**Noninvasive Inflammatory Biomarkers**

178 Inflammatory Diseases – A Modern Perspective

Yao, C., Sakata, D., Esaki, Y., Matsuoka, T., Kuroiwa, K., Sugimoto, Y. & Narumiya, S.

Th1 cell differentiation and Th17 cell expansion. *Nat. med*. 15(6) : 633-640. Yokomizo, T., Kato, K., Terawaki, K., Izumi T. & Shimizu, T. (2000). A second leukotriene

Zurier, R.B., Rossetti, R.G., Jacobson, E.W., DeMarco, D.M., Liu, N.Y., Temming, J.E., White,

A randomized, placebo-controlled trial. *Arthritis Rheum*. 39(11): 1808-1817.

disorders *J Exp. Med*. 192(3): 421-432.

(2009). Prostaglandin E2-EP4 signalling promotes immune inflammation through

B(4) receptor, BLT2. A new therapeutic target in inflammation and immunological

B.M. & Laposat, M. (1996). gamma-Linolenic acid treatment of rheumatoid arthritis.

**9** 

 *Spain* 

J. Bellido-Casado

*Institut de Recerca, Pneumology Department, Hospital Santa Creu i Sant Pau Barcelona* 

**A New Era for Assessing Airway Diseases:** 

The definition of asthma is constantly being modified and redefined. New knowledge derived from molecular biology and applied immunology is very valuable in interpreting how the airway becomes diseased. At the same time, new biomarkers useful for monitoring patients and detecting inflammatory airway disease are being identified. Currently, patients whose condition is included in the spectrum of inflammatory airway diseases may be reassessed and placed into subgroups so that the biotypes, or endotypes (disease) and phenotypes (patients), form new paradigms to delineate and integrate applied knowledge to this complex and heterogeneously-expressed disease. The definition of asthma and different

The definition of asthma may be established using a set of characteristics that are clinical (recurrent episodes of wheezing and dyspnea), pathophysiological (variability in airflow), or immunological (chronic inflammation) (Global Initiative for Astma [GINA], 2009; British Thoracic Society Scottish Intercollegiate Guidelines Network [BTSSIGN], 2008). They may be found in patients or heterogeneous groups of patients who share these symptoms to a greater or lesser extent. Depending on the emphasis placed on a more specific predominant feature of airway dysfunction, the 'nominalist' view of the concept of asthma is more relevant. On the other hand, if several features are examined together to make the diagnosis, the more 'essentialist' aspect of the specific airway disease stands out [GINA, 2009; BTSSIGN 2008]. Thus, both definitions must be kept in mind when dealing with asthma, and determining an objective system of measurement for the quantifiable aspects that that make up either definition is required [Hargreave & Nair, 2009]. For decades, different methods to quantify and measure the various components of asthma (symptoms, spirometry, maximum peak flow, bronchial provocation) have been used in situations of good health or illness, highlighting the complexity involved in studying, functionally, both the normal and altered airway. Not all the defining characteristics of asthma are present in all patients; moreover, they vary greatly and are often irregular in a single patient. The therapeutic response may also be different depending on the specific pathophysiological characteristics that are predominately found [Lotvall et al., 2011]. Therefore, the classification of asthma severity by

ways of understanding the disease undergo constant review.

**2. Asthma defined as an inflammatory airway disease** 

**1. Introduction** 

 **New Insights in the Asthma Paradigm** 

### **A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm**

J. Bellido-Casado

*Institut de Recerca, Pneumology Department, Hospital Santa Creu i Sant Pau Barcelona Spain* 

#### **1. Introduction**

The definition of asthma is constantly being modified and redefined. New knowledge derived from molecular biology and applied immunology is very valuable in interpreting how the airway becomes diseased. At the same time, new biomarkers useful for monitoring patients and detecting inflammatory airway disease are being identified. Currently, patients whose condition is included in the spectrum of inflammatory airway diseases may be reassessed and placed into subgroups so that the biotypes, or endotypes (disease) and phenotypes (patients), form new paradigms to delineate and integrate applied knowledge to this complex and heterogeneously-expressed disease. The definition of asthma and different ways of understanding the disease undergo constant review.

#### **2. Asthma defined as an inflammatory airway disease**

The definition of asthma may be established using a set of characteristics that are clinical (recurrent episodes of wheezing and dyspnea), pathophysiological (variability in airflow), or immunological (chronic inflammation) (Global Initiative for Astma [GINA], 2009; British Thoracic Society Scottish Intercollegiate Guidelines Network [BTSSIGN], 2008). They may be found in patients or heterogeneous groups of patients who share these symptoms to a greater or lesser extent. Depending on the emphasis placed on a more specific predominant feature of airway dysfunction, the 'nominalist' view of the concept of asthma is more relevant. On the other hand, if several features are examined together to make the diagnosis, the more 'essentialist' aspect of the specific airway disease stands out [GINA, 2009; BTSSIGN 2008]. Thus, both definitions must be kept in mind when dealing with asthma, and determining an objective system of measurement for the quantifiable aspects that that make up either definition is required [Hargreave & Nair, 2009]. For decades, different methods to quantify and measure the various components of asthma (symptoms, spirometry, maximum peak flow, bronchial provocation) have been used in situations of good health or illness, highlighting the complexity involved in studying, functionally, both the normal and altered airway. Not all the defining characteristics of asthma are present in all patients; moreover, they vary greatly and are often irregular in a single patient. The therapeutic response may also be different depending on the specific pathophysiological characteristics that are predominately found [Lotvall et al., 2011]. Therefore, the classification of asthma severity by

A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 183

the management of patients. Furthermore, those used routinely provide specific and limited information only. Therefore, current research being conducted focuses more on the inflammatory aspects of the lung, rather than the classical or clinical function, because it is the central pathogenic mechanism of the disease in the airway [Fabbri et al., 2005]. Severity and control disease assessment requires a multidimensional practical approach including an inflammatory view, as was previously confirmed by some authors [Fitzpatrick et al. 2011a;

Technological advancements brought about by modern molecular biology and innovative micron analysis technology applied to the study of inflammation and the pathways of oxidative, lipid, or nitric stress, for example, can allow for a better identification and definition of new parameters, inflammatory profiles, and biomarkers that are more sensitive and specific for predicting the state of progression of IAD, differences in poor outcomes, and the type of anti-inflammatory treatment best suited to manage a particular patient or patient

This section briefly discusses the most relevant parameters used in clinical practice for the monitoring of asthma, especially the most recent parameters that contribute greatly to the overall management of patients and other more promising parameters, from the perspective

From a clinical point of view, patients are typically monitored through the measurement of the symptoms present, questionnaires on the degree of control of the disease [Curtis et al., 1997] or the impact on quality of life related to health [Juniper et al., 2004], pathophysiological parameters such as the degree of bronchial obstruction [FEV1 or PEF] [Miller, 2005], and variability [Reddel, 2006] or the degree of bronchial hyper-reactivity, both specific and nonspecific [Anderson, 2008b, Cockcroft & Davis 2006; Crapo et al., 2000; Sont et al., 1999; Sterk et al., 1993]. Each of these are more or less direct methods of measuring the clinical impact of inflammatory diseases of the airways, providing complementary information to diagnostic and therapeutic management [Fuhlbrigge, 2004; Gibson & Powell, 2004; Taylor et al., 2008] and, to a lesser degree, clinical interpretation [Frey & Suki, 2008]. They have become absolutely necessary and indispensable for the classification of the patient's and establishment of control disease. In terms of study, testing of the whole response of the total airway implies knowing several pathogenic mechanisms of disease [Bousquet et al., 2000; Leuppi et al., 2001]. Understanding how control of the severity or clinical evolution of the disease is developed retains some specific limitations that result in advantages and disadvantages. For example, the main advantage of studying bronchial hyper-responsiveness to histamine or methacholine challenge is the high negative predictive value of the test [Luks et al., 2010]. On the contrary, its presence and the severity observed indicate functional impairment of the airway. This may be interpretable in either a physiological (dysfunction, with or without an associated inflammatory basal profile) or pathological context (chronic inflammation, injury, mucosal or submucosal remodelling), which may or may not be modified by treatment once established [Hargreave et al., 1981; Rosi et al., 1999; Sont et al., 1999; van Essen-Zandvliet et al., 1994]. Recent advances in the clinical use of substances, such as adenosine or mannitol, provide additional data on the association between bronchial hyper-responsiveness, as measured by these indirect stimuli,

derived by direct measurement and monitoring of inflammatory activity.

and markers of inflammation [Polosa et al., 2000; Rutgers et al., 2000].

**3.1 Global systems of measurement for airway disease** 

Haldar et al., 2008].

group.

daily medication regimen and response to treatment, as well as the different strategies and recommendations for managing patients with difficult to control asthma are essentially needed in clinical practice [Holgate & Polosa, 2006]; otherwise they are limited. New methods based on statistical physics and fluctuation analysis can be a new strategy for assessing and predicting the risk of progression of asthma [Frey & Suki, 2008], but monitoring of airway diseases also requires focus on foundations of the modern biology. Therefore, the application of new technological advancements and the disciplines applied to the study of inflammation, as well as the incorporation of new markers for diagnosis and the monitoring of patients affected by asthma, inspires optimism in the challenge to find a better conceptual understanding of asthma using a dynamic approach that continuously changes and at the same time is more significant than that achieved by mere verification.

In this chapter, specific aspects of the new contributions to the monitoring of asthma and the new research using defined groups of patients to study interrelated heterogeneous aspects of asthma will be described and discussed. These new contributions have modified the approach to grouping and reclassifying characteristics that are clinical, pathophysiological and biological as a whole, and allow for a new definition of asthma in terms of 'phenotypes' and 'endotypes' [Anderson, 2008a; Wenzel, 2006]. Although the addition of these new terms to the definition of asthma may be seen as a conceptual breakthrough, caution must be exercised. For example, new biological knowledge about the pathogenic understanding of inflammatory airway disease (IAD) requires further investigation in many aspects, including how it differs from phenotypes in chronic obstructive pulmonary disease (COPD) [Barnes, 2008], as well as in children [Spycher, 2010]. Hence, a major challenge in the field of respiratory disease today is how to adapt the definition of asthma to new scientific developments. The verification of objective 'biotypes', in terms of the development of asthma and the patients in whom they are observed, is an important conceptual advance, provided better clinical management for each patient is achieved so that the relative uniqueness of each patient with asthma can be better understood, both by the physician and the patients themselves. However, in order to define 'phenotypes' specific to patients or subgroups of patients in addition to the specific pathogenic 'endotypes' that identify them (in other words, how asthma patients become ill), it is necessary to thoroughly identify their defining characteristics. They must therefore be measured and grouped biologically and clinically in a differentiated way, even if certain aspects of the illness are the same [Lotvall et al., 2011; Moore et al., 2010].

Today's objective biological measures and markers used to better understand these biotypes are the central focus of this chapter.

#### **3. Monitoring the airway and monitoring asthma in particular**

There are a number of methods available for identifying different aspects related to the natural evolution of IAD. The contribution of each aspect is determined by providing appropriate measures and robust parameters that meet consistent methodological determinants, such as the standardization of the method used, the availability of reference values, the reproducibility of findings, and above all the application of research findings to clinical practice and the global management of patients whose situation is well-defined as well as homogeneous patients or groups of subjects. Not all biological measures studied in recent years have managed to become routinely added as a parameter with clinical value in

daily medication regimen and response to treatment, as well as the different strategies and recommendations for managing patients with difficult to control asthma are essentially needed in clinical practice [Holgate & Polosa, 2006]; otherwise they are limited. New methods based on statistical physics and fluctuation analysis can be a new strategy for assessing and predicting the risk of progression of asthma [Frey & Suki, 2008], but monitoring of airway diseases also requires focus on foundations of the modern biology. Therefore, the application of new technological advancements and the disciplines applied to the study of inflammation, as well as the incorporation of new markers for diagnosis and the monitoring of patients affected by asthma, inspires optimism in the challenge to find a better conceptual understanding of asthma using a dynamic approach that continuously changes and at the same time is more significant than that achieved by mere verification. In this chapter, specific aspects of the new contributions to the monitoring of asthma and the new research using defined groups of patients to study interrelated heterogeneous aspects of asthma will be described and discussed. These new contributions have modified the approach to grouping and reclassifying characteristics that are clinical, pathophysiological and biological as a whole, and allow for a new definition of asthma in terms of 'phenotypes' and 'endotypes' [Anderson, 2008a; Wenzel, 2006]. Although the addition of these new terms to the definition of asthma may be seen as a conceptual breakthrough, caution must be exercised. For example, new biological knowledge about the pathogenic understanding of inflammatory airway disease (IAD) requires further investigation in many aspects, including how it differs from phenotypes in chronic obstructive pulmonary disease (COPD) [Barnes, 2008], as well as in children [Spycher, 2010]. Hence, a major challenge in the field of respiratory disease today is how to adapt the definition of asthma to new scientific developments. The verification of objective 'biotypes', in terms of the development of asthma and the patients in whom they are observed, is an important conceptual advance, provided better clinical management for each patient is achieved so that the relative uniqueness of each patient with asthma can be better understood, both by the physician and the patients themselves. However, in order to define 'phenotypes' specific to patients or subgroups of patients in addition to the specific pathogenic 'endotypes' that identify them (in other words, how asthma patients become ill), it is necessary to thoroughly identify their defining characteristics. They must therefore be measured and grouped biologically and clinically in a differentiated way, even if certain aspects of the illness are the same [Lotvall et

Today's objective biological measures and markers used to better understand these biotypes

There are a number of methods available for identifying different aspects related to the natural evolution of IAD. The contribution of each aspect is determined by providing appropriate measures and robust parameters that meet consistent methodological determinants, such as the standardization of the method used, the availability of reference values, the reproducibility of findings, and above all the application of research findings to clinical practice and the global management of patients whose situation is well-defined as well as homogeneous patients or groups of subjects. Not all biological measures studied in recent years have managed to become routinely added as a parameter with clinical value in

**3. Monitoring the airway and monitoring asthma in particular** 

al., 2011; Moore et al., 2010].

are the central focus of this chapter.

the management of patients. Furthermore, those used routinely provide specific and limited information only. Therefore, current research being conducted focuses more on the inflammatory aspects of the lung, rather than the classical or clinical function, because it is the central pathogenic mechanism of the disease in the airway [Fabbri et al., 2005]. Severity and control disease assessment requires a multidimensional practical approach including an inflammatory view, as was previously confirmed by some authors [Fitzpatrick et al. 2011a; Haldar et al., 2008].

Technological advancements brought about by modern molecular biology and innovative micron analysis technology applied to the study of inflammation and the pathways of oxidative, lipid, or nitric stress, for example, can allow for a better identification and definition of new parameters, inflammatory profiles, and biomarkers that are more sensitive and specific for predicting the state of progression of IAD, differences in poor outcomes, and the type of anti-inflammatory treatment best suited to manage a particular patient or patient group.

This section briefly discusses the most relevant parameters used in clinical practice for the monitoring of asthma, especially the most recent parameters that contribute greatly to the overall management of patients and other more promising parameters, from the perspective derived by direct measurement and monitoring of inflammatory activity.

#### **3.1 Global systems of measurement for airway disease**

From a clinical point of view, patients are typically monitored through the measurement of the symptoms present, questionnaires on the degree of control of the disease [Curtis et al., 1997] or the impact on quality of life related to health [Juniper et al., 2004], pathophysiological parameters such as the degree of bronchial obstruction [FEV1 or PEF] [Miller, 2005], and variability [Reddel, 2006] or the degree of bronchial hyper-reactivity, both specific and nonspecific [Anderson, 2008b, Cockcroft & Davis 2006; Crapo et al., 2000; Sont et al., 1999; Sterk et al., 1993]. Each of these are more or less direct methods of measuring the clinical impact of inflammatory diseases of the airways, providing complementary information to diagnostic and therapeutic management [Fuhlbrigge, 2004; Gibson & Powell, 2004; Taylor et al., 2008] and, to a lesser degree, clinical interpretation [Frey & Suki, 2008]. They have become absolutely necessary and indispensable for the classification of the patient's and establishment of control disease. In terms of study, testing of the whole response of the total airway implies knowing several pathogenic mechanisms of disease [Bousquet et al., 2000; Leuppi et al., 2001]. Understanding how control of the severity or clinical evolution of the disease is developed retains some specific limitations that result in advantages and disadvantages. For example, the main advantage of studying bronchial hyper-responsiveness to histamine or methacholine challenge is the high negative predictive value of the test [Luks et al., 2010]. On the contrary, its presence and the severity observed indicate functional impairment of the airway. This may be interpretable in either a physiological (dysfunction, with or without an associated inflammatory basal profile) or pathological context (chronic inflammation, injury, mucosal or submucosal remodelling), which may or may not be modified by treatment once established [Hargreave et al., 1981; Rosi et al., 1999; Sont et al., 1999; van Essen-Zandvliet et al., 1994]. Recent advances in the clinical use of substances, such as adenosine or mannitol, provide additional data on the association between bronchial hyper-responsiveness, as measured by these indirect stimuli, and markers of inflammation [Polosa et al., 2000; Rutgers et al., 2000].

A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 185

et al., 2011]. The most important contributions of non-invasive methods currently used to measure inflammation of the airway and the most promising research being carried out with new applied technologies in molecular biology and immunology are discussed below.

