and geographic features of the Russian Federation (its Northern and Southern parts, as well as European and Asian territories)

So far there are many questions concerning monitoring and a variety of the airborne allergens, and especially their influence on the human body. The study of the airborne allergens is of theoretical and practical interest for many specialists (allergists, laboratorians, ecologists, biologists). The biological study of the airborne allergens is connected with the problem of the origin, transport, protein structure and protein interaction with the macroorganism. Under the influence of the environment the airborne allergens may be a subject to significant changes

This chapter presents the main types of the airborne allergens: their origin, transport, proper‐

The most common airborne allergens are pollen, fungal spores, house dust, house dust mites,

Fish and cancroid protein

Chitinous cover

activity

and products of insects vital

Honey

The scientific line-molecular allergology-intensively develops in modern allergology for the last 10-15 years. The modern allergen nomenclature adopted and approved in 2013 by WAO

animal allergens, insect allergens, industrial allergens, food and drug allergens.

**Animals** Epidermal Food-borne Insect

**Plants** Pollen foodstuffs

**Drugs** antibiotics, sulfanilamides, immunological drugs

**Industrial substances** polymers, pesticides, metalls **Dust** house, library, archive dust

**Table 2.** Classification of the airborne allergens.

**Phycomycetes** spores, products of fungi vital activity

is now becoming routine in practice of the allergist [3].

contribute to a different sickness rate among the population [1, 2].

and they may change their properties.

**Origin Types of the allergens**

**2. Classification of the airborne allergens**

Hair/wool epithelium feather saliva excrements

ties, monitoring, etc.

36 Allergic Diseases - New Insights

**Noninfectious allergens**

**Infectious allergens**

#### **3.1. Aeropalynology**

Aeropalynology is a part of aerobiology studying pollen grains and spores of plants passively circulating in the atmosphere. Also, it is a branch of modern biology that studies the structure and laws of formation of the pollen rain. Aeropalynology is closely connected with allergology as pollen of plants and fungal spores belong to the most widespread airborne allergens causing diseases of the upper airways [4]. Aeropalynologists are the experts engaged in identification of qualitative and quantitative structure of a pollen rain. They study features of its daily rhythms and seasonal dynamics, compile a calendar of allergenic plants dusting and sporu‐ lation of mycelial fungi. These data are necessary for various experts (allergists, otorhinolar‐ yngologists, general practitioners) and patients to predict exacerbations of allergic diseases and to take preventive measures.

Long-term observations over the years are the most informative for identifying patterns of plants dusting over large areas with different climate and landscapes.

Currently there is a single international aeropalynology service that possesses information about the dynamics of the pollen concentrations of the most common allergenic taxa in the air and the bank of these data. Since 1993, the database provides information on the dusting of different plants in Europe and forecasts the dusting based on the current and long-term aeropalynological observations [5, 6].

The program of aeropalynological research was launched in Russia in 2007. Several cities with installed pollen monitoring stations (volumetric Burkard SporeWatch sampler) had been included in this program (Fig. 1-3). These cities are Moscow, St. Petersburg, Saratov, Stavropol, Astrakhan, Rostov-on-Don, Krasnodar, Yaroslavl, Novosibirsk, Yekaterinburg, Ryazan, Smolensk, and others. The aeropalynological stations start working from 15 of April to 15 of September every year (the period of active flowering of wind-pollinated plants in Russia). The aeropalynological monitoring is carried out the year-round in Moscow and St. Petersburg. The studies of pollen are carried out by the Russian allergists and laboratory technicians, who were trained under the guidance of Severova E.A. at the Department of Palynology, Faculty of Biology of Moscow State University. The station in Moscow is the curator of a national Russian network of aeropalynological monitoring and represents Russia in the European and Interna‐ tional association of aeropalinologists.

**Figure 1.** Burkard SporeWatch sampler

**Figure 2.** Burkard SporeWatch sampler Cylinder with 7-days or 24-hours periods of selection in Krasnodar

network of aeropalynological monitoring and represents Russia in the European and Interna‐

**Figure 2.** Burkard SporeWatch sampler Cylinder with 7-days or 24-hours periods of selection in Krasnodar

tional association of aeropalinologists.

