Asthma Is the Most Common

¶ … asthma is the most common chronic disease of childhood and one of the leading causes of morbidity in children. In the United States, trends of increasing childhood asthma prevalence and morbidity in recent years have been found to disproportionately affect nonwhite children living in urban areas and children living in lower socioeconomic conditions. This study provides a review of the peer-reviewed and scholarly literature to identify normal function, damage and repair of the human bronchia, including a discussion of the normal bronchial epithelium, the bronchial epithelium in asthma, bronchial epithelial inflammation, remodeling, normal epithelial repair and cytokines, tissue factor, tissue factor pathway inhibitor, coagulation factors, tissue-type plasminogen activator, plasminogen-activator-inhibitor, epidermal growth factor, hepatocyte growth factor, and nitric oxide and bronchial epithelial damage in other diseases. A discussion concerning the need for new therapy for asthma is followed by a summary of the research and important findings in the conclusion.

Bronchial Epithelium in Asthma: Normal Function, Damage and Repair

Introduction

Today, asthma represents the most common chronic disease among young people, afflicting more than three million children in the United States. There has been an increased incidence of asthma in the United States over the last 25 years that most researchers attribute to changes in environmental conditions. This increased incidence and prevalence of asthma is especially confounding because a number of pharmacological preparations with known efficacy for asthma have been introduced during this same period and researchers continue their search for answers and improved clinical interventions. To help provide an overview of current research findings concerning bronchial epithelium in asthma, this study examines the peer-reviewed and scholarly literature to identify normal function, damage and repair of the human bronchia, including a discussion of the normal bronchial epithelium, the bronchial epithelium in asthma, bronchial epithelial inflammation, remodeling, normal epithelial repair and cytokines, tissue factor, tissue factor pathway inhibitor, coagulation factors, tissue-type plasminogen activator, plasminogen-activator-inhibitor, epidermal growth factor, hepatocyte growth factor, and nitric oxide and bronchial epithelial damage in other diseases. A discussion concerning the need for new therapy for asthma is followed by a summary of the research and important findings in the conclusion.

Review and Discussion

The normal bronchial epithelium

According to a recent study by Kercsmar, Dearborn, Schluchter, Xue, Kirchner, Sobolewski, Greenberg, Vesper and Allan (2006) asthma is the most common chronic disease affecting children today, with more than three million young people in the United States alone suffering from this condition. Moreover, the incidence of childhood asthma has continued to increase over the past several decades in spite of the increasing availability of efficacious medications that have been shown to control chronic symptoms and treating exacerbations (Kercsmar et al. 2006).

Furthermore, the prevalence of asthma and its associated morbidity are inordinately elevated among inner-city children compared to their suburban counterparts, the majority of whom are racial minorities (Wright & Steinbach 2001). For instance, Kercsmar and his associates emphasize that, "African-American children in the United States have a higher prevalence of asthma and greater morbidity as measured by acute care visits and hospitalizations compared with white children" (p. 1574). Notwithstanding the growing body of research into asthma and its associated risk factors, it remains unclear how differences in generally known asthma risk factors including chemical and particulate air pollutants, environmental and in utero tobacco smoke exposure, viral respiratory infections, and home allergen exposure explain recent increases in the prevalence of this disease. In this regard, Wright and Steinbach (2001, p. 1086) add that, "As yet unidentified unique factors may contribute to the higher asthma morbidity and mortality rates seen in inner-city poor minority populations."

There are some age-related changes that typically occur in the normal bronchial epithelium. In this regard, Burke and Laramie report that, "There is a gradual age-related decline in pulmonary function beginning at about age 40. The elastic recoil of the lungs decreases, owing to changes in elastin and collagen. The lung weight is decreased by approximately one fifth, the bronchi harden, and the bronchial epithelium and mucous glands degenerate" (2000, p. 161). The histology of the bronchial epithelium in a normal human lung and the histology of the bronchiolar epithelium of a normal human lung are shown in Figure 1 below.