This semi-invasive technique is used to obtain a representative sample of bronchial secretions in the airway. Some methodological variations in the induction procedure may produce samples from a more central or peripheral compartment of the airway, providing versatility in IAD screening in terms of both central and peripheral orientation. This is characterized as such because the differential inflammatory profile obtained is reproducible and can be correctly interpreted in a clinical context. However, this is not the case in the study of the various markers of inflammation and oxidative stress obtained from the supernatant, which have strict methodological considerations and limitations in the interpretation of results [Nicholas, 2009]. Bearing this in mind, induced sputum has become the gold standard non-invasive method for measuring bronchial cell inflammation and for certain soluble markers that are identifiable and of specific dilution [Bakakos, 2011; Djukanovic 2004]. The standardization of the method and procedure, adequate safeness of the technique, good toleration by the patient, ease of use, and the obtainment of reference values have made it an essential technique in the study of complex patients who require the characterization of the bronchial inflammatory pattern in order to be managed correctly [Djukanovic, 2002]. The current characterization of the endotypes and phenotypes of patients with inflammatory airway disease, based on inflammatory cell patterns [Balzar et al., 2011; Haldar et al., 2008], and the combination of different biomarkers, such as those derived from 'esputoma' [Gray et al., 2008; Nicholas, 2006] and oxidative stress [Louhelainen et al., 2008a; Louhelainen et al., 2008b], 15-lipoxygenase pathway [Chu, et al., 2002], gluthation oxidation [Fitzpatrick et al., 2011b] or genetics and protein identification [Baines et al., 2011; Hastie et al, 2010] make sputum an indisputable protagonist in the new definition of phenotypes, the classification of patients with asthma and COPD [Fabbri et al., 2003; Fatj et al., 2009; Louis et al., 2002; Wang et al., 2011], the therapeutic management of these patients, the prediction of therapeutic response [Green et al., 2002; Jayaram et al., 2006, Caramori et al., 2005], and the evaluation of efficacy in the most recent anti-inflammatory molecules [Haldar et al, 2009; Pavord et al. 2009, van Rensen et al. 2009]. Currently, new techniques in molecular biology may be applied to the study of sputum in order to study the expression shown in the cellular response of certain subtypes of cell lines, as found for example, by studying toll-like cell receptors or local innate immunity measured by flow cytometry [Lay et al, 2011], or those derived from cell cultures [Bettiol et al., 2002], the cellular response to markers of cell migration [Dent et al., 2004], or even those obtained from the analysis of proteomics traces or esputoma [Park & Rim, 2011; Nicholas & Djukanovic,

2009], as well as the genome [Baines et al., 2011; Bisgaard et al., 2011].

**3.2.2 The exhaled fraction determined singularly or in combination** 

The use of exhaled markers capable of reflecting a clinically useful measurement of the inflammation and oxidative stress present in the airway currently involves bronchial nitric oxide (NO) as the main marker [Barnes et al., 2010]. This measurement is reproducible and the method has been standardized [ATS/ERS, 2005]. Because its concentration is dependent on the flow and the source of production, a compartmental model of alveolar or bronchial

**3.2.1 Induced sputum** 

Other findings arising from the current radiological spectrum of diseases of the airway also provide useful data to study dynamic airway inflammation, expressed as the degree of trapped air and bronchial wall thickness [Gupta et al., 2010].

It is therefore possible to say that although the parameters and measurements discussed in this section can be applied to both asthma and COPD in order to achieve better clinical management, its meaning and clinical interpretation are often heterogeneous or variable depending on the patient, the therapy administered at the time of measurement, and the spectrum of IADs identified in the subject, both initially and over time [Avital et al., 1995; Dima et al., 2010]. Discrimination of all the spectrum of obstructive airway diseases is the goal to achieve at present time.

#### **3.2 Direct systems of injury and repair measurement in pulmonary biology**

The pathology of the airway has a clearly inflammatory pathophysiological basis, although the role this plays, both in the short term and in the long term, in the biological continuum of integrity, dysfunction, injury, and repair continues to undergo constant research. Traditionally, the gold standard in inflammatory activity has been to use various measurements and markers, both inflammatory and those of oxidative stress, obtained invasively by bronchial biopsy and bronchoalveolar lavage [Bergeron et al., 2007; Brasier et al., 2010; Hallstrand et al., 2011]. Although various parameters and histological patterns of inflammation have been identified with these techniques, many of them are shared by the anatomopathological spectrum of IAD. This causes ambiguity, both in the various pathogenic contexts in which pathophysiological interpretation is difficult and, consequently, non-pathognomonic situations in which discrimination regarding asthma or COPD occurs. Only some of these findings have demonstrated applicability to clinical care and a certain ability to differentiate or discriminate between the inflammatory pathogenic states of an underlying pathologic lesion, as in the study of bronchial remodelling [Sont et al., 1999; Sont et al., 2003], or the prevalence of the cellular profile of bronchial infiltration, as in the case of life-threatening asthma [Mauad et al., 2004; Mauad et al., 2008]. Furthermore, both techniques also have some significant limitations when carried out routinely. The first is that only one compartment of the airway is represented unless both procedures are done at the same time. The second limitation is the invasiveness of the procedure. This is an obstacle when routinely performed on the patient, despite the fact that these methods have been standardized and allow for a better visualization of the type of inflammation and anatomical injury caused by IAD in certain patients. They are also useful in studying the pathogenic mechanisms involved, as well as classifying and identifying the stage of disease [Fabbri et al., 2003; Fabbri et al., 2005; Moore et al., 2011], but the requirements of the procedure do not make them suitable for routine monitoring of the patient and are therefore generally reserved today for the systematic study of inflammation in pulmonary biology research.

The new semi-invasive methods, such as induced sputum, or non-invasive methods, such as the measurement of nitric oxide, condensation, or exhaled temperature, provide new and useful data directly from the airway that may be used in the classification of IADs and the management of patients [Popov, 2011]. Some markers derived from blood samples can also be used in assessing inflammatory disease and its systemic impact. These include eosinophil cationic proteins, as well as cationic peroxidase and leucotriens [Koh et al., 2007; Rabinovitch, 2007], but other novel systemic blood biomarkers are also promising [Verrills et al., 2011]. The most important contributions of non-invasive methods currently used to measure inflammation of the airway and the most promising research being carried out with new applied technologies in molecular biology and immunology are discussed below.

#### **3.2.1 Induced sputum**

184 Inflammatory Diseases – A Modern Perspective

Other findings arising from the current radiological spectrum of diseases of the airway also provide useful data to study dynamic airway inflammation, expressed as the degree of

It is therefore possible to say that although the parameters and measurements discussed in this section can be applied to both asthma and COPD in order to achieve better clinical management, its meaning and clinical interpretation are often heterogeneous or variable depending on the patient, the therapy administered at the time of measurement, and the spectrum of IADs identified in the subject, both initially and over time [Avital et al., 1995; Dima et al., 2010]. Discrimination of all the spectrum of obstructive airway diseases is the

The pathology of the airway has a clearly inflammatory pathophysiological basis, although the role this plays, both in the short term and in the long term, in the biological continuum of integrity, dysfunction, injury, and repair continues to undergo constant research. Traditionally, the gold standard in inflammatory activity has been to use various measurements and markers, both inflammatory and those of oxidative stress, obtained invasively by bronchial biopsy and bronchoalveolar lavage [Bergeron et al., 2007; Brasier et al., 2010; Hallstrand et al., 2011]. Although various parameters and histological patterns of inflammation have been identified with these techniques, many of them are shared by the anatomopathological spectrum of IAD. This causes ambiguity, both in the various pathogenic contexts in which pathophysiological interpretation is difficult and, consequently, non-pathognomonic situations in which discrimination regarding asthma or COPD occurs. Only some of these findings have demonstrated applicability to clinical care and a certain ability to differentiate or discriminate between the inflammatory pathogenic states of an underlying pathologic lesion, as in the study of bronchial remodelling [Sont et al., 1999; Sont et al., 2003], or the prevalence of the cellular profile of bronchial infiltration, as in the case of life-threatening asthma [Mauad et al., 2004; Mauad et al., 2008]. Furthermore, both techniques also have some significant limitations when carried out routinely. The first is that only one compartment of the airway is represented unless both procedures are done at the same time. The second limitation is the invasiveness of the procedure. This is an obstacle when routinely performed on the patient, despite the fact that these methods have been standardized and allow for a better visualization of the type of inflammation and anatomical injury caused by IAD in certain patients. They are also useful in studying the pathogenic mechanisms involved, as well as classifying and identifying the stage of disease [Fabbri et al., 2003; Fabbri et al., 2005; Moore et al., 2011], but the requirements of the procedure do not make them suitable for routine monitoring of the patient and are therefore generally reserved today for the systematic study of

The new semi-invasive methods, such as induced sputum, or non-invasive methods, such as the measurement of nitric oxide, condensation, or exhaled temperature, provide new and useful data directly from the airway that may be used in the classification of IADs and the management of patients [Popov, 2011]. Some markers derived from blood samples can also be used in assessing inflammatory disease and its systemic impact. These include eosinophil cationic proteins, as well as cationic peroxidase and leucotriens [Koh et al., 2007; Rabinovitch, 2007], but other novel systemic blood biomarkers are also promising [Verrills

**3.2 Direct systems of injury and repair measurement in pulmonary biology** 

trapped air and bronchial wall thickness [Gupta et al., 2010].

goal to achieve at present time.

inflammation in pulmonary biology research.

This semi-invasive technique is used to obtain a representative sample of bronchial secretions in the airway. Some methodological variations in the induction procedure may produce samples from a more central or peripheral compartment of the airway, providing versatility in IAD screening in terms of both central and peripheral orientation. This is characterized as such because the differential inflammatory profile obtained is reproducible and can be correctly interpreted in a clinical context. However, this is not the case in the study of the various markers of inflammation and oxidative stress obtained from the supernatant, which have strict methodological considerations and limitations in the interpretation of results [Nicholas, 2009]. Bearing this in mind, induced sputum has become the gold standard non-invasive method for measuring bronchial cell inflammation and for certain soluble markers that are identifiable and of specific dilution [Bakakos, 2011; Djukanovic 2004]. The standardization of the method and procedure, adequate safeness of the technique, good toleration by the patient, ease of use, and the obtainment of reference values have made it an essential technique in the study of complex patients who require the characterization of the bronchial inflammatory pattern in order to be managed correctly [Djukanovic, 2002]. The current characterization of the endotypes and phenotypes of patients with inflammatory airway disease, based on inflammatory cell patterns [Balzar et al., 2011; Haldar et al., 2008], and the combination of different biomarkers, such as those derived from 'esputoma' [Gray et al., 2008; Nicholas, 2006] and oxidative stress [Louhelainen et al., 2008a; Louhelainen et al., 2008b], 15-lipoxygenase pathway [Chu, et al., 2002], gluthation oxidation [Fitzpatrick et al., 2011b] or genetics and protein identification [Baines et al., 2011; Hastie et al, 2010] make sputum an indisputable protagonist in the new definition of phenotypes, the classification of patients with asthma and COPD [Fabbri et al., 2003; Fatj et al., 2009; Louis et al., 2002; Wang et al., 2011], the therapeutic management of these patients, the prediction of therapeutic response [Green et al., 2002; Jayaram et al., 2006, Caramori et al., 2005], and the evaluation of efficacy in the most recent anti-inflammatory molecules [Haldar et al, 2009; Pavord et al. 2009, van Rensen et al. 2009]. Currently, new techniques in molecular biology may be applied to the study of sputum in order to study the expression shown in the cellular response of certain subtypes of cell lines, as found for example, by studying toll-like cell receptors or local innate immunity measured by flow cytometry [Lay et al, 2011], or those derived from cell cultures [Bettiol et al., 2002], the cellular response to markers of cell migration [Dent et al., 2004], or even those obtained from the analysis of proteomics traces or esputoma [Park & Rim, 2011; Nicholas & Djukanovic, 2009], as well as the genome [Baines et al., 2011; Bisgaard et al., 2011].

#### **3.2.2 The exhaled fraction determined singularly or in combination**

The use of exhaled markers capable of reflecting a clinically useful measurement of the inflammation and oxidative stress present in the airway currently involves bronchial nitric oxide (NO) as the main marker [Barnes et al., 2010]. This measurement is reproducible and the method has been standardized [ATS/ERS, 2005]. Because its concentration is dependent on the flow and the source of production, a compartmental model of alveolar or bronchial

A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 187

performed one by one [Baraldi et al., 2009]. However, recent specific phenotypes of asthma

Calculating the nasal exhaled fraction may be useful for the study and diagnosis of primary ciliary dyskinesia and is virtually abolished in this disease [Horváth et al., 2003]. On the

**3.2.3 The multiple molecular studies of biological lung samples, or systemic samples,** 

Since the detection of a single marker of inflammation or oxidative stress does not identify a specific inflammatory disease of the airway as defined today, but rather proves the heterogeneity of inflammatory conditions and diseases of the airway, current research on biological markers focuses on a combination of identifying patterns of proteins and volatile compounds that can be identified by molecular marks or traces they contain. At the molecular level, many omic compounds (proteomic, metabolomic, genomic) may be identified. Within the field of respiratory medicine, these compounds will remain an enigma until the wealth of empirical molecular information is organized and interpreted, free of *a priori* hypotheses, and the subsequent translation to clinical practice can be carried out. Such information would have the great advantage of producing a custom 'fingerprint' of inflammation during a specific time point in the evolution of the disease process in a particular patient. The correct interpretation of molecular information will allow the clinician to identify and predict the patient and disease biotypes (phenotype and endotype), the severity of the inflammatory process in progress, the clinical evolution, the type of treatment to be applied, the response to the therapy administered, and the prognosis for each individual patient [Crameri, 2005; Vijverberg et al., 2011]. The new molecular application technology based on bioinformatics, cluster analysis, and artificial intelligence algorithms that are being developed at present provide all this information for the purpose of prediction and interpretation at a biological level. This is useful in understanding the new biology of systems integration [Perpiñá, 2010; Scott et al., 2007; Thaler & Hanson, 2005]. The

First, chromatography and mass spectrometry have been used to identify hundreds of volatile exhaled organic compounds originating from the metabolic pathways involved in pulmonary biology. However, their use as biomarkers with a clinical application is still under study [Freidrich, 2009]. Secondly, the addition of other specific technology for determining the spectrum of metabolites, proteins or organic compounds in different biological samples (cellular, fluid, or gas) in blood, sputum, or bronchoalveolar lavage, and developed on line, such as the application a multiple set of nanosensors (arrays) or the application of high-resolution nuclear magnetic resonance, has achieved rapid progress in identifying potential profiles and patterns of disease-specific biomarkers. For example, different patterns of compounds originating in breathing and exhaled breath evaluated by the so-called *electronic nose* identify molecules of different size, volume and dipole [Lewis, 2004]. These patterns have shown good sensitivity and discrimination capabilities for this combination and may be as helpful as those already described for the identification of certain odours, producing an odoriferous mark or *smellprint*. This would specifically identify the type of inflammation present and could be useful in the differential diagnosis of specific IADs [Dragonieri et al., 2007] in addition to the particular diagnostic and therapeutic

patients, like aspirin intolerant asthmatics can be identified [Sanak et al, 2011].

contrary nasal NO levels are higher in rhinitis [Struben et al., 2006]

following is a description of the research currently being conducted.

**significant to pulmonary disease** 

strategies for each patient.

origin of exhaled nitric oxide has been developed in order to study of the origin of the alteration and the lung injury, situating it at a more central or peripheral level in the airway [Puckett et al., 2010]. This model allows the production variability within the spectrum of IAD and the anti-inflammatory modulation produced by the therapy administered to be studied in depth. While this method is certainly advantageous when carrying out clinical monitoring within a timeframe because of its non-invasivity, it may only be used in certain patients, i.e. those in whom the main source of NO has been identified as a clearly modifiable and dependent element of the course of inflammation with therapeutic antiinflammatory management, or for the screening of type of activated inflammation [Anderson et al., 2011]. The use of NO in the research of no homogeneously selected patients leads to a clinically confusing interpretation in terms of its identification and therefore must be considered limited or biased under these circumstances [Dweik et al., 2010]. In addition, certain methodological considerations conditioned by the design of NO research studies regarding the cut-off used to make a management decision must be taken into account during diagnosis and treatment, i.e. conditioning the patient management strategy based on the levels of NO [Gibson, 2009; Quaedvlieg et al., 2009; Schleich et al., 2009; Schneider et al., 2009].

Other exhaled markers of the bronchial airway, such as the detection of carbon monoxide and other volatile hydrocarbon compounds, products of lipid peroxidation, have also been studied [Antczak et al., 2011]. However, a definitive standardization of methods for immediate application in a clinical context has not been achieved. Similarly, the measurement of exhaled temperature increase at the start of breathing with regard to the reference point marked during the entire period of measurement of the increase is associated with the presence of active inflammation and airway remodelling [Paredi, 2005]. This also occurs with the measurement of bronchial blood flow, estimated by mass spectrometry using the Fick principle, and the calculation of the dilution of exhaled acetylene [the initial concentration inhaled is known]. Both methods may have their place in the spectrum of non-invasive monitoring of bronchial inflammation if the optimal exhaled flow is standardized methodologically for the purpose of measurement [Paredi & Barnes, 2010].