38 Allergic Diseases - New Insights

**Figure 1.** Burkard SporeWatch sampler

**Figure 3.** Burkard-Hirst SporeWatch in Moscow, Saratov, Pyatigorsk, Rostov-on-Don, St. Petersburg, Stavropol

The measurement of the concentration of mycelial fungi spores in the atmospheric air is carried out by means of the automatic Burkard sampler on the Petri dish with Czapek agarized medium (Fig. 4).

Figure 4. **Burkard SporeWatch sampler and sampler (St. Petersburg)** PU-1B The determination of the spores concentration (colony forming units - CFU) in the atmospheric air of the premises, parks and industrial areas is also very informative for **Figure 4.** Burkard SporeWatch sampler and sampler (St. Petersburg) PU-1B The determination of the spores concentra‐ tion (colony forming units-CFU) in the atmospheric air of the premises, parks and industrial areas is also very informa‐ tive for collecting the patient's medical history and selection of diagnostic tests in vivo and in vitro.

collecting the patient's medical history and selection of diagnostic tests in vivo and in vitro.

**Pollen-grain morphology**

For allergology the greatest interest is represented by pollen grains of wind-pollinated plants (angiosperms and gymnosperms) because pollen is spread by wind over hundreds of kilometers from the habitats of plants. Most of the entomogenous plants are not allergenic

Figure 5. The pollen grain development and its participation in fertilization

for human.

#### **3.2. Pollen-grain morphology**

For allergology the greatest interest is represented by pollen grains of wind-pollinated plants (angiosperms and gymnosperms) because pollen is spread by wind over hundreds of kilome‐ ters from the habitats of plants. Most of the entomogenous plants are not allergenic for human.

**Figure 5.** The pollen grain development and its participation in fertilization

Pollen grains are reduced male gametogophytes developing inside the microspore shell. Pollen grains of the angiosperms are formed from microspores (mother cells) in microsporangia which represent anther nests.

Each pollen grain consists of the vegetative cell, and the generative cell is immersed in the cytoplasm of the vegetative cell (Fig. 5). During the germination the vegetative cell forms a pollen tube, and the nucleus of the generative cell forms 2 sperm cells involved in fertilization [4].

#### **3.3. Sporoderma**

Sporoderma is a shell of pollen grains (Fig. 6).

Sporoderma is a set of morphologically different layers: perispory (Perina=trifina), exine (exosporium), and intine (endosporium) which protect the cytoplasm of the pollen grain from the external influence. The sporoderma layer structure is specific to orders, families, genera and species of higher plants.

Pollen grains are reduced male gametogophytes developing inside the microspore shell. Pollen grains of the angiosperms are formed from microspores (mother cells) in

Each pollen grain consists of the vegetative cell, and the generative cell is immersed in the cytoplasm of the vegetative cell (Fig. 5). During the germination the vegetative cell forms a pollen tube, and the nucleus of the generative cell forms 2 sperm cells involved in

**Sporoderma**

microsporangia which represent anther nests.

Sporoderma is a shell of pollen grains (Fig. 6).

Figure 6. Pollen grain structure **Figure 6.** Pollen grain structure

fertilization [4].

processing.

respiratory tract [4].

concentration.

pollen of the entomophilous plants.

**3.2. Pollen-grain morphology**

40 Allergic Diseases - New Insights

**Figure 5.** The pollen grain development and its participation in fertilization

which represent anther nests.

and species of higher plants.

Sporoderma is a shell of pollen grains (Fig. 6).

fertilization [4].

**3.3. Sporoderma**

For allergology the greatest interest is represented by pollen grains of wind-pollinated plants (angiosperms and gymnosperms) because pollen is spread by wind over hundreds of kilome‐ ters from the habitats of plants. Most of the entomogenous plants are not allergenic for human.

Pollen grains are reduced male gametogophytes developing inside the microspore shell. Pollen grains of the angiosperms are formed from microspores (mother cells) in microsporangia

Each pollen grain consists of the vegetative cell, and the generative cell is immersed in the cytoplasm of the vegetative cell (Fig. 5). During the germination the vegetative cell forms a pollen tube, and the nucleus of the generative cell forms 2 sperm cells involved in

Sporoderma is a set of morphologically different layers: perispory (Perina=trifina), exine (exosporium), and intine (endosporium) which protect the cytoplasm of the pollen grain from the external influence. The sporoderma layer structure is specific to orders, families, genera

The exine is the outer part of the pollen grain shell of most seed plants. The structure of the exine includes sporopollenin. It is a biopolymer that can withstand the high temperature, and is insoluble in alkalis and acids (Fig. 7). Sporoderma is a set of morphologically different layers: perispory (Perina=trifina), exine (exosporium), and intine (endosporium) which protect the cytoplasm of the pollen grain from the external influence. The sporoderma layer structure is specific to orders, families, genera and species of higher plants.