Figure 1. (a) Histology of the bronchial epithelium of a normal human lung; and (B) Histology of the bronchiolar epithelium of a normal human lung

Source: The National Academy of Sciences 2009 at http://books.nap.edu/books/0309044847 / xhtml/images/img00107.gif

The bronchial epithelium shown as (a) in Figure 1 above is organized as a pseudostratified columnar epithelium that has ciliated and secretory mucous goblet cells which line the surface; in addition, basal cells can be discerned from time to time along the basement membrane (Comparative dosimetry of radon in mines and homes 1991). According to these authorities, "The bronchiolar epithelium is thinner and varies in organization from pseudostratified columnar to cuboidal epithelium, depending on how peripheral it is in the branching of bronchioles. The surface is lined by ciliated cells and goblet cells and/or Clara cells, depending on the level of branching of the bronchioles" (Comparative dosimetry of radon in mines and homes 1991, p. 169).

According to a study by Demoly, Simony-Lafontaine, Chanez, Pujol, Lequeux, Michel and Bousquet (1994), significant changes occur in normal bronchial epithelium cells in response to a variety of antagonists. For instance, these researchers report that, "In chronic inflammatory diseases, cells are recruited but may also derive from local proliferation. In normal bronchial epithelium, under 5% of cells are in cycle but in asthma and chronic bronchitis, proliferation may occur" (Demoly et al. 1994, p. 214). Identification of cycling cells can be achieved through immunohistochemistry using PC10 monoclonal antibody (Demoly et al. 1994). In this regard, in this study, these researchers enumerated PCNA-positive cells (labeling index = LI) in bronchial biopsies of seven healthy smokers (HS), 11 healthy non-smokers (HNS), 30 non-smoking asthmatics (NSA), six smoking asthmatics (SA) and 18 chronic bronchitics (CB) (Demoly et al. 1994). A positive control group was comprised of 20 non-small cell lung cancer patients (Demoly et al. 1994).

These researchers note that ciliated and secretory cells were proliferating cell nuclear antigen-negative, but basal cells were found to be proliferating cell nuclear antigen-positive in one member of the 11 HNS (LI = 0.18 +/- 0.60) (Demoly et al. 1994). None of the seven HS were proliferating cell nuclear antigen-postive, but two of the 30 NSA (LI = 0.05 +/- 0.20), two of the six SA (LI = 2.4 +/- 4.3) and 11 of the 18 CB (LI = 12 +/- 20) were positive for proliferating cell nuclear antigen (Demoly et al. 1994). In sum, these researchers conclude that, "In smokers, proliferating cell nuclear antigen positivity correlated with tobacco consumption (Rho = 0.62, p < 0.0008) and in patients with chronic bronchitis, with the degree of metaplasia (tau = 0.815, p < 0.0001). The submucosa of most subjects showed no proliferating cell nuclear antigen immunoreactivity. These findings suggest that the bronchial mucosa of nonsmokers is not hyperproliferative, even in asthmatics. Tobacco smoking increases proliferating cell nuclear antigen immunoreactivity, possibly leading to the metaplasia of chronic bronchitis" (Demoly et al. 1994, p. 215).

The bronchial epithelium in asthma

According to Spannhake, Reddy, Jacoby, Yu, Saatian and Tian (2002), a number of factors are thought to exacerbate the symptoms associated with asthma, with air pollution and viral infections being regarded as especially significant. These authors add that, "Evidence indicates that each of these respiratory insults individually can increase asthma severity in susceptible individuals" (Spannhake et al. 2002, p. 665). In addition, the growing body of research concerning the causes and incidence of asthma has resulted in a need for interpreting epidemiologic findings concerning the impact of long-term exposure to ozone which is known to cause chronic health effects. In this regard, Levy, Carrothers, Tuomisto, Hammitt and Evans report that, "Evidence from animal and human exposure studies shows that long-term exposure could produce sustained decrements in lung function, particularly in small airway measures. Studies of chronic ozone exposures in monkeys and rats found thickening of the epithelium and interstitium of bronchioles" (2001, p. 1215).

In these experiments, the respiratory epithelium was shown to be replaced by bronchial epithelium in the central acinar region in rats following long-term exposure to ozone (Levy et al. 2001). Consequently, long-term exposure to ozone is believed to cause bronchiolitis with remodeling of the bronchiolar epithelium resulting in a decreased diffusion capacity, fibrosis resulting in a reduction of lung elasticity, and proliferation of type II pneumocytes; these changes in have also been identified in autopsied human lungs following long-term ozone exposure (Levy et al. 2001).