The identification of different volatile compounds produced by oxidative stress, nitrosative stress, inflammation and metallic elements, and obtained from the exhaled condensate has also been possible through the use of chromatography and mass spectrometry [Corradi et al. 2007; Corradi et al, 2010]. Some of these compounds may be considered biomarkers in clinical practice [Baraldi et al., 2009 Kostikas et al., 2008; Loukides et al, 2011], such as pH determination [Kostikas et al., 2011; Antus et al, 2010]. However, it is necessary to simplify the instrumentation of the procedure for routine use in clinical practice. The standardization of methodology and applicability to clinical practice of other compounds under investigation as potential biomarkers has yet to be sufficiently achieved due, among other things, to certain limitations, such as contamination of condensate compounds of the mouth (especially if concomitant oral inflammation occurs), difficulty in calculating the optimal dilution of the selected parameter, or instability, volatility, and interaction of the mixture of soluble compounds that can take place during this process [Horvàth et al. 2005]. Comparisons with other biomarkers obtained and already standardized are needed to establish the utility of the different compounds of exhaled condensate, especially if

origin of exhaled nitric oxide has been developed in order to study of the origin of the alteration and the lung injury, situating it at a more central or peripheral level in the airway [Puckett et al., 2010]. This model allows the production variability within the spectrum of IAD and the anti-inflammatory modulation produced by the therapy administered to be studied in depth. While this method is certainly advantageous when carrying out clinical monitoring within a timeframe because of its non-invasivity, it may only be used in certain patients, i.e. those in whom the main source of NO has been identified as a clearly modifiable and dependent element of the course of inflammation with therapeutic antiinflammatory management, or for the screening of type of activated inflammation [Anderson et al., 2011]. The use of NO in the research of no homogeneously selected patients leads to a clinically confusing interpretation in terms of its identification and therefore must be considered limited or biased under these circumstances [Dweik et al., 2010]. In addition, certain methodological considerations conditioned by the design of NO research studies regarding the cut-off used to make a management decision must be taken into account during diagnosis and treatment, i.e. conditioning the patient management strategy based on the levels of NO [Gibson, 2009; Quaedvlieg et al., 2009; Schleich et al.,

Other exhaled markers of the bronchial airway, such as the detection of carbon monoxide and other volatile hydrocarbon compounds, products of lipid peroxidation, have also been studied [Antczak et al., 2011]. However, a definitive standardization of methods for immediate application in a clinical context has not been achieved. Similarly, the measurement of exhaled temperature increase at the start of breathing with regard to the reference point marked during the entire period of measurement of the increase is associated with the presence of active inflammation and airway remodelling [Paredi, 2005]. This also occurs with the measurement of bronchial blood flow, estimated by mass spectrometry using the Fick principle, and the calculation of the dilution of exhaled acetylene [the initial concentration inhaled is known]. Both methods may have their place in the spectrum of non-invasive monitoring of bronchial inflammation if the optimal exhaled flow is standardized methodologically for the purpose of measurement [Paredi &

The identification of different volatile compounds produced by oxidative stress, nitrosative stress, inflammation and metallic elements, and obtained from the exhaled condensate has also been possible through the use of chromatography and mass spectrometry [Corradi et al. 2007; Corradi et al, 2010]. Some of these compounds may be considered biomarkers in clinical practice [Baraldi et al., 2009 Kostikas et al., 2008; Loukides et al, 2011], such as pH determination [Kostikas et al., 2011; Antus et al, 2010]. However, it is necessary to simplify the instrumentation of the procedure for routine use in clinical practice. The standardization of methodology and applicability to clinical practice of other compounds under investigation as potential biomarkers has yet to be sufficiently achieved due, among other things, to certain limitations, such as contamination of condensate compounds of the mouth (especially if concomitant oral inflammation occurs), difficulty in calculating the optimal dilution of the selected parameter, or instability, volatility, and interaction of the mixture of soluble compounds that can take place during this process [Horvàth et al. 2005]. Comparisons with other biomarkers obtained and already standardized are needed to establish the utility of the different compounds of exhaled condensate, especially if

2009; Schneider et al., 2009].

Barnes, 2010].

performed one by one [Baraldi et al., 2009]. However, recent specific phenotypes of asthma patients, like aspirin intolerant asthmatics can be identified [Sanak et al, 2011].

Calculating the nasal exhaled fraction may be useful for the study and diagnosis of primary ciliary dyskinesia and is virtually abolished in this disease [Horváth et al., 2003]. On the contrary nasal NO levels are higher in rhinitis [Struben et al., 2006]

#### **3.2.3 The multiple molecular studies of biological lung samples, or systemic samples, significant to pulmonary disease**

Since the detection of a single marker of inflammation or oxidative stress does not identify a specific inflammatory disease of the airway as defined today, but rather proves the heterogeneity of inflammatory conditions and diseases of the airway, current research on biological markers focuses on a combination of identifying patterns of proteins and volatile compounds that can be identified by molecular marks or traces they contain. At the molecular level, many omic compounds (proteomic, metabolomic, genomic) may be identified. Within the field of respiratory medicine, these compounds will remain an enigma until the wealth of empirical molecular information is organized and interpreted, free of *a priori* hypotheses, and the subsequent translation to clinical practice can be carried out. Such information would have the great advantage of producing a custom 'fingerprint' of inflammation during a specific time point in the evolution of the disease process in a particular patient. The correct interpretation of molecular information will allow the clinician to identify and predict the patient and disease biotypes (phenotype and endotype), the severity of the inflammatory process in progress, the clinical evolution, the type of treatment to be applied, the response to the therapy administered, and the prognosis for each individual patient [Crameri, 2005; Vijverberg et al., 2011]. The new molecular application technology based on bioinformatics, cluster analysis, and artificial intelligence algorithms that are being developed at present provide all this information for the purpose of prediction and interpretation at a biological level. This is useful in understanding the new biology of systems integration [Perpiñá, 2010; Scott et al., 2007; Thaler & Hanson, 2005]. The following is a description of the research currently being conducted.

First, chromatography and mass spectrometry have been used to identify hundreds of volatile exhaled organic compounds originating from the metabolic pathways involved in pulmonary biology. However, their use as biomarkers with a clinical application is still under study [Freidrich, 2009]. Secondly, the addition of other specific technology for determining the spectrum of metabolites, proteins or organic compounds in different biological samples (cellular, fluid, or gas) in blood, sputum, or bronchoalveolar lavage, and developed on line, such as the application a multiple set of nanosensors (arrays) or the application of high-resolution nuclear magnetic resonance, has achieved rapid progress in identifying potential profiles and patterns of disease-specific biomarkers. For example, different patterns of compounds originating in breathing and exhaled breath evaluated by the so-called *electronic nose* identify molecules of different size, volume and dipole [Lewis, 2004]. These patterns have shown good sensitivity and discrimination capabilities for this combination and may be as helpful as those already described for the identification of certain odours, producing an odoriferous mark or *smellprint*. This would specifically identify the type of inflammation present and could be useful in the differential diagnosis of specific IADs [Dragonieri et al., 2007] in addition to the particular diagnostic and therapeutic strategies for each patient.

A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 189

Inflammatory diseases of the airway, and asthma in particular, are complex and heterogenous, both in terms of the biological expression of inflammation they produce and in terms of the 'biotypes' (endotypes and phenotypes) that can be objectively translated from this condition. New information provided by new technology applied to modern molecular biology and immunology requires the current concept and definition of asthma to be modified and adapted. The attainment of this important progressive scientific knowledge can help to address how and why this condition occurs and may contribute to a better understanding of the classification of each asthma patient, the proper diagnosis of the type of asthma presented, the monitoring approach, the personalized treatment required, and the method to determine prognosis. A wide spectrum of biomarkers is currently being incorporated as clinically useful parameters. What remains is to gradually adapt them to comprehensive of multidimensional approaches and medical procedures, and establish the

[1] Adcock, IM.; Caramori, G. & Chung, KF. (2008). New targets for drug development in

[2] American Thoracic Society; & European Respiratory Society. (2005). ATS/ERS

[3] Anderson, GP. (2008a). Endotyping asthma: new insights into key pathogenic

[4] Anderson, JT.; Zeng, M.; Li, Q.; Stapley, R.; Moore, DR 2nd.; Chenna, B.; Fineberg, N.;

[6] Antczak, A.; Ciebiada, M.; Kharitonov, SA.; Gorski, P. & Barnes, PJ. (2011).

[7] Antus, B.; Barta, I.; Kullmann, T.; Lazar, Z.; Valyon, M.; Horvath, I. & Csiszer, E. (2010).

[8] Avital, A.; Springer, C.; Bar-Yishay, E. & Godfrey, S. (1995). Adenosine, methacholine,

Ovarian Cycle. *Inflammation*. May 18. [Epub ahead of print].

*Med*. Vol.182, No.12, pp.1492-1497, ISSN 1073-449X.

asthma. *Lancet*. Vol. 372, No. 9643, (September 2008), pp.1073-1087, ISSN 0140-6736.

recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide, 2005. *Am J Respir Crit Care Med*. Vol.171, No 8, (April 2005), pp.912-930, ISSN 1073-449X.

mechanisms in a complex, heterogenous disease. *Lancet.* Vol. 372, No. 9643,

Zmijewski, J.; Eltoum, IE.; Siegal, GP.; Gaggar, A.; Barnes, S.; Velu, SE.; Thannickal, VJ.; Abraham, E.; Patel, RP.; Lancaster, JR Jr.; Chaplin, DD.; Dransfield, MT. & Deshane, JS. (2011). Elevated levels of NO are localized to distal airways in asthma. *Free Radic Biol Med*. Vol. 50, No.11, (Juny 2011), pp.1679-1688, ISSN 0891-5849. [5] Anderson, SD. (2008b). Provocative challenges to help diagnose and monitor asthma:

exercise, methacholine, adenosine, and mannitol. *Curr Opin Pulm Med*. Vol. 14, No.

Inflammatory Markers: Exhaled Nitric Oxide and Carbon Monoxide During the

Assessment of exhaled breath condensate pH in exacerbations of asthma and chronic obstructive pulmonary disease: A longitudinal study. *Am J Respir Crit Care* 

and exercise challenges in children with asthma or paediatric chronic obstructive pulmonary disease. *Thorax*. Vol. 50, No.5, (May 1995), pp.511-516, ISSN 0040-6376.

appropriate indications and clinical applications in respiratory disease.

(September 2008), pp. 1107-1119, ISSN 0140-6736.

1, (January 2008), pp. 39-45, ISSN 1070-5287.

**5. Conclusion** 

**6. References** 

#### **4. The future of targeted and individualized biological respiratory therapy**

The new concept of biotypes (endotypes and phenotypes) of IAD is transforming the definition of asthma to the point where it will soon be possible to obtain a more accurate simplification of what is understood today as asthma in order to better adapt to patient management and the clinical reality. However, the definition of asthma that currently exists is complex and will remain so, despite continuous modification and rehabilitation, given that although the heterogeneity of the IAD is better understood and the subtypes of the disease and patient subgroups are better classified, the multifactorial and dynamic aspects of the biological responses involved make applying reductionist criteria very difficult. It is therefore necessary to maintain a flexible and mentally versatile attitude that is as dynamic as biology itself. This attitude will facilitate the understanding of respiratory medicine of the future, which in turn will affect therapeutic management. This approach may be considered the cornerstone of individualized therapy for respiratory patients. It will aid in the progressive incorporation of biopharmaceuticals capable of regulating or altering inflammatory pathways, the remodelling process and the smooth muscle response. Both strategies are complementary and briefly commented.

First, the action and biological and immunological mechanisms will modulate the degree of response obtained at the molecular level and will result clinically in a very specific action. Being able to have these modulating biopharmaceuticals will be crucial and improve the quality of life for patients with IAD and asthma [Adock et al., 2008; Casale & Stokes, 2008]. However, the availability of these drugs will lead to a major challenge at the clinical level that they will be reflected in trying to establish well-defined therapeutic indications to ensure safe, efficient, and cost-effective use. Some examples of modulating biopharmaceuticals in the context of eosinophilic inflammation that have been used in patients with poor control and greater severity of symptoms are those that interfere with the biological action of IgE, IL-5 or TNF. The attainment of adequate control and improvement of outcome in these patients is a sign of success in the development of new molecules, such as the monoclonal antibodies, mepolizumab, etarnercept or omalizumab [Holgate et al, 2011; Pavord et al., 2010; Pelaia et al., 2011]. Other biological modulating drugs acting at the neutrophilic, mast o lymphocite cells or other relevant molecules in the pathways and the inflammatory response, are being tested for the purpose of incorporation into the therapeutic arsenal available for IAD [Chung & Marwick, 2010, Barnes, 2009]. Pharmacogenetics and understanding of innate immunty pathways are promising areas of research to discover determined mechanisms and specific molecules to reverse the altered inflammatory response [Caramori et al., 2004; Kanagaratham et al., 2011; Gupta & Agrawal, 2010; Slager, 2010].

Second, the important thing to considering the therapy of releaving symptoms in the asthma clinic course is the understanding of the mechanical obstruction in the airways and the air-trapping compensatory consequence [Sorkness et al., 2008]. New long-acting smooth muscle relaxant molecules are been incorporated alone or in combination to the inhaler therapy [Cazzola et al., 2011; Chung et al., 2009; Kiyokawa et al., 2011; Postma et al., 2011], but recent knowing of the genetics of airway smooth muscle points out a new strategies to develop asthma targeted molecules [Hai, 2008]. Another specific therapy focus on smooth muscle of the airways, like bronchial thermoplasty, is still under evaluation [Thompson et al., 2011].

#### **5. Conclusion**

188 Inflammatory Diseases – A Modern Perspective

The new concept of biotypes (endotypes and phenotypes) of IAD is transforming the definition of asthma to the point where it will soon be possible to obtain a more accurate simplification of what is understood today as asthma in order to better adapt to patient management and the clinical reality. However, the definition of asthma that currently exists is complex and will remain so, despite continuous modification and rehabilitation, given that although the heterogeneity of the IAD is better understood and the subtypes of the disease and patient subgroups are better classified, the multifactorial and dynamic aspects of the biological responses involved make applying reductionist criteria very difficult. It is therefore necessary to maintain a flexible and mentally versatile attitude that is as dynamic as biology itself. This attitude will facilitate the understanding of respiratory medicine of the future, which in turn will affect therapeutic management. This approach may be considered the cornerstone of individualized therapy for respiratory patients. It will aid in the progressive incorporation of biopharmaceuticals capable of regulating or altering inflammatory pathways, the remodelling process and the smooth muscle response. Both

First, the action and biological and immunological mechanisms will modulate the degree of response obtained at the molecular level and will result clinically in a very specific action. Being able to have these modulating biopharmaceuticals will be crucial and improve the quality of life for patients with IAD and asthma [Adock et al., 2008; Casale & Stokes, 2008]. However, the availability of these drugs will lead to a major challenge at the clinical level that they will be reflected in trying to establish well-defined therapeutic indications to ensure safe, efficient, and cost-effective use. Some examples of modulating biopharmaceuticals in the context of eosinophilic inflammation that have been used in patients with poor control and greater severity of symptoms are those that interfere with the biological action of IgE, IL-5 or TNF. The attainment of adequate control and improvement of outcome in these patients is a sign of success in the development of new molecules, such as the monoclonal antibodies, mepolizumab, etarnercept or omalizumab [Holgate et al, 2011; Pavord et al., 2010; Pelaia et al., 2011]. Other biological modulating drugs acting at the neutrophilic, mast o lymphocite cells or other relevant molecules in the pathways and the inflammatory response, are being tested for the purpose of incorporation into the therapeutic arsenal available for IAD [Chung & Marwick, 2010, Barnes, 2009]. Pharmacogenetics and understanding of innate immunty pathways are promising areas of research to discover determined mechanisms and specific molecules to reverse the altered inflammatory response [Caramori et al., 2004; Kanagaratham et al., 2011; Gupta & Agrawal,

Second, the important thing to considering the therapy of releaving symptoms in the asthma clinic course is the understanding of the mechanical obstruction in the airways and the air-trapping compensatory consequence [Sorkness et al., 2008]. New long-acting smooth muscle relaxant molecules are been incorporated alone or in combination to the inhaler therapy [Cazzola et al., 2011; Chung et al., 2009; Kiyokawa et al., 2011; Postma et al., 2011], but recent knowing of the genetics of airway smooth muscle points out a new strategies to develop asthma targeted molecules [Hai, 2008]. Another specific therapy focus on smooth muscle of the airways, like bronchial thermoplasty, is still under

**4. The future of targeted and individualized biological respiratory therapy** 

strategies are complementary and briefly commented.

2010; Slager, 2010].

evaluation [Thompson et al., 2011].

Inflammatory diseases of the airway, and asthma in particular, are complex and heterogenous, both in terms of the biological expression of inflammation they produce and in terms of the 'biotypes' (endotypes and phenotypes) that can be objectively translated from this condition. New information provided by new technology applied to modern molecular biology and immunology requires the current concept and definition of asthma to be modified and adapted. The attainment of this important progressive scientific knowledge can help to address how and why this condition occurs and may contribute to a better understanding of the classification of each asthma patient, the proper diagnosis of the type of asthma presented, the monitoring approach, the personalized treatment required, and the method to determine prognosis. A wide spectrum of biomarkers is currently being incorporated as clinically useful parameters. What remains is to gradually adapt them to comprehensive of multidimensional approaches and medical procedures, and establish the appropriate indications and clinical applications in respiratory disease.

#### **6. References**


A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 191

[22] Caramori, G.; Adcock, IM. & Ito, K. (2004). Anti-inflammatory inhibitors of IkappaB

[23] Caramori, G.; Pandit, A. & Papi, A. (2005). Is there a difference between chronic airway

[24] Casale, TB, & Stokes, JR. (2008). Immunomodulators for allergic respiratory disorders. *J Allergy Clin Immunol*. Vo.121. No. 2, (February 2008), pp. 288-296, ISSN 0091-6749. [25] Cazzola, M.; Calzetta, L. & Matera, MG. (2011). β(2) -adrenoceptor agonists: current

[26] Chu, HW.; Balzar, S.; Westcott, JY.; Trudeau, JB.; Sun, Y.; Conrad, DJ. & Wenzel, SE.

[27] Chung, KF, & Marwick, JA. (2010). Molecular mechanisms of oxidative stress in

2004), pp.1141-1147, ISSN 1472-4472.

Vol. 32, No 11, pp.1558-1565, ISSN 1365-2222.

2, (April 2010), pp.93-98, ISSN 1528-4050.

2000), pp. 309-329, ISSN 1073-449X.

1997), pp.1032-1039, ISSN 1073-449X.

ISSN 1528-4050.

ISSN 1871-5281.