The internal structure of the exine forms texture and sculpture. And that is a main point for the aeropalynologists in determining the morphological type of pollen grains. exine includes sporopollenin. It is a biopolymer that can withstand the high temperature, and is insoluble in alkalis and acids (Fig. 7).

The exine is the outer part of the pollen grain shell of most seed plants. The structure of the

The internal structure of the exine forms texture and sculpture. And that is a main point for

the aeropalynologists in determining the morphological type of pollen grains.

Figure 7. **The structure of angiosperm sporoderma:** 1- endexine, 2 – intine, 3- styles, 4- integument, 5- underlaying layer. **Figure 7.** The structure of angiosperm sporoderma: 1-endexine, 2 – intine, 3-styles, 4-integument, 5-underlaying layer.

Intine is the inner layer of sporoderma bordering with cytoplasm and collapsing in acytolysis

Trifina is a thin squamous layer of wind-pollinated plants. It is poorly visible in contrast to the

The complex morphology and molecular composition of pollen helps to activate the immune system of a human for the purpose of elimination of airborne allergens from mucosa of the

**The main plants the pollen of which causes an allergy**

In the central Europe, the most common cause of pollinosis is a pollen of wind-pollinated plants: trees, cereals and weeds. Their pollen is volatile, and during flowering of these plants the pollen accumulates in the air in quantity sufficient enough to create a relatively high

As a result of the aeropalynological researches the calendars of dusting are compiled annually. The calendars containing 15 taxa of plants are compiled for Europe. . However, in Intine is the inner layer of sporoderma bordering with cytoplasm and collapsing in acytolysis processing.

Trifina is a thin squamous layer of wind-pollinated plants. It is poorly visible in contrast to the pollen of the entomophilous plants.

The complex morphology and molecular composition of pollen helps to activate the immune system of a human for the purpose of elimination of airborne allergens from mucosa of the respiratory tract [4].

#### **3.4. The main plants the pollen of which causes an allergy**

In the central Europe, the most common cause of pollinosis is the pollen of wind-pollinated plants: trees, cereals and weeds. Their pollen is volatile, and during flowering of these plants the pollen accumulates in the air in quantity sufficient enough to create a relatively high concentration.

As a result of the aeropalynological researches the calendars of dusting are compiled annually. The calendars containing 15 taxa of plants are compiled for Europe.. However, in the North and the South of Russia (the European part of it) a range of airborne allergens is larger and depends on the trajectory of the distribution of pollen (Fig. 8) [4].

During the examination and management of patients with pollinosis, the seasonal and daily changes of the pollen content should also be taken into account.

The allergy to pollen of grasses and trees grows only in the period of their flowering. So each region has its own seasonal peaks of incidence. The maximum concentration of pollen in the air is in the early morning hours, from dawn until 12 p.m.

Among deciduous trees the allergic diseases are caused most often by pollen of birch, alder, hazel, maple, oak, and others. We present the main taxa of trees which are determined in the atmospheric air:

**Figure 8.** The trajectory of the distribution of pollen from Moscow. 7. Populus deltoides (Cottonwood) (Fig.14) 8. Acer negundo (Fig.15)

9. Fraxinus americana (White ash) (Fig.16)

Figure 8. Alnus incana **Figure 9.** Alnus incana

Figure 9. Betula verrucosa

Intine is the inner layer of sporoderma bordering with cytoplasm and collapsing in acytolysis

Trifina is a thin squamous layer of wind-pollinated plants. It is poorly visible in contrast to the

The complex morphology and molecular composition of pollen helps to activate the immune system of a human for the purpose of elimination of airborne allergens from mucosa of the

In the central Europe, the most common cause of pollinosis is the pollen of wind-pollinated plants: trees, cereals and weeds. Their pollen is volatile, and during flowering of these plants the pollen accumulates in the air in quantity sufficient enough to create a relatively high

As a result of the aeropalynological researches the calendars of dusting are compiled annually. The calendars containing 15 taxa of plants are compiled for Europe.. However, in the North and the South of Russia (the European part of it) a range of airborne allergens is larger and

During the examination and management of patients with pollinosis, the seasonal and daily

The allergy to pollen of grasses and trees grows only in the period of their flowering. So each region has its own seasonal peaks of incidence. The maximum concentration of pollen in the

Among deciduous trees the allergic diseases are caused most often by pollen of birch, alder, hazel, maple, oak, and others. We present the main taxa of trees which are determined in the

processing.

respiratory tract [4].