5381.

kinase in asthma and COPD. *Curr Opin Investig Drugs*. Vol.5, No.11, (November

inflammation in chronic severe asthma and chronic obstructive pulmonary disease?. *Curr Opin Allergy Clin Immunol*. Vol. 5, No.1, (February 2005), pp.77-83.

and future direction. *Br J Pharmacol*. Vol.163, No.1, (May 2011) pp.4-17, ISSN 1476-

(2002). Expression and activation of 15-lipoxygenase pathway in severe asthma: relationship to eosinophilic phenotype and collagen deposition. *Clin Exp Allergy*.

airways and lungs with reference to asthma and chronic obstructive pulmonary disease. *Ann N Y Acad Sci*. Vol. 1203, (August 2010), pp. 85-91, ISSN 0077-8923. [28] Chung, KF.; Caramori, G. & Adcock, IM. (2009). Inhaled corticosteroids as combination

therapy with beta-adrenergic agonists in airways disease: present and future. *Eur J Clin Pharmacol*. Vol. 65, No.9, (Sepetember 2009), pp.853-871, ISSN 0031-6970. [29] Cockcroft, DW. & Davis, BE. (2006). Mechanisns of airway hyperresponsiveness. *J* 

*Allergy Clin immunol*. Vol. 118, No. 3 (Setember 2006), pp. 551-559, ISSN 0091-6749.

investigate occupational lung diseases. *Curr Opin Allergy Clin Immunol*. Vol. 10, No.

children. *Inflamm Allergy Drug Targets*. Vol. 6, No. 3, (Sepetember 2007), pp.150-159,

MacIntyre, NR.; McKay, RT.; Wanger, JS.; Anderson, SD.; Cockcroft, DW.; Fish, JE. & Sterk, PJ. (2000). Guidelines for methacoline and exercise challenge testing-1999. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July 1999. *Am J Respir Cri Care Med*. Vol.161, No.1, (January

Chronic Lung Disease. *Am J Respir Crit Care Med*. Vol. 156, No. 4 pt1, (October

Louis, R.; Davies, DE. & Djukanovic, R. (2004). Contribution of eotaxin-1 to

[30] Corradi, M.; Gergelova, P. & Mutti, A. (2010). Use of exhaled breath condensate to

[31] Corradi, M.; Zinelli, C. & Caffarelli, C. (2007). Exhaled breath biomarkers in asthmatic

[32] Crameri, R. (2005). The potential of proteomics and peptidomics fro allergy asnd asthma. *Allergy*. Vol. 60, No.10, (October 2005), pp. 1227-1237, ISSN 0105-4538. [33] Crapo, RO.; Casaburi, R.; Coates, AL.; Enright, PL.; Hankinson, JL.; Irvin, CG.;

[34] Curtis, JR.; Martin, DP. & Martin, TR. (1997). Patient-assessed Health Outcomes in

[35] Dent, G.; Hadjicharalambous, C.; Yoshikawa, T.; Handy, RL.; Powell, J.; Anderson, IK.;


[9] Baines, KJ.; Simpson, JL.; Wood, LG.; Scott, RJ. & Gibson, PG. (2011). Transcriptional

[10] Bakakos, P.; Schleich, F.; Alchanatis, M. & Louis, R. (2011). Induced sputum in asthma:

[11] Balzar, S.; Fajt, ML.; Comhair, SA.; Erzurum, SC.; Bleecker, E.; Busse, WW.; Castro, M.;

[12] Baraldi, E.; Carraro, S.; Giordano, G.; Reniero, F.; Perilongo, G. & Zacchello, F. (2009).

[13] Barnes, PJ. (2008). Immunology of asthma and chronic obstructive pulmonary disease. *Nat Rev Immunol*. Vol. 8, No. 3, (March 2008), pp. 183–192, ISSN 1474-1733. [14] Barnes, PJ. (2009). Histone deacetylase-2 and airway disease. *Ther Adv Respir Dis*. Vol.3,

[15] Barnes, PJ.; Dweik, RA.; Gelb, AF.; Gibson, PG.; George, SC.; Grasemann, H.; Pavord,

[16] Bergeron, C.; Tulic, MK. & Hamid, Q. (2007). Tools used to measure airway

[17] Bettiol, J.; Sele, J.; Henket, M.; Louis, E.; Malaise, M.; Bartsch, P. & Louis, R. (2002).

[19] Bousquet, J.; Jeffery, PK.; Busse, WW.; Johnson, M. & Vignola, AM. (2000). Asthma.

[21] British Thoracic Society Scottish Intercollegiate Guidelines Network. (2008) British

*Crit Care Med.* Vol.161, No.5, (May 2000), pp.1720-1745, ISSN 1073-449X. [20] Brasier, AR.; Victor, S.; Ju, H.; Busse, WW.; Curran-Everett, D.; Bleecker, E.; Castro, M.;

0091-6749.

0903-1936.

pp.299-309, ISSN 1073-449X.

(October 2009), p.30, ISSN 1720-8424.

2010), pp.682-692, ISSN 0012-3692.

2011), pp.1155-1164, ISSN 0091-6749.

2010), pp.147-157, ISSN 1752-8054.

ISSN 0040-6376.

No.5, (October 2009), pp.235-243, ISSN 1753-4658.

8673.

phenotypes of asthma defined by gene expression profiling of induced sputum samples. *J Allergy Clin Immunol*. Vol. 127. No.1, (January 2011), pp.153-160, ISSN

from bench to bedside. *Curr Med Chem*. Vol. 18, No. 10, pp.1415-1422. ISSN 0929-

Gaston, B.; Israel, E.; Schwartz, LB.; Curran-Everett, D.; Moore, CG. & Wenzel, SE. (2011). Mast cell phenotype, location, and activation in severe asthma: data from the severe asthma research program. *Am J Respir Crit Care Med*. Vol.183, Vol.3,

Metabolomics: moving towards personalized medicine*. Ital J Pediatr*. Vol. 35, No.1,

ID.; Ratjen, F.; Silkoff, PE.; Taylor, DR. & Zamel, N. (2010). Exhaled nitric oxide in pulmonary diseases: a comprehensive review. *Chest*. Vol. 138, No. 3, (September

remodelling in research. *Eur Respir J*. Vol. 29, No.3, (March 2007), pp.596-604, ISSN

Cytokine production from sputum cells after allergenic challenge in IgE-mediated asthma. *Allergy*. Vol.57, No.12, (December 2002), pp.1145-1150, ISSN 0105-4538. [18] Bisgaard, H.; Pipper, CB. & Bønnelykke, K. (2011). Endotyping early childhood asthma

by quantitative symptom assessment. *J Allergy Clin Immunol*. Vol. 127, No. 5, (May

From bronchoconstriction to airways inflammation and remodeling. *Am J Respir* 

Chung, KF.; Gaston, B.; Israel, E.; Wenzel, SE.; Erzurum, SC.; Jarjour, NN. & Calhoun, WJ. (2010). Predicting intermediate phenotypes in asthma using bronchoalveolar lavage-derived cytokines. *Clin Transl Sci*. Vol. 3, No.4, (August

guideline on the management of Asthma. *Thorax*. Vol.63, No. Suppl. 4, pp. iv1–121,


A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 193

[47] Friedrich, MJ. (2009). Scientists seek to sniff out diseases: electronic "noses" may

[48] Fuhlbrigge, AL. (2004). Asthma severity and asthma control: symptoms, pulmonary

[49] Gibson, PG. & Powell, H. (2004) Written actions plans for asthma: an evidence-based

[50] Gibson, PG. (2009). Using fractional exhaled nitric oxide to guide asthma therapy:

[51] Global Initiative for Asthma. Global strategy for asthma management and prevention.

[52] Gray, RD.; MacGregor, G.; Noble, D.; Imrie, M.; Dewar, M.; Boyd, AC.; Innes, JA.;

[53] Green, RH.; Brightling, CE.; McKenna, S.; Hargadon, B.; Parker, D.; Bradding, P.;

[54] Gupta, GK. & Agrawal, DK. (2010). CpG oligodeoxynucleotides as TLR9 agonists:

[55] Gupta, S.; Siddiqui, S.; Haldar, P.; Entwisle, JJ.; Mawby, D.; Wardlaw, AJ.; Bradding, P.;

[56] Hai, CM. Mechanistic systems biology of inflammatory gene expression in airway

[57] Haldar, P.; Brightling, CE.; Hargadon, B.; Gupta, S.; Monteiro, W.; Sousa, A.; Marshall,

Vol. 65, No.9, (September 2010), pp.775-781, ISSN 0040-6376.

*Technol*. Vol.5, No.4, (December 2008), pp.279-288, ISSN 1570-1638.

*Exp Allergy*. Vol.39, No.4, (April 2009), pp.478-490, ISSN 1365-2222.

Available at http//www.ginasthma.org [accessed 8 March 2009].

(September 2008), pp.444-452, ISSN 1073-449X.

ISSN 0098-7484.

ISSN 0040-6376.

2004), pp. 1-6, ISSN 1070-5287.

pp.1715-1721, ISSN 0140-6736.

2010), pp.225-35, ISSN 1173-8804.

p.588, ISSN 1533-4406.

*Pediatr Res*. Vol. 69, No.2, (February 2011), pp.154-159. ISSN 0031-3998. [46] Frey, U. & Suky, B. (2008). Complexity of chronic asthma and chronic obstructive

with airway macrophage functional impairment in children with severe asthma.

pulmonary disease: implications for risk assessment, and disease progression and control. *Lancet.* Vol. 372, No. 9643, (September 2008), pp.1088-1099, ISSN 0140-6736.

someday be diagnostic tools. *JAMA*. Vol. 301, No.6, (February 2009), pp.585-586,

function, and inflammatory markers. *Curr Opin Pulm Med*. Vol. 10, No. 1, (January

review of the key components. *Thorax*. Vol. 59, No. 2, (February 2004), pp. 94-99,

design and methodological issues for ASthma TReatment ALgorithm studies. *Clin* 

Porteous, DJ. & Greening, AP. (2008). Sputum proteomics in inflammatory and suppurative respiratory diseases. *Am J Respir Crit Care Med*. Vol. 178, No. 5,

Wardlaw, AJ. & Pavord, ID. (2002). Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. *Lancet*. Vol. 360, No. 9347, (November 2002),

therapeutic application in allergy and asthma. *BioDrugs*. Vol. 24, No. 4, (August

Pavord, ID.; Green, RH. & Brightling, CE. (2010). Quantitative analysis of highresolution computed tomography scans in severe asthma subphenotypes. *Thorax*.

smooth muscle as tool for asthma drug development. (2008). *Curr Drug Discov* 

RP.; Bradding, P.; Green, RH.; Wardlaw, AJ. & Pavord, ID. (2009). Mepolizumab and exacerbations of refractory eosinophilic asthma. *N Engl J Med*. Vol. 360, No. 10, (March 2009), pp. 973-984. Erratum in: *N Engl J Med* Vol. 364, No.6, (February 2011),

eosinophil chemotactic activity of moderate and severe asthmatic sputum. *Am J Respir Crit Care Med*. Vol. 169, No.10, (May 2004), pp.1110-1117, ISSN 1073-449X.


[37] Djukanovi,c R.; Sterk, PJ.; Fahy, JC. & Hargreave, FE. (2002). Standardised

[38] Djukanovic, R. & Sterk, PJ. (2004). *An atlas of induced sputum: an aid for research and diagnosis.* The Partenon Publishing Group, ed. ISBN 1842140051. London. [39] Dragonieri, S.; Schot, R.; Mertens, BJ.; Le Cessie, S.; Gauw, SA.; Spanevello, A.; Resta,

[41] Fabbri, L.; Peters, SP.; Pavord, I.; Wenzel, SE.; Lazarus, SC.; Macnee, W.; Lemaire, F. &

[42] Fabbri, LM.; Romagnoli, M.; Corbetta, L.; Casoni, G.; Busljetic, K.; Turato, G.; Ligabue,

[43] Fajt, ML. & Wenzel, SE. (2009). Asthma phenotypes in adults and clinical implications. *Expert Rev Respir Med*. Vol.3, No. 6, (December 2009), pp.607-625, ISSN 1747-6348. [44] Fitzpatrick, AM, Teague, WG, Meyers, DA, Peters, SP, Li, X, Li, H, Wenzel, SE, Aujla,

*Immunol*. Vol.127, No.2, (Febreuary 2011), pp.382-389. ISSN 0091-6749. [45] Fitzpatrick, AM.; Teague, WG.; Burwell, L.; Brown, MS.; Brown, LA; & NIH/NHLBI

Vol.171, No.7, (April 2005), pp.686-698, ISSN 1073-449X.

*Immunol*. Vol. 120, No.4, (October 2007), pp.856-862, ISSN 0091-6749. [40] Dweik, RA.; Sorkness, RL.; Wenzel, S.; Hammel, J.; Curran-Everett, D.; Comhair, SA.;

37, (September 2002), pp. 1s-55s. ISSN 0903-1936.

1176-9106.

1073-449X.

pp.418-424, ISSN 1073-449X.

eosinophil chemotactic activity of moderate and severe asthmatic sputum. *Am J Respir Crit Care Med*. Vol. 169, No.10, (May 2004), pp.1110-1117, ISSN 1073-449X. [36] Dima, E.; Rovina, N.; Gerassimou, C.; Roussos, C. & Gratziou, C. (2010). Pulmonary

function tests, sputum induction, and bronchial provocation tests: diagnostic tools in the challenge of distinguishing asthma and COPD phenotypes in clinical practice. *Int J Chron Obstruct Pulmon Dis*. Vol. 5, (September 2010), pp.287-296, ISSN

methodology of sputum induction and processing. *Eur Respir J*. Vol. 20, No. suppl

O.; Willard, NP.; Vink, TJ.; Rabe, KF.; Bel, EH. & Sterk, PJ. (2007). An electronic nose in the discrimination of patients with asthma and controls. *J Allergy Clin* 

Bleecker, E.; Busse, W.; Calhoun, WJ.; Castro, M.; Chung, KF.; Israel, E.; Jarjour, N.; Moore, W.; Peters, S.; Teague, G.; Gaston, B.; Erzurum, SC. & National Heart, Lung, and Blood Institute Severe Asthma Research Program. (2010). Use of exhaled nitric oxide measurement to identify a reactive, at-risk phenotype among patients with asthma. *Am J Respir Crit Care Med.* Vol.181, No.10, (May 2010), pp.1033-1341. ISSN

Abraham, E. (2005). Allergic rhinitis, asthma, airway biology, and chronic obstructive pulmonary disease in AJRCCM in 2004. *Am J Respir Crit Care Med*.

G.; Ciaccia, A.; Saetta, M. & Papi, A. (2003). Differences in airway inflammation in patients with fixed airflow obstruction due to asthma or chronic obstructive pulmonary disease. *Am J Respir Crit Care Med*. Vol.167, No. 3, (Frebruary 2003),

S, Castro, M, Bacharier, LB, Gaston, BM, Bleecker, ER, Moore, WC; National Institutes of Health/National Heart, Lung, and Blood Institute Severe Asthma Research Program. Heterogeneity of severe asthma in childhood: confirmation by cluster analysis of children in the National Institutes of Health/National Heart, Lung, and Blood Institute Severe Asthma Research Program. (2011a). *J Allergy Clin* 

Severe Asthma Research Program. (2011b). Glutathione oxidation is associated

with airway macrophage functional impairment in children with severe asthma. *Pediatr Res*. Vol. 69, No.2, (February 2011), pp.154-159. ISSN 0031-3998.


A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 195

[68] Juniper, EF.; Wisniewski, ME.; Cox, FM.; Emmett, AH.; Nielsen, KE. & O'Byrne, PM.

[70] Kiyokawa, H.; Matsumoto, H.; Nakaji, H.; Niimi, A, Ito, I.; Ono, K.; Takeda, T.; Oguma,

[71] Koh, G C-H.; Shek, L P-C.; Goh, D Y-T.; Van Bever, H. & Koh, D S-Q. (2007). Eosinophil

[72] Kostikas, K.; Koutsokera, A.; Papiris, S.; Gourgoulianis, KI. & Loukides, S. (2008).

[73] Kostikas, K.; Papaioannou, AI.; Tanou, K.; Giouleka, P.; Koutsokera, A.; Minas, M.;

[74] Lay, JC.; Peden, DB. & Alexis, NE. (2011). Flow cytometry of sputum: assessing

[75] Leuppi, JD.; Salome, CM.; Jenkins, CR.; Koskela, H.; Brannan, JD.; Anderson, SD.;

[77] Lötvall, J.; Akdis, CA.; Bacharier, LB.; Bjermer, L.; Casale, TB.; Custovic, A.; Lemanske,

[79] Louhelainen, N.; Rytilä, P.; Obase, Y.; Mäkelä, M.; Haahtela, T.; Kinnula, VL. &

*Inflamm Allergy Drug Discov*. Vol.5, No.1, pp.45-56, ISSN 1872-213X.

No. 4, (April 2007), pp.696-705, ISSN 0954-6111.

Vol.105, No.4, (April 2011), pp.526-532, ISSN 0954-6111.

No.9, (September 2004), pp.663-672, ISSN 1520-4898.

585-603, ISSN 1176-9106.

4303.

*Toxicol*. Vol. 23, No. 7, (Juny 2011), pp.392-406, ISSN 0895-8378.

ISSN 1323-8930.

2222.

(2004). Relationship between quality of life and clinical status in asthma: a factor analysis. *Eur Respir J*. Vol. 23, No. 2, (February 2004), pp.287-291, ISSN 0903-1936. [69] Kanagaratham, C.; Camateros, P.; Flaczyk, A. & Radzioch, D. (2011). Polymorphisms in

TOLL-like receptor genes and their roles in allergic asthma and atopy. *Recent Pat* 

T.; Otsuka, K. & Mishima, M. (2011). Centrilobular Opacities in the Asthmatic Lung Successfully Treated with Inhaled Ciclesonide and Tiotropium: With Assessment of Alveolar Nitric Oxide Levels. *Allergol Int.* February 25*.* [Epub ahead of print],

cationic protein: is it useful in asthma?. A systematic review. *Respir Med*. Vol. 101,

Exhaled breath condensate in patients with asthma: implications for application in clinical practice. *Clin Exp Allergy*. Vol. 38, No.4, (April 2008), pp.557-565, ISSN 1365-

Papiris, S.; Gourgoulianis, KI.; Taylor, DR.& Loukides, S. (2011). Exhaled NO and exhaled breath condensate pH in the evaluation of asthma control. *Respir Med*.

inflammation and immune response elements in the bronchial airways. *Inhal* 

Andersson, M.; Chan, HK. & Woolcock, AJ. (2001). Markers of airway inflammation and airway hyperresponsiveness in patients with well-controlled asthma. *Eur Respir J*. Vol.18, (No.3, Sepetember 2001), pp.444-450, ISSN 0903-1936. [76] Lewis, NS. (2004). Comparisons between mammalian and artificial olfaction based on

arrays of carbon black-polymer composite vapor detectors. *Acc Chem Res*. Vol. 37,

RF Jr.; Wardlaw, AJ.; Wenzel, SE. & Greenberger, PA. Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome. (2011). *J Allergy Clin Immunol*. Vol.127, No.2, (February 2011), pp.355-360, ISSN 0091-6749. [78] Louhelainen, N.; Myllärniemi, M.; Rahman, I. & Kinnula, VL. Airway biomarkers of

the oxidant burden in asthma and chronic obstructive pulmonary disease: current and future perspectives. (2008a). *Int J Chron Obstruct Pulmon Dis*. Vol. 3, No. 4, Pp.

Pelkonen, A. (2008b). The value of sputum 8-isoprostane in detecting oxidative stress in mild asthma. *J Asthma*. Vol. 45, No.2, (March 2008), pp.149-154, ISSN 1532-


[58] Haldar, P.; Pavord, ID.; Shaw, DE.; Berry, MA.; Thomas, M.; Brightling, CE.; Wardlaw,

[60] Hargreave, FE. & Nair, P. (2009). The definition and diagnosis of asthma. *Clin Exp Allergy*, Vol. 39, No.11, (November 2009), pp.1652-1658, ISSN 1365-2222. [61] Hargreave, FE.; Ryan, G.; Thomson, NC.; O'Byrne, PM.; Latimer, K.; Juniper, EF &

[62] Hastie, AT.; Moore, WC.; Meyers, DA.; Vestal, PL.; Li, H.; Peters, SP.; Bleecker, ER. &

[63] Holgate, ST. & Polosa, R. (2006). The mechanisms, diagnosis, and management of

[64] Holgate, ST.; Noonan, M.; Chanez, P.; Busse, W.; Dupont, L.; Pavord, I.; Hakulinen, A.;

[66] Horváth, I.; Loukides, S.; Wodehouse, T.; Csiszér, E.; Cole, PJ.; Kharitonov, SA. &

[67] Jayaram, L.; Pizzichini, MM.; Cook, RJ.; Boulet, LP.; Lemière, C.; Pizzichini, E.; Cartier,

Barnes, PJ. (2003). Comparison of exhaled and nasal nitric oxide and exhaled carbon monoxide levels in bronchiectatic patients with and without primary ciliary dyskinesia. *Thorax*. Vol. 58, No.1, (January 2003), pp.68-72. Erratum in: (2004)

A.; Hussack, P.; Goldsmith, CH.; Laviolette, M.; Parameswaran, K. & Hargreave, FE. (2006). Determining asthma treatment by monitoring sputum cell counts: effect on exacerbations. *Eur Respir J*. Vol. 27, No.3, (March 2006), pp.483-494, ISSN 0903-

*Eur Respir J*. Vol. 37, No. 6, (Juny 2011), pp.1352-1359, ISSN 0903-1936. [65] Horváth, I.; Hunt, J.; Barnes, PJ.; Alving, K.; Antczak, A.; Baraldi, E.; Becher, G.; van

2011), pp.801-810, ISSN 1365-2222.

5, (November 1981), pp.347-355, ISSN 0091-6749.

(May 2010), pp:1028-1036, ISSN 0091-6749.

*Thorax*. Vol. 59, No.6, p.543, ISSN 0040-6376.

0140-6736.

ISSN 0903-1936.

1936.

AJ. & Green, RH. (2008). Cluster analysis and clinical asthma phenotypes. *Am J Respir Crit Care Med*. Vol. 178, No.3, (August 2008), pp.218-224, ISSN 1073-449X. [59] Hallstrand, TS.; Lai, Y.; Ni, Z.; Oslund, RC.; Henderson, WR Jr.; Gelb, MH. & Wenzel,

SE. (2011). Relationship between levels of secreted phospholipase A[2] groups IIA and X in the airways and asthma severity. *Clin Exp Allergy*. Vol. 41, No. 6, (June

Dolovich, J. (1981). Bronchial responsiveness to histamine or methacholine in asthma: measurement and clinical significance. *J Allergy Clin Immunol*. Vol. 68, No.

National Heart, Lung, and Blood Institute Severe Asthma Research Program. Analyses of asthma severity phenotypes and inflammatory proteins in subjects stratified by sputum granulocytes. (2010). *J Allergy Clin Immunol*. Vol. 125, No. 5,

severe asthma in adults. *Lancet*. Vol. 368, No. 9537, (August 2006), pp.780-93, ISSN

Paolozzi, L.; Wajdula, J.; Zang, C.; Nelson, H. & Raible, D. (2011). Efficacy and safety of etanercept in moderate-to-severe asthma: a randomised, controlled trial.

Beurden, WJ.; Corradi, M.; Dekhuijzen, R.; Dweik, RA.; Dwyer, T.; Effros, R.; Erzurum, S.; Gaston, B.; Gessner, C.; Greening, A.; Ho, LP.; Hohlfeld, J.; Jöbsis, Q.; Laskowski, D.; Loukides, S.; Marlin, D.; Montuschi, P.; Olin, AC.; Redington, AE.; Reinhold, P.; van Rensen, EL.; Rubinstein, I.; Silkoff, P.; Toren, K.; Vass, G.; Vogelberg, C.; Wirtz, H. & ATS/ERS Task Force on Exhaled Breath Condensate. (2005). Exhaled breath condensate: methodological recommendations and unresolved questions. *Eur Respir J*. Vol.26, No.3, (September 2005), pp. 523-548,


A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 197

[89] Nicholas, B.; Skipp, P.; Mould, R.; Rennard, S.; Davies, DE.; O'Connor, CD. &

[92] Park, CS. & Rhim, T. (2011). Application of proteomics in asthma research. *Expert Rev* 

[93] Pavord, ID.; Haldar, P.; Bradding, P. & Wardlaw, AJ. (2010). Mepolizumab in

[94] Pavord, ID.; Jeffery, PK.; Qiu, Y.; Zhu, J.; Parker, D.; Carlsheimer, A.; Naya, I. & Barnes,

[95] Pelaia, G.; Gallelli, L.; Renda, T.; Romeo, P.; Busceti, MT.; Grembiale, LD.; Maselli, R.;

[98] Popov, TA. Human exhaled breath analysis. (2011). *Ann Allergy Asthma Immunol*.

[99] Postma, DS.; O'Byrne, PM. & Pedersen, S. (2011). Comparison of the effect of low-dose

[100] Puckett, JL.; Taylor, RW.; Leu, SY.; Guijon, OL.; Aledia, AS.; Galant, SP. & George, SC.

[101] Quaedvlieg, V.; Sele, J.; Henket, M. & Louis, R. (2009). Association between asthma

[102] Rabinovitch, N. (2007). Urinary leukotriene E4. *Immunol Allergy Clin N Am*. Vol. 27. No.

[103] Reddel, HK. Peak flow monitoring in clinical practice and clinical asthma trials. (2006). *Curr Opin Pulm Med*. Vol. 12, No. 1, (January 2006), pp. 75–81, ISSN 1070-5287.

categories. *Respir Res*. Vol. 11, (April 2010), p.47, ISSN 1465-993X.

Vol.106, No.6, (June 2011), pp.451-456, ISSN 1081-1206.

*Proteomics*. Vol. 8, No. 2, (April 2011), pp.221-230, ISSN 1478-9450.

*Proteomics*. Vol. 6, No.15, (August 2006), pp.4390-4401, ISSN 1615-9853. [90] Paredi, P. & Barnes, PJ. (2009). The airway vasculature: recent advances and clinical implications. *Thorax*. Vol. 64, No. 5, (May 2009), pp.444-450, ISSN 0040-6376. [91] Paredi, P.; Kharitonov, SA. & Barnes, PJ. (2005). Correlation of exhaled breath

2005), p.15, ISSN 1465-993X.

5, pp.1083-1089, ISSN 0091-6749.

pp.30-35, ISSN 0903-1936.

pp.1822-1829, ISSN 1365-2222.

6, (June 2007), pp. 451-464, ISSN 0889-8561.

0012-3692.

0040-6376.

Djukanović, R. (2006). Shotgun proteomic analysis of human-induced sputum.

temperature with bronchial blood flow in asthma. *Respir Res*. Vol.6, (February

refractory eosinophilic asthma. *Thorax.* Vol. 65, No.4, (April 2010), p. 370, ISSN

NC. (2009). Airway inflammation in patients with asthma with high-fixed or lowfixed plus as-needed budesonide/formoterol. *J Allergy Clin Immunol*. Vol.123, No.

Marisco, SA. & Vatrella, A. (2011). Update on optimal use of omalizumab in management of asthma. *J Asthma and Allergy.* Vol.4, pp.49-59 ISSN 1178-6965. [96] Perpiñá Tordera, M. (2010). Why do we look at asthma through the keyhole?. *Arch Bronconeumol*. Vol. 46, No. 8, (Augus 2010), pp.433-438, ISSN 0300-2896. [97] Polosa, R.; Ciamarra, I.; Mangano, G.; Prosperini, G.; Pistorio, MP.; Vancheri, C. &

Crimi, N. (2000). Bronchial hyperresponsiveness and airway inflammation markers in nonasthmatics with allergic rhinitis. *Eur Respir J*. Vol.15, No.1, (January 2000),

ciclesonide and fixed-dose fluticasone propionate and salmeterol combination on long-term asthma control. *Chest*. Vol.139, No.2, (February 2011), pp.311-318, ISSN

(2010). Clinical patterns in asthma based on proximal and distal airway nitric oxide

control and bronchial hyperresponsiveness and airways inflammation: a crosssectional study in daily practice. *Clin Exp Allergy*. Vol. 39, No. 12, (December 2009),


[80] Louis, RE.; Cataldo, D.; Buckley, MG.; Sele, J.; Henket, M.; Lau, LC.; Bartsch, P.; Walls,

[81] Loukides S.; Kontogianni K.; Hillas G.& Horvath I. (2011). Exhaled breath condensate

[82] Luks, VP.; Vandemheen, KL. & Aaron, SD. (2010). Confirmation of asthma in an era of

[83] Mauad, T.; Ferreira, DS.; Costa, MB.; Araujo, BB.; Silva, LF.; Martins, MA.; Wenzel, SE.

[84] Mauad, T.; Silva, LF.; Santos, MA.; Grinberg, L.; Bernardi, FD.; Martins, MA.; Saldiva,

[85] Miller, MR.; Hankinson, J.; Brusasco, V.; Burgos, F.; Casaburi, R.; Coates, A.; Crapo, R.;

[86] Moore, WC.; Evans, MD.; Bleecker, ER.; Busse, WW.; Calhoun, WJ.; Castro, M.; Fan

[87] Moore, WC.; Meyers, DA.; Wenzel, SE.; Teague, WG.; Li, H.; Li, X.; D'Agostino, R

[88] Nicholas, B. & Djukanovic, R. (2009). Induced sputum: a window to lung

No.2, (August 2002), pp.325-331, ISSN 0903-1936.

(October 2004), pp.857-862, ISSN 1073-449X.

Vol. 26, No. 2, (August 2005), pp. 319-338, ISSN 0903-1936.

ISSN 1568-0118.

1936.

1020-4989.

print].

0300-5127.

pp.315-323, ISSN 1073-449X.

AF. & Djukanovic, R. (2002). Evidence of mast-cell activation in a subset of patients with eosinophilic chronic obstructive pulmonary disease*. Eur Respir J*. Vol. 20,

in asthma: from bench to bedside. *Curr Med Chem*. Vol.18, No.10, pp.1432-1443,

overdiagnosis. *Eur Respir J*. Vol.36, No. 2, (August 2010), pp.255-260, ISSN 0903-

& Dolhnikoff, M. (2008). Characterization of autopsy-proven fatal asthma patients in São Paulo, Brazil. *Rev Panam Salud Publica*. Vol. 23, No.6, (June 2008), pp.418-423,

PH. & Dolhnikoff, M. (2004). Abnormal alveolar attachments with decreased elastic fiber content in distal lung in fatal asthma. *Am J Respir Crit Care Med*. Vol.170, No.8,

Enright, P.; van der Grinten, CP.; Gustafsson, P.; Jensen, R.; Johnson, DC.; MacIntyre, N.; McKay, R.; Navajas, D.; Pedersen, OF.; Pellegrino, R.; Viegi, G.; Wanger, J. & ATS/ERS Task Force. Series "Standardisation of spirometry. ATS/ERS task force, standardization of lung function testing". (2005). *Eur Respir J*.

Chung, K.; Erzurum, SC.; Curran-Everett, D.; Dweik, RA.; Gaston, B.; Hew, M.; Israel, E.; Mayse, ML.; Pascual, RM.; Peters, SP.; Silveira, L.; Wenzel, SE. & Jarjour, NN; for the National Heart, Lung, and Blood Institute's Severe Asthma Research Program. Safety of investigative bronchoscopy in the Severe Asthma Research Program. (2011). *J Allergy Clin Immunol*. Apr 13. [Epub ahead of

Jr.; Castro, M.; Curran-Everett, D.; Fitzpatrick, AM.; Gaston, B.; Jarjour, NN.; Sorkness, R.; Calhoun, WJ.; Chung, KF.; Comhair, SA.; Dweik, RA.; Israel, E.; Peters, SP.; Busse, WW.; Erzurum, SC.; Bleecker, ER. & The National Heart, Lung, and Blood Institute's Severe Asthma Research Program. Identification of asthma phenotypes using cluster analysis in the Severe Asthma Research Program. (2010). *Am J Respir Crit Care Med*. Vol.181, No.4, (February 2010),

pathology. *Biochem Soc. Trans*. Vol. 37, No Pt4, (August 2009), pp.868-872, ISSN


A New Era for Assessing Airway Diseases: New Insights in the Asthma Paradigm 199

[114] Spycher, BD.; Silverman, M. & Kuehni, CE. (2010). Phenotypes of childhood asthma:

[115] Sterk, PJ.; Fabbri, LM.; Quanjer, PH.; Cockcroft, DW.; O'Byrne, PM.; Anderson, SD.;

[116] Struben, VM.; Wieringa, MH.; Feenstra, L. & de Jongste, JC. (2006). Nasal nitric oxide and nasal allergy. *Allergy*. Vol. 61, No. 6, pp.665-670, ISSN 0105-4538. [117] Taylor, DR.; Bateman, ED.; Boulet, LP.; Boushey, HA.; Busse, WW.; Casale, TB.;

[118]Thaler, ER. & Hanson, CW. (2005). Medical applications of electronic nose

[119] Thomson, NC.; Rubin, AS.; Niven, RM.; Corris, PA.; Siersted, HC.; Olivenstein, R.;

[120]van Essen-Zandvliet, EE.; Hughes, MD.; Waalkens, HJ.; Duiverman, EJ. &

[121] van Rensen, EL.; Evertse, CE.; van Schadewijk, WA.; van Wijngaarden, S.; Ayre, G.;

[122] Verrills, NM.; Irwin, JA.; He, XY.; Wood, LG.; Powell, H.; Simpson, JL.; McDonald,

[123] Vijverberg, SJ.; Koenderman, L.; Koster, ES.; van der Ent, CK.; Raaijmakers, JA. &

*Med*. Vol.183, No. 12, (June 2011), pp.1633-1643, ISSN 1073-449X.

*Suppl*. Vol. 16, (March 1993), pp.53-83, ISSN 0106-4347.

No. 3, (September 2008), pp.545-554, ISSN 0903-1936.

Vol. 64, No.1, (January 2009), pp.72-80, ISSN 0105-4538.

8750-7587.

1365-2222.

ISSN 1743-4440.

ISSN 1471-2466.

629, ISSN 1365-2222.

1936.

bronchodilation. *J Appl Physiol*. Vol.104, No.2, (February 2008), pp.394-403, ISSN

are they real?. *Clin Exp Allergy*. Vol. 40, No.8, (August 2010), pp.1130-1141, ISSN

Juniper, EF. & Malo, JL. (1993). Airway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. *Eur Respir J*

Chanez, P.; Enright, PL.; Gibson, PG.; de Jongste, JC.; Kerstjens, HA.; Lazarus, SC.; Levy, ML.; O'Byrne, PM.; Partridge, MR.; Pavord, ID.; Sears, MR.; Sterk, PJ.; Stoloff, SW.; Szefler, SJ.; Sullivan, SD.; Thomas, MD.; Wenzel, SE. & Reddel, HK. (2008). A new perspective on concepts of asthma severity and control. *Eur Respir J*. Vol. 32,

technology. *Exert Rev Med Devices*. Vol. 2, No. 5, (September 2005), pp. 559-566,

Pavord, ID.; McCormack, D.; Laviolette, M.; Shargil,l NS.; Cox, G. & AIR Trial Study Group. (2011). Long-term (5 year) safety of bronchial thermoplasty: Asthma Intervention Research (AIR) trial. *BMC Pulm Med*. Vol.11, (February 2011), p.8,

Kerrebijn, KF. (1994). Remission of childhood asthma after long-term treatment with an inhaled corticosteroid [budesonide]: can it be achieved? Dutch CNSLD Study Group. *Eur Respir J*. Vol.7, No.1, (January 1994), pp.63-68. ISSN 0903-

Mauad, T.; Hiemstra, PS.; Sterk, PJ. & Rabe, KF. (2009). Eosinophils in bronchial mucosa of asthmatics after allergen challenge: effect of anti-IgE treatment. *Allergy*.

VM.; Sim, A. & Gibson, PG. Identification of Novel Diagnostic Biomarkers for Asthma and Chronic Obstructive Pulmonary Disease. (2011). *Am J Respir Crit Care* 

Maitland-van der Zee, AH. (2011). Biomarkers of therapy responsiveness in asthma: pitfalls and promises. *Clin Exp Allergy*. Vol. 41, No.5, (May 2011), pp.615-


[104] Rosi, E.; Ronchi, MC.; Grazzini, M.; Duranti, R. & Scano, G. (1999). Sputum analysis,

[105] Rutgers, SR.; Timens, W.; Tzanakis, N.; Kauffman, HF.; van der Mark, TW.; Koëter,

[106] Sanak, M.; Gielicz, A.; Bochenek, G.; Kaszuba, M.; Niżankowska-Mogilnicka, E. &

[107] Schleich, FN.; Seidel, L.; Sele, J.; Manise, M.; Quaedvlieg, V.; Michils, A. & Louis, R.