42 Allergic Diseases - New Insights

concentration.

atmospheric air:

**1.** Alnus incana (Grey alder) (Fig.9)

**3.** Corylus avellana (Hazel) (Fig.11)

**5.** Ulmus americana (Elm) (Fig.13)

**7.** Populus deltoides (Cottonwood) (Fig.15)

**9.** Fraxinus americana (White ash) (Fig.17)

**6.** Salix caprea (Willow) (Fig.14)

**8.** Acer negundo (Fig.16)

**4.** Quercus alba (Oak) (Fig.12)

pollen of the entomophilous plants.

**3.4. The main plants the pollen of which causes an allergy**

depends on the trajectory of the distribution of pollen (Fig. 8) [4].

changes of the pollen content should also be taken into account.

air is in the early morning hours, from dawn until 12 p.m.

**2.** Betula verrucosa (Common silver birch) (Fig.10)

Figure 8. Alnus incana

the atmospheric air:

1. Alnus incana (Grey alder) (Fig.8)

3. Corylus avellana (Hazel) (Fig.10) 4. Quercus alba (Oak) (Fig.11) 5. Ulmus americana (Elm) (Fig.12) 6. Salix caprea (Willow) (Fig.13)

8. Acer negundo (Fig.15)

7. Populus deltoides (Cottonwood) (Fig.14)

9. Fraxinus americana (White ash) (Fig.16)

2. Betula verrucosa (Common silver birch) (Fig.9)

Among deciduous trees the allergic diseases are caused most often by pollen of birch, alder, hazel, maple, oak, and others. We present the main taxa of trees which are determined in

#### **Figure 10.** Figure 9. Betula verrucosa Betula verrucosa

Figure 10. Corylus avellana **Figure 11.** Corylus avellana

Figure 11. Quercus alba **Figure 12.** Quercus alba

Figure 11. Quercus alba

Figure 12. Ulmus Americana

Figure 12. Ulmus Americana

**Figure 13.** Figure 12. Ulmus Americana Ulmus Americana

Figure 10. Corylus avellana

Figure 11. Quercus alba

Among deciduous trees the allergic diseases are caused most often by pollen of birch, alder, hazel, maple, oak, and others. We present the main taxa of trees which are determined in

the atmospheric air:

Figure 8. Alnus incana

44 Allergic Diseases - New Insights

**Figure 10.** Figure 9. Betula verrucosa Betula verrucosa

Figure 10. Corylus avellana

Figure 10. Corylus avellana

**Figure 11.** Corylus avellana

Figure 11. Quercus alba

Figure 11. Quercus alba

**Figure 12.** Quercus alba

Figure 12. Ulmus Americana

Figure 12. Ulmus Americana

1. Alnus incana (Grey alder) (Fig.8)

3. Corylus avellana (Hazel) (Fig.10) 4. Quercus alba (Oak) (Fig.11) 5. Ulmus americana (Elm) (Fig.12) 6. Salix caprea (Willow) (Fig.13)

8. Acer negundo (Fig.15)

7. Populus deltoides (Cottonwood) (Fig.14)

9. Fraxinus americana (White ash) (Fig.16)

2. Betula verrucosa (Common silver birch) (Fig.9)

Figure 13. Salix caprea **Figure 14.** Salix caprea Figure 13. Salix caprea

Figure 14 Populus deltoids **Figure 15.** Populus deltoids

Figure 14 Populus deltoids

Figure 15. Acer negundo

Figure 15. Acer negundo

Figure 14 Populus deltoids

Figure 13. Salix caprea

**Figure 16.** Figure 15. Acer negundo Acer negundo

Figure 16. Fraxinus Americana **Figure 17.** Fraxinus Americana

There is a structural homology between allergens of trees pollen, and it is expressed much more weakly, than the existing affinity of allergens of grass pollen. Therefore, the patients who have an increased sensitivity to the birch pollen react at the same time to the pollen of hazel and alder. There is a structural homology between allergens of trees pollen, and it is expressed much more weakly, than the existing affinity of allergens of grass pollen. Therefore, the patients who have an increased sensitivity to the birch pollen react at the same time to the pollen of hazel and alder.