[108] Schneider, A.; Tilemann, L.; Schermer, T.; Gindner, L.; Laux, G.; Szecsenyi, J. & Meyer,

[109] Scott, SM.; James, D. & Ali, Z. (2007). Data analysis for electronic nose systems.

[110] Slager, RE.; Hawkins, GA.; Ampleford, EJ.; Bowden, A.; Stevens, LE.; Morton, MT.;

[111] Sont, JK.; De Boer, WI.; van Schadewijk, WA.; Grünberg, K.; van Krieken, JH.;

[112] Sont, JK.; Willems, LN.; Bel, EH.; van Krieken, JH.; Vandenbroucke, JP. & Sterk PJ.

[113] Sorkness, RL.; Bleecker, ER.; Busse, WW.; Calhoun, WJ.; Castro, M.; Chung, KF.;

Tomkinson, A.; Wenzel, SE.; Longphre, M.; Bleecker, ER. & Meyers, DA. (2010). IL-4 receptor α polymorphisms are predictors of a pharmacogenetic response to a novel IL-4/IL-13 antagonist. *J Allergy Clin Immunol*. Vol.126, No.4, (October 2010),

Hiemstra, PS.; Sterk, PJ. & Asthma Management Project University of Leiden Study Group. (2003). Fully automated assessment of inflammatory cell counts and cytokine expression in bronchial tissue. *Am J Respir Crit Care Med*. Vol.167, No.11,

(1999). Clinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment. The AMPUL Study Group. *Am J Respir Crit Care Med*. Vol. 159, No. 4 pt 1, (April 1999), pp. 1043–

Curran-Everett, D.; Erzurum, SC.; Gaston, BM.; Israel, E.; Jarjour, NN.; Moore, WC.; Peters, SP.; Teague, WG.; Wenzel, SE. & National Heart, Lung, and Blood Institute Severe Asthma Research Program. (2008). Lung function in adults with stable but severe asthma: air trapping and incomplete reversal of obstruction with

*Allergy*. Vol.30, No.5, (May 2000), pp.657-662, ISSN 1365-2222.

(December 2010), pp.1039-1044, ISSN 0040-6376.

*Microchim Acta*. Vol. 156, pp. 183-207, ISSN 0026-3672.

*Immunol*. Vol. 127, No.5, (May 2011), pp.1141-1147, ISSN 0091-6749.

0091-6749.

1465-993X.

pp.875-878, ISSN 0091-6749.

1051, ISSN 1073-449X.

(June 2003), pp.1496-1503, ISSN 1073-449X.

bronchial hyperresponsiveness, and airway function in asthma: results of a factor analtysis. *J Allergy Clin Immunol*. Vol. 103, No. 2 pt1, (February), pp.232-237, ISSN

GH. & Postma, DS. (2000). Airway inflammation and hyperresponsiveness to adenosine 5'-monophosphate in chronic obstructive pulmonary disease. *Clin Exp* 

Szczeklik, A. (2011). Targeted eicosanoid lipidomics of exhaled breath condensate provide a distinct pattern in the aspirin-intolerant asthma phenotype. *J Allergy Clin* 

(2010). Exhaled nitric oxide thresholds associated with a sputum eosinophil count ≥3% in a cohort of unselected patients with asthma. *Thorax*. Vol. 65, No.12,

FJ. (2009). Diagnosing asthma in general practice with portable exhaled nitric oxide measurement--results of a prospective diagnostic study: FENO < or = 16 ppb better than FENO < or =12 ppb to rule out mild and moderate to severe asthma. *Respir Res*. Vol. 10, p.15. Erratum in (2009). *Respir Res*. Vol.10, (March 2009), p. 64, ISSN bronchodilation. *J Appl Physiol*. Vol.104, No.2, (February 2008), pp.394-403, ISSN 8750-7587.


**Part 10** 

**Role of AC0T7** 


**Part 10** 

**Role of AC0T7** 

200 Inflammatory Diseases – A Modern Perspective

[124] Wang, F.; He, XY, Baines, KJ.; Gunawardhana, LP.; Simpson, JL.; Li, F. & Gibson, PG.

[125] Ward, C.; Reid, DW.; Orsida, BE.; Feltis, B.; Feltis, B.; Ryan, VA.; Johns, DP. & Walters,

[126] Wenzel, SE. (2006). Asthma: defining of the persistent adult phenotypes. *Lancet.* Vol.

Vol. 35, No.12, (December 2005), pp. 1565-1571, ISSN 1365-2222.

368, No. 9537, (August 2006), pp. 804-813, ISSN 0140-6736.

asthma. *Eur Respir J*. Jan 13. [Epub ahead of print].

(2011). Different inflammatory phenotypes in adults and children with acute

EH. (2005). Inter-relationships between airway inflammation, reticular basement memebrane thikening and bronchial hyper-reactivity to methacholine in asthma: a systematic bronchoalveolar lavage and airway biopsy analysis. *Clin Exp Allergy*.

**10** 

*Australia* 

*Charles Sturt University* 

**Role of ACOT7 in Arachidonic Acid** 

Crystall Swarbrick, Noelia Roman and Jade K. Forwood

Acyl-CoA Thioesterases (ACOTs) perform a wide range of cellular functions by catalysing the thiolytic cleavage of activated fatty acyl-CoAs. Substrates of ACOTs include short to long-chain acyl-CoAs as well as a range of methyl-branched, and dicarboxylic bile acid-CoAs (M. C. Hunt & Alexson, 2008). Expression of ACOTs have been detected in both prokaryotes and eukaryotes with expression in higher organisms being detected in cytosol,

Within the ACOT enzyme family, one member in particular, ACOT7 has recently been identified as playing a role inflammation through the production of arachidonic acid (AA). It was recently proposed that ACOT7-mediated AA production may provide a complementary source of AA to the well characterised phospholipase A2 (PLA2) pathway (Satoru Sakuma, Usa, & Fujimoto, 2006); see Table 1 for review of Acot substrate specificity. Evidence for these observations was through experimental data showing that ACOT7 possessed high substrate specificity for AA-CoA; the gene encoding ACOT7 was highly expressed in macrophages and up-regulated when stimulated by lipopolysaccharide (LPS); and that over-expression of the enzyme lead to an increase in prostaglandin production. Together, these observations highlight a novel role of ACOT7 in inflammation through the production of arachidonic acid from the thiolytic cleavage of activated polyunsaturated

Arachidonic acid has a number of important cellular roles, namely in cell signalling, regulation of metabolic and signalling enzymes, and inflammation. Despite its importance, its methods of cellular production are not fully understood. The most well characterised cellular pathway for arachidonic acid involves its release from membrane phospholipids via the action of PLA2, an enzyme responsible for the catalysis and hydrolysis of phospholipids

Expression of PLA2 is regulated according to the requirement for arachidonic acid and well understood. Rapid activation of PLA2 is achieved via posttranslational modification, and enzyme activity is activated by phosphorylation (controlled by mitogen-activated protein), while prolonged expression is regulated at a transcriptional level by cytokines and growth factors such as macrophage colony stimulating factor, tumor necrosis stimulating factor-

mitochondria, peroxisomes and endoplasmic reticulum (J. Yamada, 2005).

omega-6 fatty acid C20:4-CoA (Forwood et al., 2007).

at the *sn*-2 position (Sakuma et al, 2006) (see Figure 1).

**2. Cellular production of arachidonic acid (C20:4)** 

**1. Introduction** 

**Production and Inflammation** 

## **Role of ACOT7 in Arachidonic Acid Production and Inflammation**

Crystall Swarbrick, Noelia Roman and Jade K. Forwood *Charles Sturt University Australia* 

#### **1. Introduction**

Acyl-CoA Thioesterases (ACOTs) perform a wide range of cellular functions by catalysing the thiolytic cleavage of activated fatty acyl-CoAs. Substrates of ACOTs include short to long-chain acyl-CoAs as well as a range of methyl-branched, and dicarboxylic bile acid-CoAs (M. C. Hunt & Alexson, 2008). Expression of ACOTs have been detected in both prokaryotes and eukaryotes with expression in higher organisms being detected in cytosol, mitochondria, peroxisomes and endoplasmic reticulum (J. Yamada, 2005).

Within the ACOT enzyme family, one member in particular, ACOT7 has recently been identified as playing a role inflammation through the production of arachidonic acid (AA). It was recently proposed that ACOT7-mediated AA production may provide a complementary source of AA to the well characterised phospholipase A2 (PLA2) pathway (Satoru Sakuma, Usa, & Fujimoto, 2006); see Table 1 for review of Acot substrate specificity. Evidence for these observations was through experimental data showing that ACOT7 possessed high substrate specificity for AA-CoA; the gene encoding ACOT7 was highly expressed in macrophages and up-regulated when stimulated by lipopolysaccharide (LPS); and that over-expression of the enzyme lead to an increase in prostaglandin production. Together, these observations highlight a novel role of ACOT7 in inflammation through the production of arachidonic acid from the thiolytic cleavage of activated polyunsaturated omega-6 fatty acid C20:4-CoA (Forwood et al., 2007).

#### **2. Cellular production of arachidonic acid (C20:4)**

Arachidonic acid has a number of important cellular roles, namely in cell signalling, regulation of metabolic and signalling enzymes, and inflammation. Despite its importance, its methods of cellular production are not fully understood. The most well characterised cellular pathway for arachidonic acid involves its release from membrane phospholipids via the action of PLA2, an enzyme responsible for the catalysis and hydrolysis of phospholipids at the *sn*-2 position (Sakuma et al, 2006) (see Figure 1).

Expression of PLA2 is regulated according to the requirement for arachidonic acid and well understood. Rapid activation of PLA2 is achieved via posttranslational modification, and enzyme activity is activated by phosphorylation (controlled by mitogen-activated protein), while prolonged expression is regulated at a transcriptional level by cytokines and growth factors such as macrophage colony stimulating factor, tumor necrosis stimulating factor-

Role of ACOT7 in Arachidonic Acid Production and Inflammation 205

an increase in prostaglandin production, is consistent with the role of ACOT7 in

Fig. 1. The cellular pathway of arachidonic acid release involves the phospholipaseA2, and

Cellular production of arachidonic acid is utilized in a range of pathways, including the generation of potent mediators to initiate an inflammatory response. Two well characterised pathways important in inflammation include the cyclooxygenase (COX) and 5- Lipoxygnease pathways, and involve the conversion of arachidonic acid into prostanoids (prostaglandins, thromboxans), and leukotrienes respectively. The prostanoids include a range of arachidonic acid-derived metabolites that function to maintain body homeostasis by acting in a paracrine and autocrine fashion on cells within the vicinity of their release and are targets for the anti-inflammatory drugs including aspirin and derivatives. They typically exert their effect through activation of cell surface specific G-protein coupled receptors (GPCR), of which there are several subtypes for each prostanoid: PGD receptor (DP); PGE receptors EP1, EP2, EP3 and EP4 subtypes; PGF receptor (FP); PGI receptor (IP); and TX receptor (TP). There is also a receptor found on Th2 cells (CRTH2) that reacts to PGD2 but belongs to the chemokine receptor family (Narumiya, 2009; Wang, Honn, & Nie, 2007). Prostanoid production is increased during the inflammation response, particularly during acute inflammation, prior to the recruitment of leukocytes. There are a number of different

other AA-producing pathways.

**2.1 Role of arachidonic acid in inflammation** 

inflammation through the production of AA-derived inflammatory mediators.


Table 1. Substrate specificities for acyl-coA thioesterases.

alpha and glucocorticoid (Jiang et al., 2001; Satoru Sakuma, et al., 2006). While PLA2 is well known for its role in generating AA from the cleavage of the acyl bond of membrane phospholipids in a range of cell types, several lines of evidence have recently developed to suggest that PLA2 may not be solely responsible for controlling AA levels. For example, it was shown that AA-CoA can supply AA for prostaglandin (PG) synthesis (S. Sakuma et al., 1994), and that a novel enzymatic pathway exists whereby thioesterase cleavage of AA-CoA is responsible for supplying free AA to be utilised in the synthesis of prostaglandins (S. Sakuma, et al., 1994). A similar mechanism has also been described for controlling cellular levels of AA where it was demonstrated that AA levels were under the control of competing actions of an acyl-CoA thioesterase and synthetase, independent of the classical PLA2 cascade (Maloberti et al., 2005). In further support of these observations, when competition on AA levels were reduced through inhibition of an AA acyl-CoA synthetase (ACS; the opposite reaction that is catalysed by Acot7), substantial increases in PG levels were observed (Castilla et al., 2004); and moreover, in an independent study, overexpression of an acyl-CoA synthetase was been shown to cause a marked increase in synthesis of AA-CoA, increased 20:4 incorporation into membrane phospholipids, reduced cellular levels of unesterified 20:4, and reduced secretion of prostaglandin E2 (PGE2) while inhibition of the ACS resulted in increased release of PGE2 (Golej et al., 2011). Thus, it is emerging that inflammation is a complex cellular process, and PLA2 is unlikely to be the sole enzymatic pathway responsible for regulating AA levels, typically kept low due to the potent biological actions of the eicosanoids (Flesch, Schonhardt, & Ferber, 1989; Irvine, 1982), and that complementary pathways exist to contribute to AA generation and synthesis of eicosanoid inflammatory mediators during an immune response. That ACOT7 is abundantly expressed in macrophages and upregulated during an immune response; has high specificity for AA-CoA; and its over-expression in LPS-simulated macrophages cause

**Acot Homologue Preferred acyl-CoA substrate**  Acot1 Lauroyl and palmitoyl-CoA Acot2 Lauroyl and palmitoyl-CoA

Acot3 Palmitoyl-CoA Acot4 Succinyl-CoA Acot5 Decanoyl-CoA Acot6 Not determined Acot7 Arachidonoyl-CoA

Acot8 Bile acids

Table 1. Substrate specificities for acyl-coA thioesterases.

Acot9 Myristoyl-CoA Acot10 Myristoyl-CoA Acot11 Not determined Acot12 Acetyl-CoA

Acot13 Aromatic acyl-CoAs

alpha and glucocorticoid (Jiang et al., 2001; Satoru Sakuma, et al., 2006). While PLA2 is well known for its role in generating AA from the cleavage of the acyl bond of membrane phospholipids in a range of cell types, several lines of evidence have recently developed to suggest that PLA2 may not be solely responsible for controlling AA levels. For example, it was shown that AA-CoA can supply AA for prostaglandin (PG) synthesis (S. Sakuma et al., 1994), and that a novel enzymatic pathway exists whereby thioesterase cleavage of AA-CoA is responsible for supplying free AA to be utilised in the synthesis of prostaglandins (S. Sakuma, et al., 1994). A similar mechanism has also been described for controlling cellular levels of AA where it was demonstrated that AA levels were under the control of competing actions of an acyl-CoA thioesterase and synthetase, independent of the classical PLA2 cascade (Maloberti et al., 2005). In further support of these observations, when competition on AA levels were reduced through inhibition of an AA acyl-CoA synthetase (ACS; the opposite reaction that is catalysed by Acot7), substantial increases in PG levels were observed (Castilla et al., 2004); and moreover, in an independent study, overexpression of an acyl-CoA synthetase was been shown to cause a marked increase in synthesis of AA-CoA, increased 20:4 incorporation into membrane phospholipids, reduced cellular levels of unesterified 20:4, and reduced secretion of prostaglandin E2 (PGE2) while inhibition of the ACS resulted in increased release of PGE2 (Golej et al., 2011). Thus, it is emerging that inflammation is a complex cellular process, and PLA2 is unlikely to be the sole enzymatic pathway responsible for regulating AA levels, typically kept low due to the potent biological actions of the eicosanoids (Flesch, Schonhardt, & Ferber, 1989; Irvine, 1982), and that complementary pathways exist to contribute to AA generation and synthesis of eicosanoid inflammatory mediators during an immune response. That ACOT7 is abundantly expressed in macrophages and upregulated during an immune response; has high specificity for AA-CoA; and its over-expression in LPS-simulated macrophages cause an increase in prostaglandin production, is consistent with the role of ACOT7 in inflammation through the production of AA-derived inflammatory mediators.

Fig. 1. The cellular pathway of arachidonic acid release involves the phospholipaseA2, and other AA-producing pathways.

#### **2.1 Role of arachidonic acid in inflammation**

Cellular production of arachidonic acid is utilized in a range of pathways, including the generation of potent mediators to initiate an inflammatory response. Two well characterised pathways important in inflammation include the cyclooxygenase (COX) and 5- Lipoxygnease pathways, and involve the conversion of arachidonic acid into prostanoids (prostaglandins, thromboxans), and leukotrienes respectively. The prostanoids include a range of arachidonic acid-derived metabolites that function to maintain body homeostasis by acting in a paracrine and autocrine fashion on cells within the vicinity of their release and are targets for the anti-inflammatory drugs including aspirin and derivatives. They typically exert their effect through activation of cell surface specific G-protein coupled receptors (GPCR), of which there are several subtypes for each prostanoid: PGD receptor (DP); PGE receptors EP1, EP2, EP3 and EP4 subtypes; PGF receptor (FP); PGI receptor (IP); and TX receptor (TP). There is also a receptor found on Th2 cells (CRTH2) that reacts to PGD2 but belongs to the chemokine receptor family (Narumiya, 2009; Wang, Honn, & Nie, 2007). Prostanoid production is increased during the inflammation response, particularly during acute inflammation, prior to the recruitment of leukocytes. There are a number of different

Role of ACOT7 in Arachidonic Acid Production and Inflammation 207

Fig. 2. Domain organisation of Acots.

prostanoids produced from the COX pathway, including prostaglandin (PG) D2, prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2α), prostacyclin (PGI2) and thromboxane (TX) A2 (Narumiya, 2009). Gilroy et al. found that PGE2 levels are raised only during the initial phases of inflammation whilst PGD2 becomes the predominant prostanoid during the final stages of the inflammatory response (Gilroy et al., 1999; Tilley, Coffman, & Koller, 2001).

5-lipoxygenase utilises free arachidonic acid in conjunction with 5-lipoxygenase activatingprotein (FLAP), the 5-lipoxygenase activating protein, to catalyse the oxygenation of arachidonic acid into hydroperoxy-eicosatetraenoic acid (HPETE). FLAP selectively transfers arachidonic acid to 5-lipoxygenase and enhances the sequential oxygenation of this substrate to produce 5(S)-hydroperoxyeicosatetraenoic acid (5HpETE), as well as dehydration of arachidonic acid to leukotriene A4 (LTA4). LTA4 can then be exported from the cell and undergo transcellular metabolism or be converted into either the proinflammatory LTB4 or into a cysteinyl leukotrienes (cysLTs) LTC4, LTD4 or LTE4. The cysteinyl leukotrienes are a family of bronchoconstrictive, vasoconstrictive proinflammatory molecules. The primary signalling method for these leukotrienes is the activation of GPCRs on cell surfaces, namely BLT1 and BLT2 forLTB4, and CysLT1 and CysLT2 for CysLT's. Leukotrienes are thought to play a role in innate immune defence as well as a role in antimicrobial host defence. Importantly, they have been shown to play a role in respiratory diseases, such as asthma, allergies, such as anaphylaxis (Ferreira et al., 2008), as well as cardiovascular disease (Evans, Ferguson, Mosley, & Hutchinson, 2008).

Cytochrome P450 is thought to act on endogenous arachidonic acid converting it into epoxyeicosatrienoic acids (EETs) (Piomelli, 2000). Although found primarily in the liver cytochrome P450 has also been detected in a number of different tissues such as lungs, kidney, skin, adrenal cortex and brain tissues. The implications of P450 in the brain were demonstrated by Nicholson & Renton (2005) by removing astrocytes from rat brains and demonstrating that levels of P450 are modulated by inflammation using LPS stimulation.

Peroxisome-proliferator-activated receptors (PPARs) α and γ regulate the transcription of target genes through agonist binding to the ligand-binding domain (LBD) of these genes. PPARα plays a role in fatty acid regulation, through modulating the expression of target genes, and are characterised by a high lipid catabolic activity. Jiang et al. (2001) found that clofibrate, which activates PPARα, up-regulates the expression of cPLA2 and COX-2 in preadipocytes. PPARγ has been shown to play a role in the regulation of differentiation of preadipocytes into adipocytes. It was found by Murakami et al. (2001) that fatty acyl-CoA's function as antagonists for PPARα and PPARγ.

#### **3. Acyl-CoA thioesterase activity**

Acyl-CoA's perform a wide range of important cellular functions, serving as primary substrates for fatty acid degradation and lipid synthesis as well as regulators of cellular mechanisms such as ion fluxes, vesicle trafficking, protein phosphorylation and gene expression (see Figure 2 for domain organisation of Acot family members).

Most recently, the action of a specific enzyme within the ACOT enzyme family was demonstrated to act on arachidonoyl-CoA, and therefore possibly play a role in arachidonic acid production for the generation of prostanoids and leukotrienes. This is achieved by cleaving the thioester bond of activated C20:4-CoA in the general reaction described in Figure 3.

prostanoids produced from the COX pathway, including prostaglandin (PG) D2, prostaglandin E2 (PGE2), prostaglandin F2alpha (PGF2α), prostacyclin (PGI2) and thromboxane (TX) A2 (Narumiya, 2009). Gilroy et al. found that PGE2 levels are raised only during the initial phases of inflammation whilst PGD2 becomes the predominant prostanoid during the final stages of the inflammatory response (Gilroy et al., 1999; Tilley, Coffman, & Koller,

5-lipoxygenase utilises free arachidonic acid in conjunction with 5-lipoxygenase activatingprotein (FLAP), the 5-lipoxygenase activating protein, to catalyse the oxygenation of arachidonic acid into hydroperoxy-eicosatetraenoic acid (HPETE). FLAP selectively transfers arachidonic acid to 5-lipoxygenase and enhances the sequential oxygenation of this substrate to produce 5(S)-hydroperoxyeicosatetraenoic acid (5HpETE), as well as dehydration of arachidonic acid to leukotriene A4 (LTA4). LTA4 can then be exported from the cell and undergo transcellular metabolism or be converted into either the proinflammatory LTB4 or into a cysteinyl leukotrienes (cysLTs) LTC4, LTD4 or LTE4. The cysteinyl leukotrienes are a family of bronchoconstrictive, vasoconstrictive proinflammatory molecules. The primary signalling method for these leukotrienes is the activation of GPCRs on cell surfaces, namely BLT1 and BLT2 forLTB4, and CysLT1 and CysLT2 for CysLT's. Leukotrienes are thought to play a role in innate immune defence as well as a role in antimicrobial host defence. Importantly, they have been shown to play a role in respiratory diseases, such as asthma, allergies, such as anaphylaxis (Ferreira et al., 2008), as well as cardiovascular disease (Evans, Ferguson, Mosley, & Hutchinson, 2008). Cytochrome P450 is thought to act on endogenous arachidonic acid converting it into epoxyeicosatrienoic acids (EETs) (Piomelli, 2000). Although found primarily in the liver cytochrome P450 has also been detected in a number of different tissues such as lungs, kidney, skin, adrenal cortex and brain tissues. The implications of P450 in the brain were demonstrated by Nicholson & Renton (2005) by removing astrocytes from rat brains and demonstrating that levels of P450 are modulated by inflammation using LPS stimulation. Peroxisome-proliferator-activated receptors (PPARs) α and γ regulate the transcription of target genes through agonist binding to the ligand-binding domain (LBD) of these genes. PPARα plays a role in fatty acid regulation, through modulating the expression of target genes, and are characterised by a high lipid catabolic activity. Jiang et al. (2001) found that clofibrate, which activates PPARα, up-regulates the expression of cPLA2 and COX-2 in preadipocytes. PPARγ has been shown to play a role in the regulation of differentiation of preadipocytes into adipocytes. It was found by Murakami et al. (2001) that fatty acyl-CoA's

Acyl-CoA's perform a wide range of important cellular functions, serving as primary substrates for fatty acid degradation and lipid synthesis as well as regulators of cellular mechanisms such as ion fluxes, vesicle trafficking, protein phosphorylation and gene

Most recently, the action of a specific enzyme within the ACOT enzyme family was demonstrated to act on arachidonoyl-CoA, and therefore possibly play a role in arachidonic acid production for the generation of prostanoids and leukotrienes. This is achieved by cleaving the thioester bond of activated C20:4-CoA in the general reaction described in

expression (see Figure 2 for domain organisation of Acot family members).

2001).

function as antagonists for PPARα and PPARγ.

**3. Acyl-CoA thioesterase activity** 

Figure 3.

Fig. 2. Domain organisation of Acots.

Role of ACOT7 in Arachidonic Acid Production and Inflammation 209

Fig. 4. Classical pathway of inflammation with role of ACOT7 in arachidonic acid

barrel with 25% of Acot7 residues involved in interdomain contacts (see Figure 5).

Structural insights into the function of ACOT7 have recently been undertaken through the cloning and high-level recombinant expression of ACOT7. Pioneering characterisation of ACOT7 was undertaken by Yamada et al. (1994) through the isolation and purification of rat liver, however high level over-expression and isolation of the enzyme required cloning of cDNA into bacterial expression vectors (Broustas, Larkins, Uhler, & Hajra, 1996; Junji Yamada et al., 1999). Serek et al. (2006) elucidated the structure of the C-terminal of the ACOT7 protein purified from *Mus musculis* by crystallising the C-terminal domain and analysing the high resolution structure using X-Ray diffraction techniques. From this data it was identified that the C-terminal domain of ACOT7 exists in a hexameric form. Forwood et al (2007) isolated and expressed both the N- and C-terminals of ACOT7. These domains were then separately crystallised and the resulting structures (in 1.8 and 2.5 Å resolution respectively) were superimposed to determine the structure of the full length ACOT7. Within the hexamer of Acot7 β-sheets from each domain form a semicontinuous antiparallel

production.

**4. Structure of ACOT7** 

Fig. 3. ACOTs hydrolyse acyl-CoA esters to form free fatty acids and CoASH

#### **3.1 Activity of ACOT7**

It has been demonstrated that ACOT7 cleaves arachidonoyl-CoA to release CoA as CoASH and the free fatty acid arachidonic acid. Arachidonic acid is the precursor for a number of eicanosoids that enable the activation of macrophages (Kirkby, Roman, Kobe, Kellie, & Forwood, 2010), and it has been shown that ACOT7 expression is upregulated in macrophages in the presence of LPS and colony-stimulating factor 1 (CSF-1) (2007). It is from this data that the putative role of ACOT7 in inflammation was recognised. As shown in figure 4 below, ACOT7 may provide an alternative pathway for inflammation to the well characterised PLA2-mediated pathway and a possible target for a new class of antiinflammation therapies.

Fujita et al. (2011) found that levels of both cytosolic and mitochondrial ACOT7 within mammalian heart muscle increase in response to a high fat diet, and induce inflammation. The increased levels of ACOT7 are in response to the increasing levels of acyl-CoA imported across the mitochondrial membranes from the cytosol via the action of carnitine plamitoyltransferase (CPT). This mechanism is thought to reduce the "lipotoxic" effects of insulin resistance which leads to contractile dysfunction of the heart as the cells accumulate proinflammatory molecules such as acyl-CoA, diacylglycerol and ceramide (Fujita, et al., 2011).

ACOT7 is also known as brain acyl-CoA hydrolase (BACH) and has been purified from the brain cytosol of rats and humans and is believed to be responsible for acyl-CoA hydrolytic activity in the brain. Given the highly toxic nature of long chain acyl-CoA's, as a detergent, the activity of ACOT7 within the brain may be to reduce the levels of these within neurons (Kuramochi et al., 2002). Furthermore Takagi et al. (2006) found that ACOT7 is expressed in mouse testis and may play a role in spermatogenesis. The results of this research suggest that the regulation of ACOT7 occurs at a posttranscriptional level or that the rate of turnover in the testis is higher than in the brain. The level of ACOT7 protein was higher in the brain than the testis however the mRNA level was higher in the adult testis than the brain. The physiological significance of ACOT7 within the testis has not been determined however it is thought to scavenge cytosolic free long-chain acyl-CoA's.

Fig. 3. ACOTs hydrolyse acyl-CoA esters to form free fatty acids and CoASH

It has been demonstrated that ACOT7 cleaves arachidonoyl-CoA to release CoA as CoASH and the free fatty acid arachidonic acid. Arachidonic acid is the precursor for a number of eicanosoids that enable the activation of macrophages (Kirkby, Roman, Kobe, Kellie, & Forwood, 2010), and it has been shown that ACOT7 expression is upregulated in macrophages in the presence of LPS and colony-stimulating factor 1 (CSF-1) (2007). It is from this data that the putative role of ACOT7 in inflammation was recognised. As shown in figure 4 below, ACOT7 may provide an alternative pathway for inflammation to the well characterised PLA2-mediated pathway and a possible target for a new class of anti-

Fujita et al. (2011) found that levels of both cytosolic and mitochondrial ACOT7 within mammalian heart muscle increase in response to a high fat diet, and induce inflammation. The increased levels of ACOT7 are in response to the increasing levels of acyl-CoA imported across the mitochondrial membranes from the cytosol via the action of carnitine plamitoyltransferase (CPT). This mechanism is thought to reduce the "lipotoxic" effects of insulin resistance which leads to contractile dysfunction of the heart as the cells accumulate proinflammatory molecules such as acyl-CoA, diacylglycerol and ceramide

ACOT7 is also known as brain acyl-CoA hydrolase (BACH) and has been purified from the brain cytosol of rats and humans and is believed to be responsible for acyl-CoA hydrolytic activity in the brain. Given the highly toxic nature of long chain acyl-CoA's, as a detergent, the activity of ACOT7 within the brain may be to reduce the levels of these within neurons (Kuramochi et al., 2002). Furthermore Takagi et al. (2006) found that ACOT7 is expressed in mouse testis and may play a role in spermatogenesis. The results of this research suggest that the regulation of ACOT7 occurs at a posttranscriptional level or that the rate of turnover in the testis is higher than in the brain. The level of ACOT7 protein was higher in the brain than the testis however the mRNA level was higher in the adult testis than the brain. The physiological significance of ACOT7 within the testis has not been determined however it is

thought to scavenge cytosolic free long-chain acyl-CoA's.

**3.1 Activity of ACOT7** 

inflammation therapies.

(Fujita, et al., 2011).

Fig. 4. Classical pathway of inflammation with role of ACOT7 in arachidonic acid production.

#### **4. Structure of ACOT7**

Structural insights into the function of ACOT7 have recently been undertaken through the cloning and high-level recombinant expression of ACOT7. Pioneering characterisation of ACOT7 was undertaken by Yamada et al. (1994) through the isolation and purification of rat liver, however high level over-expression and isolation of the enzyme required cloning of cDNA into bacterial expression vectors (Broustas, Larkins, Uhler, & Hajra, 1996; Junji Yamada et al., 1999). Serek et al. (2006) elucidated the structure of the C-terminal of the ACOT7 protein purified from *Mus musculis* by crystallising the C-terminal domain and analysing the high resolution structure using X-Ray diffraction techniques. From this data it was identified that the C-terminal domain of ACOT7 exists in a hexameric form. Forwood et al (2007) isolated and expressed both the N- and C-terminals of ACOT7. These domains were then separately crystallised and the resulting structures (in 1.8 and 2.5 Å resolution respectively) were superimposed to determine the structure of the full length ACOT7. Within the hexamer of Acot7 β-sheets from each domain form a semicontinuous antiparallel barrel with 25% of Acot7 residues involved in interdomain contacts (see Figure 5).

Role of ACOT7 in Arachidonic Acid Production and Inflammation 211

The acyl-CoA thioesterase gene (*ACOT)* family encodes for two specific types of enzyme, acyl-CoA thioesterase type I and type II, which are determined by differences in structure and sequence. These two types catalyse similar reactions but share no similarity in structure or function, demonstrating that they are analogous and not homologous. They are an example of convergent evolution, whereby two molecules have evolved to fill the same need within the cell. Type I ACOT proteins are members of the α/β hydrolase fold enzyme superfamily. This superfamily also includes a number of esterase-activity-inhibiting enzymes such as carboxyl-esterase's and lipases. This group is comprised of only four genes; *ACOT1, ACOT2, ACOT4* and *ACOT6*. These proteins share a high degree of sequence homology, all forming an 80 kilobase gene cluster on chromosome 14q24.3, demonstrating that they have arisen as a result of gene duplication. Within the mouse and rat orthologues there is a similar phenomenon; *Acot1, Acot2, Acot3, Acot4, Acot5* and *Acot6* are clustered on chromosomes 12 D3 within the mouse and 6q31 within the rat. This can be seen in figure 8 below which also demonstrates the cellular compartments in which each is expressed

Type II ACOTs are members of the 'hot dog' fold enzyme superfamily. The type II ACOTs are far less related than the type I ACOTs. There is only one type II ACOT that does not contain a double 'hot dog' domain suggesting that they may have evolved as a gene duplication event, allowing for the accommodation of bulky substrates. Type II ACOTs show highly divergent sequences making evolutionary comparisons difficult without threedimensional structures, as structural interaction conservation does not directly correspond with residue conservation. Within the mouse genome there is an additional type II ACOT, known as *Acot10*, which shares 95% mRNA identity with ACOT9. The other seven type II ACOT genes are highly conserved among human, mouse and rat indicating that they were all present in the ancestor preceding mammalian radiation (Brocker, et al., 2010; M. C. Hunt

The ACOT7 enzyme is highly conserved, exhibiting greater than 95% sequence homology at the amino acid level between human, mice and rats (Kuramochi et al., 2002). Transcription start sites for ACOT7 were characterised by Takagi et al. in 2004 and shown to encode a 43kDa subunit, located in the cytosol; and six isoforms comprised of 50kDa subunits, expressed at trace levels and located in the mitochondria. Independent studies have confirmed that the ACOT7 gene can generate up to seven different protein isoforms as can be seen below in figure 6 (J. Yamada, 2005). The human ACOT7 gene consists of 13 exons, with the first four of these able to be used as first exons. The most well characterised of the ACOT7 isoforms, ACOT7a, is derived from the sequence corresponding to transcription initiation at exon 2 (M. Hunt et al., 2007; Kirkby, et al.,

Expression of ACOT7 has been detected in the developing mouse embryo brain as early as embryonic 11.5 days although in very low concentrations and increases until day seven following birth. Thereafter the level declined until day 28 following birth when it reached a steady state which was about 70% of its highest expression (on day 7) and identical to

**5. Genetic regulation of ACOTs** 

(Brocker, Carpenter, Nebert, & Vasiliou, 2010).

& Alexson, 2008; Kirkby, et al., 2010).

2010).

**5.1 Expression and regulation of ACOT7** 

Fig. 5. Structure of full-length Acot7 showing monomer and trimer arrangement.

Wedged between the two monomers that make up the protomer are six CoA molecules, making contacts with residues from each domain. Opposite this binding site is a large hydrophobic tunnel, conserved within thioesterases that may be involved in the fatty-acid recognition and release. The individual domains are inactive when in homomeric complexes however when combined the activity can be restored to half that of the wild type enzyme. The arrangement of the N- and C-domains within ACOT7 and the positioning of the CoA molecules within the N-domain suggest that the full molecule contains three copies each of two distinct active sites in ACOT7. There are two potential active sites within ACOT7 (sites I and II, see Figure 6); these were determined via sequence analysis of mammalian ACOT7s. To assess the role that each of these active sites play in catalysis each residue was mutated to Ala and the recombinant mutant enzymes were isolated and the activity of the mutant residues determined. The mutations in site I resulted in dramatic reductions in catalytic activity, whereas the analogous mutations in site II did not affect activity. These findings demonstrated that site II were not directly involved in catalysis. Furthermore the introduction of the key catalytic residues from site I into site II resulted in a four-fold increase in catalytic activity when compared with the wild-type Acot7. Thus, Acot7 (structures of each domain presented in figure 7) is believed to contain a "half-of-sites" activity, which may regulate the enzyme by placing an upper limit on enzyme efficiency and allows the cell to regulate the cellular concentrations of AA-CoA and arachidonic acid.