The coniferous plants produce the pollen in large quantities, but its allergenicity is lower because the diameter of pollen grains is 30 to 100 microns. The coniferous plants produce the pollen in large quantities, but its allergenicity is lower because the diameter of pollen grains is 30 to 100 microns.

The birch pollen has the most evident allergenic activity among the analogs – the pollen of deciduous wind-pollinated plants - because it contains about 40 proteins, the 6 of which have the properties of allergens [11]. The birch pollen has the most evident allergenic activity among the analogs – the pollen of deciduous wind-pollinated plants-because it contains about 40 proteins, the 6 of which have the properties of allergens [11].

The development of technology of various recombinant allergens production has revealed that the common allergens of protein nature (e.g. "panallergens", Bet v 1 protein and Bet v 2 birch profilin) are responsible for cross-reacting of allergens of spring trees pollen [11]. The development of technology of various recombinant allergens production has revealed that the common allergens of protein nature (e.g. "panallergens", Bet v 1 protein and Bet v 2 birch profilin) are responsible for cross-reacting of allergens of spring trees pollen [11].

The main characteristic of the cereals pollen is its spherical, oval or elliptical shape. There are no many detailed species and genera definitions. But pollen grain has always one pore (Fig. 17) and a smooth surface. The size of the cultivated cereals pollen is usually larger

11 groups of major and minor allergens could be defined among the cereals [12,13].

The main taxa of cereals (Poaceae) (Fig.17): The main taxa of cereals (Poaceae) (Fig.18):

> 1. Dactylis glomerata (Cocksfoot) 2. Festuca elatior (Meadow fescue) 3. Phleum pratense (Timothy) 4. Poa pratensis (Meadow grass)

6. Bromus inermis (Brome grass) 7. Secale cereale (Cultivated rye) 8. Holcus lanatus (Velvet grass)

10. Maize (Corn)

11. Arrhenatherum elatius

5. Agrostis stolonifera (Redtop, Bentgrass)

9. Alopecurus pratensis (Meadow foxtail)

than of wild one and ranges from 25 to 75 microns [4].

Figure 13. Salix caprea

Figure 14 Populus deltoids

46 Allergic Diseases - New Insights

**Figure 16.** Figure 15. Acer negundo Acer negundo

Figure 16. Fraxinus Americana

have the properties of allergens [11].

the properties of allergens [11].

The main taxa of cereals (Poaceae) (Fig.17):

The main taxa of cereals (Poaceae) (Fig.18):

1. Dactylis glomerata (Cocksfoot) 2. Festuca elatior (Meadow fescue) 3. Phleum pratense (Timothy) 4. Poa pratensis (Meadow grass)

6. Bromus inermis (Brome grass) 7. Secale cereale (Cultivated rye) 8. Holcus lanatus (Velvet grass)

10. Maize (Corn)

11. Arrhenatherum elatius

5. Agrostis stolonifera (Redtop, Bentgrass)

9. Alopecurus pratensis (Meadow foxtail)

than of wild one and ranges from 25 to 75 microns [4].

hazel and alder.

and alder.

**Figure 17.** Fraxinus Americana

There is a structural homology between allergens of trees pollen, and it is expressed much more weakly, than the existing affinity of allergens of grass pollen. Therefore, the patients who have an increased sensitivity to the birch pollen react at the same time to the pollen of

There is a structural homology between allergens of trees pollen, and it is expressed much more weakly, than the existing affinity of allergens of grass pollen. Therefore, the patients who have an increased sensitivity to the birch pollen react at the same time to the pollen of hazel

The coniferous plants produce the pollen in large quantities, but its allergenicity is lower

The coniferous plants produce the pollen in large quantities, but its allergenicity is lower

The birch pollen has the most evident allergenic activity among the analogs – the pollen of deciduous wind-pollinated plants-because it contains about 40 proteins, the 6 of which have

The development of technology of various recombinant allergens production has revealed that the common allergens of protein nature (e.g. "panallergens", Bet v 1 protein and Bet v 2 birch