#### **5. Genetic regulation of ACOTs**

210 Inflammatory Diseases – A Modern Perspective

Fig. 5. Structure of full-length Acot7 showing monomer and trimer arrangement.

concentrations of AA-CoA and arachidonic acid.

Wedged between the two monomers that make up the protomer are six CoA molecules, making contacts with residues from each domain. Opposite this binding site is a large hydrophobic tunnel, conserved within thioesterases that may be involved in the fatty-acid recognition and release. The individual domains are inactive when in homomeric complexes however when combined the activity can be restored to half that of the wild type enzyme. The arrangement of the N- and C-domains within ACOT7 and the positioning of the CoA molecules within the N-domain suggest that the full molecule contains three copies each of two distinct active sites in ACOT7. There are two potential active sites within ACOT7 (sites I and II, see Figure 6); these were determined via sequence analysis of mammalian ACOT7s. To assess the role that each of these active sites play in catalysis each residue was mutated to Ala and the recombinant mutant enzymes were isolated and the activity of the mutant residues determined. The mutations in site I resulted in dramatic reductions in catalytic activity, whereas the analogous mutations in site II did not affect activity. These findings demonstrated that site II were not directly involved in catalysis. Furthermore the introduction of the key catalytic residues from site I into site II resulted in a four-fold increase in catalytic activity when compared with the wild-type Acot7. Thus, Acot7 (structures of each domain presented in figure 7) is believed to contain a "half-of-sites" activity, which may regulate the enzyme by placing an upper limit on enzyme efficiency and allows the cell to regulate the cellular The acyl-CoA thioesterase gene (*ACOT)* family encodes for two specific types of enzyme, acyl-CoA thioesterase type I and type II, which are determined by differences in structure and sequence. These two types catalyse similar reactions but share no similarity in structure or function, demonstrating that they are analogous and not homologous. They are an example of convergent evolution, whereby two molecules have evolved to fill the same need within the cell. Type I ACOT proteins are members of the α/β hydrolase fold enzyme superfamily. This superfamily also includes a number of esterase-activity-inhibiting enzymes such as carboxyl-esterase's and lipases. This group is comprised of only four genes; *ACOT1, ACOT2, ACOT4* and *ACOT6*. These proteins share a high degree of sequence homology, all forming an 80 kilobase gene cluster on chromosome 14q24.3, demonstrating that they have arisen as a result of gene duplication. Within the mouse and rat orthologues there is a similar phenomenon; *Acot1, Acot2, Acot3, Acot4, Acot5* and *Acot6* are clustered on chromosomes 12 D3 within the mouse and 6q31 within the rat. This can be seen in figure 8 below which also demonstrates the cellular compartments in which each is expressed (Brocker, Carpenter, Nebert, & Vasiliou, 2010).

Type II ACOTs are members of the 'hot dog' fold enzyme superfamily. The type II ACOTs are far less related than the type I ACOTs. There is only one type II ACOT that does not contain a double 'hot dog' domain suggesting that they may have evolved as a gene duplication event, allowing for the accommodation of bulky substrates. Type II ACOTs show highly divergent sequences making evolutionary comparisons difficult without threedimensional structures, as structural interaction conservation does not directly correspond with residue conservation. Within the mouse genome there is an additional type II ACOT, known as *Acot10*, which shares 95% mRNA identity with ACOT9. The other seven type II ACOT genes are highly conserved among human, mouse and rat indicating that they were all present in the ancestor preceding mammalian radiation (Brocker, et al., 2010; M. C. Hunt & Alexson, 2008; Kirkby, et al., 2010).

#### **5.1 Expression and regulation of ACOT7**

The ACOT7 enzyme is highly conserved, exhibiting greater than 95% sequence homology at the amino acid level between human, mice and rats (Kuramochi et al., 2002). Transcription start sites for ACOT7 were characterised by Takagi et al. in 2004 and shown to encode a 43kDa subunit, located in the cytosol; and six isoforms comprised of 50kDa subunits, expressed at trace levels and located in the mitochondria. Independent studies have confirmed that the ACOT7 gene can generate up to seven different protein isoforms as can be seen below in figure 6 (J. Yamada, 2005). The human ACOT7 gene consists of 13 exons, with the first four of these able to be used as first exons. The most well characterised of the ACOT7 isoforms, ACOT7a, is derived from the sequence corresponding to transcription initiation at exon 2 (M. Hunt et al., 2007; Kirkby, et al., 2010).

Expression of ACOT7 has been detected in the developing mouse embryo brain as early as embryonic 11.5 days although in very low concentrations and increases until day seven following birth. Thereafter the level declined until day 28 following birth when it reached a steady state which was about 70% of its highest expression (on day 7) and identical to

Role of ACOT7 in Arachidonic Acid Production and Inflammation 213

Fig. 7. (A) (B) Quaternary structure of Acot7 (N terminus) and C terminus respectively

Fig. 6. Active sites of Acot7: Active site I is comprised of Asn24 from the N-domain and Asp213 from the C-domain; the analogous site (later determined to be inactive) is comprised of Glu39 from the N-domain and Thr198 from the C-domain

Fig. 6. Active sites of Acot7: Active site I is comprised of Asn24 from the N-domain and Asp213 from the C-domain; the analogous site (later determined to be inactive) is comprised

of Glu39 from the N-domain and Thr198 from the C-domain

Fig. 7. (A) (B) Quaternary structure of Acot7 (N terminus) and C terminus respectively

Role of ACOT7 in Arachidonic Acid Production and Inflammation 215

Human Chromosome 14

Fig. 9. The type I acyl-CoA thioesterase gene cluster is found on chromosome 14q24.3 in the human genome and chromosome 12 D3 in the mouse genome, adapted from Hunt &

Mouse Chromosome 12

Fig. 10. Structural organisation of the human BACH gene, exons are designated by blue

Inflammation is a complex immune response that involves the production of eicosanoids via AA. The cellular role of ACOT7 has been extended to include the cleavage of arachidonoyl:CoA to yield arachidonic acid, and therefore may provide a mechanism for the supply of arachidonic acid from intracellular arachidonoyl-CoA. This is supported by a number of lines of evidence: ACOT7 is highly expressed in macrophages and upregulated by proinflammatory stimuli; the preferred substrate of ACOT7 is arachidonoyl-CoA and the reaction product is the central precursor for lipid inflammatory mediators; and finally, overexpression of ACOT7 in activated macrophages increases prostaglandin production. Thus, ACOT7 is able to complement the well-characterised PLA2 AA-producing pathway, and

boxes and introns by red segments, adapted from Yamada et al. (2005)

Alexson (2008)

**6. Conclusion** 

Fig. 8. Full length Acot7 demonstrating the N terminus domain (in green) and C terminus domain (in purple)

levels recorded at birth. The expression of *ACOT7* was located only in cells committed to neuronal lineage, and continues to be expressed in these cells resulting in the high expression of *ACOT7* in the adult brain (Junji Yamada, Kuramochi, Takagi, & Suga, 2004).

Research by Takagi, Suto, Suga & Yamada (2005) showed that ACOT7 gene expression is regulated by Sterol Regulatory Element-Binding Proteins (SREBPs). SREBPs form a few transcription factors which play a critical role in the regulation of cholesterol and fatty acids. Within the cell SREBPs are located in the membrane, to enter the nucleus they undergo proteolytic cleavage and their N-terminals are released as nSREBPs. Within the nucleus SREBPs bind to the sterol regulatory element (SRE) of target genes. The BACH gene promoter region contains two SRE motifs providing a binding partner for nSREBPs stimulating the production of cDNA of Acot7 (Takagi, et al., 2005).

Fig. 8. Full length Acot7 demonstrating the N terminus domain (in green) and C terminus

levels recorded at birth. The expression of *ACOT7* was located only in cells committed to neuronal lineage, and continues to be expressed in these cells resulting in the high expression of *ACOT7* in the adult brain (Junji Yamada, Kuramochi, Takagi, & Suga,

Research by Takagi, Suto, Suga & Yamada (2005) showed that ACOT7 gene expression is regulated by Sterol Regulatory Element-Binding Proteins (SREBPs). SREBPs form a few transcription factors which play a critical role in the regulation of cholesterol and fatty acids. Within the cell SREBPs are located in the membrane, to enter the nucleus they undergo proteolytic cleavage and their N-terminals are released as nSREBPs. Within the nucleus SREBPs bind to the sterol regulatory element (SRE) of target genes. The BACH gene promoter region contains two SRE motifs providing a binding partner for nSREBPs

stimulating the production of cDNA of Acot7 (Takagi, et al., 2005).

domain (in purple)

2004).

Human Chromosome 14

Mouse Chromosome 12

Fig. 9. The type I acyl-CoA thioesterase gene cluster is found on chromosome 14q24.3 in the human genome and chromosome 12 D3 in the mouse genome, adapted from Hunt & Alexson (2008)

Fig. 10. Structural organisation of the human BACH gene, exons are designated by blue boxes and introns by red segments, adapted from Yamada et al. (2005)

### **6. Conclusion**

Inflammation is a complex immune response that involves the production of eicosanoids via AA. The cellular role of ACOT7 has been extended to include the cleavage of arachidonoyl:CoA to yield arachidonic acid, and therefore may provide a mechanism for the supply of arachidonic acid from intracellular arachidonoyl-CoA. This is supported by a number of lines of evidence: ACOT7 is highly expressed in macrophages and upregulated by proinflammatory stimuli; the preferred substrate of ACOT7 is arachidonoyl-CoA and the reaction product is the central precursor for lipid inflammatory mediators; and finally, overexpression of ACOT7 in activated macrophages increases prostaglandin production. Thus, ACOT7 is able to complement the well-characterised PLA2 AA-producing pathway, and

Role of ACOT7 in Arachidonic Acid Production and Inflammation 217

Jiang, Y. J., Hatch, G. M., Mymin, D., Dembinski, T., Kroeger, E. A., & Choy, P. C. (2001).

Kirkby, B., Roman, N., Kobe, B., Kellie, S., & Forwood, J. K. (2010). Functional and structural

Kunishima, N., Asada, Y., Sugahara, M., Ishijima, J., Nodake, Y., Sugahara, M., . . . Sugahara,

Kuramochi, Y., Takagi-Sakuma, M., Kitahara, M., Emori, R., Asaba, Y., Sakaguchi, R., . . .

Maloberti, P., Castilla, R., Castillo, F., Maciel, F. C., Mendez, C. F., Paz, C., & Podestá, E. J.

Murakami, K., Ide, T., Nakazawa, T., Mochizuki, T., & Kaowaki, T. (2001). Fatty-acyl-CoA

Narumiya, S. (2009). Prostanoids and inflammation: a new concept arising from receptor

Piomelli, D. (2000). Neurophsychopharmacology: the Fifth Generation of Progress

Sakuma, S., Fujimoto, Y., Doi, K., Nagamatsu, S., Nishida, H., & Fujita, T. (1994). Existence of

Sakuma, S., Usa, K., & Fujimoto, Y. (2006). The regulation of formation of prostaglandins

Serek, R., Forwood, J., Hume, D., Martin, J., & Kobe, B. (2006). Crystallisation of the C-

Takagi, M., Ohtomo, T., Hiratsuka, K., Kuramochi, Y., Suga, T., & Yamada, J. (2006).

Takagi, M., Suto, F., Suga, T., & Yamada, J. (2005). Sterol Regulatory Element-Binding

*and Cellular Biochemistry, 275*(1), 199-206. doi: 10.1007/s11010-005-1990-y

*Structural Biology and Crystallisation Communications, 62*, 133-135.

preadipocytes. *Journal of Lipid Research, 42*(5), 716.

*49*(4), 366-377. doi: 10.1016/j.plipres.2010.04.001

*98*(1-2), 81-92. doi: 10.1016/s0169-328x(01)00323-0

10.1016/j.jmb.2005.07.008

*Biochemical Journal, 353*, 231-238.

*Research Communications, 202*(2), 1054-1059.

277. doi: 10.1016/j.prostaglandins.2006.02.005

*Archives of Biochemistry and Biophysics, 446*, 161-166.

1804-1814.

009-0500-1

*Arachidonic Acid* 

Modulation of cytosolic phospholipase A2 by PPAR activators in human

properties of mammalian acyl-coenzyme A thioesterases. *Progress in Lipid Research,* 

M. (2005). A Novel Induced-fit Reaction Mechanism of Asymmetric Hot Dog Thioesterase PaaI. *Journal of Molecular Biology, 352*(1), 212-228. doi:

Yamada, J. (2002). Characterization of mouse homolog of brain acyl-CoA hydrolase: molecular cloning and neuronal localization. *Molecular Brain Research,* 

(2005). Silencing the expression of mitochondrial acyl CoA thioesterase I and acyl CoA synthetase 4 inhibits hormone induced steroidogenesis. *FEBS Journal, 272*(7),

thioesters inhibit recruitment of steroid receptor co-activator 1 to α and γ isoforms of peroxisome-proliferator-activated receptors by competing with agonists.

knockout mice. *Journal of Molecular Medicine, 87*(10), 1015-1022. doi: 10.1007/s00109-

an enzymatic pathway furnishing arachidonic acid for prostaglandin synthesis from arachidonoyl CoA in rabbit kidney medulla. *Biochemical and Biophysical* 

and arachidonoyl-CoA from arachidonic acid in rabbit kidney medulla microsomes by linoleic acid hydroperoxide. *Prostaglandins & Other Lipid Mediators, 79*(3-4), 271-

terminal domain of the mouse brain cytosolic long-chain acyl-CoA thioesterase.

Localization of a long-chain acyl-CoA hydrolase in spermatogenic cells in mice.

Protein-2 modulates human brain acyl-CoA hydrolase gene transcription. *Molecular* 

may play a role in inflammation by producing sufficient levels of AA for eicosanoid production.

#### **7. References**


may play a role in inflammation by producing sufficient levels of AA for eicosanoid

Brocker, C., Carpenter, C., Nebert, D., & Vasiliou, V. (2010). Evolutionary divergence and

Broustas, C., Larkins, L., Uhler, M., & Hajra, A. (1996). Molecular cloning and expression of

Castilla, R., Maloberti, P., Castillo, F., Duarte, A., Cano, F., Cornejo Maciel, F., . . . Podesta, E.

Flesch, I., Schonhardt, T., & Ferber, E. (1989). Phospholipases and acyltransferases in

Forwood, J. K., Thakur, A. S., Guncar, G., Marfori, M., Mouradov, D., Meng, W., . . . Martin,

Fujita, M., Momose, A., Ohtomo, T., Nishinosono, A., Tanonaka, K., Toyoda, H., . . .

Golej, D. L., Askari, B., Kramer, F., Barnhart, S., Vivekanandan-Giri, A., Pennathur, S., &

Hunt, M., Greene, S., Hultenby, K., Svensson, L., Engberg, S., & Alexson, S. (2007).

*Molecular Life Sciences, 64*(12), 1558-1570. doi: 10.1007/s00018-007-7062-6 Hunt, M. C., & Alexson, S. E. H. (2008). Novel functions of acyl-CoA thioesterases and

Irvine, R. F. (1982). How is the level of free arachidonic acid controlled in mammalian cells?

*Lipid Research, 47*(6), 405-421. doi: 10.1016/j.plipres.2008.05.001

macrophages. *Journal of Molecular Medicine, 67*(3), 119-122.

*Journal of Biological Chemistry, 271*(18), 10470-10476.

functions of the human acyl-CoA thioesterase gene (ACOT) family. *Human* 

cDNA encoding rat brain cytosolic Acyl-Coenzyme A thioester hydrolase. *The* 

(2004). Arachidonic acid regulation of steroid synthesis: new partners in the signaling pathway of steroidogenic hormones. *Endocrine research, 30*(4), 599-606. Evans, J. F., Ferguson, A. D., Mosley, R. T., & Hutchinson, J. H. (2008). What's all the FLAP

about?: 5-lipoxygenase-activating protein inhibitors for inflammatory diseases. *Trends in Pharmacological Sciences, 29*(2), 72-78. doi: 10.1016/j.tips.2007.11.006 Ferreira, G. B., Overbergh, L., van Etten, E., Lage, K., D'Hertog, W., Hansen, D. A., . . .

Waelkens, E. (2008). Protein induced changes during the maturation process of human dendritic cells: A 2 D DIGE approach. *PROTEOMICS–Clinical Applications,* 

J. L. (2007). Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation. *Proceedings of the National Academy of* 

Yamada, J. (2011). Upregulation of fatty acyl-CoA thioesterases in the heart and skeletal muscle of rats fed a high-fat diet. *Biological Pharmacy Bulletin, 34*(1), 87-91. Gilroy, D. W., Colville-Nash, P., Willis, D., Chivers, J., Paul-Clark, M., & Willoughby, D.

(1999). Inducible cyclooxygenase may have anti-inflammatory properties. *Nature* 

Bornfeldt, K. E. (2011). Long-chain acyl-CoA synthetase 4 modulates prostaglandin E2 release from human arterial smooth muscle cells. *Journal of Lipid Research, 52*(4),

Alternative exon usage selectively determines both tissue distribution and subcellular localization of the acyl-CoA thioesterase 7 gene products. *Cellular and* 

acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism. *Progress in* 

production.

**7. References** 

*Genomics, 4*(6), 411-420.

*2*(9), 1349-1360.

*Sciences, 104*(25), 10382.

*medicine, 5*(6), 698-701.

*Biochemical Journal, 204*(1), 3.

782.


**Part 11** 

**Inflammatory Bowel Disease** 