In their study, "Airway mucosal inflammation even in patients with newly diagnosed asthma," Laitinen, Laitinen and Haahtela (1993) analyzed bronchial biopsies from 14 patients who were newly diagnosed with asthma (n=four males; 10 females); on average, the 14 subjects had exhibited asthma symptoms for 7.4 months (range, 2 to 12 months); besides these 14 subjects, these researchers also examined bronchial biopsies from four control subjects (Laitinen et al. 1993). According to these researchers, none of the subjects had received corticosteroids, disodium cromoglycate, or theophylline prior to the conduct of their study (Laitinen et al. 1993). Using a rigid-tube bronchoscope, the bronchial biopsies were taken under local anaesthesia from two different airway levels: (a) inside the right upper lobe bronchus, and (b) at the opening of the right middle lobe (Laitinen et al. 1993).

Preparations of the specimens thereby obtained were completed for light as well as electron microscopy; the researchers also used slot grids 1 x 2 mm which facilitated photography of a large area of the thin sections and analysis through an application of a graphic Autocad software program (Laitinen et al. 1993). In the 14 subjects with newly diagnosed asthma, Laitinen and his associates identified an increase in the numbers of mast cells (p < 0.001), eosinophils (p < 0.05), lymphocytes (p < 0.05), and macrophages (p < 0.05) in the epithelium compared to those found in the control subjects. According to Laitinen and his associates, "In the lamina propria, these asthmatic patients had more eosinophils (p < 0.001), lymphocytes (p < 0.001), macrophages (p < 0.001), and plasma cells (p < 0.001) than did the control subjects. We conclude that, in asthma, an airway inflammatory process is present even at a clinically early stage of the disease. In the asthmatic airways, there are signs of a general inflammatory response caused by more than one cell type" (1993, p. 697).

Based on their observation that there remains a dearth of timely studies that have evaluated the asthmatic airway in childhood, Coku-ra?, Seckin, Camcio-lu, Sarimurat and Aksoy (2001) assessed the histopathological changes that take place in the bronchi of children with moderate asthma using light and electron microscopy. These researchers used bronchial biopsy specimens taken from 10 children who suffered from moderate asthma (n = seven boys; three girls) of mean (SD) age 9.3 (3.8) years (range 5-14); the specimens were analyzed using both light and electron microscopy (Coku-ra? et al. 2001). According to these researchers, "Patients had not had a respiratory infection for at least one month and they had not been treated with steroids or sodium cromoglycate for four weeks before the study. Bronchoscopy was performed under general anaesthesia using a Karl Storz rigid paediatric bronchoscope. Biopsy materials were stained with uracyl acetate and lead citrate and evaluated under a Zeiss-10 electron microscope and light microscope" (Coku-ra? et al. 2001, p. 25).

Based on their light and electron microscopic analysis of the specimens, Coku-ra? And his associates found thickening and hyalinization of the basement membrane in nine of the subjects; in addition, in some cases, the ciliated epithelial cells exhibited loss of cilia (Coku-ra? et al. 2001). In addition, these researchers report that overactive fibroblasts were identified consistently throughout the samples of all ten subjects and six of the subjects were shown to have degranulating mast cells and lymphocyte infiltration in the submucosa; however, eosinophils was identified in just one biopsy sample (Coku-ra? et al. 2001). Based on their findings, these researchers conclude that, "Children with moderate asthma develop bronchial inflammation similar to the reaction observed in adults. However, in our study the inflammation was rich in lymphocytes rather than eosinophils" (Coku-ra? et al. 2001, p. 26).

Bronchial epithelial inflammation

According to Zhao, Vaszar, Qiu, Shi and Kao (2000), "Bronchial epithelial cell expression of inflammatory cytokines and growth factors contributes to the airway inflammation that is characteristic of asthma, chronic bronchitis, and bronchiectasis. Structural changes can develop in airways because of the chronic inflammation in cystic fibrosis or atypical mycobacterial pulmonary disease or because of chronic allograft rejection after lung transplantation, which can manifest as obliterative bronchiolitis" (p. 958). Drugs that are administered either systemically or topically can, in some cases, provide suppression of inappropriate airway inflammation and therapeutic benefits in diseases such as asthma and chronic bronchitis; certain pharmacological preparations may also serve to diminish the airway destruction that occurs in bronchiectasis and obliterative bronchiolitis (Zhao et al. 2000).

An early study by Konradova, Hlouskova and Tomanek (1977) examined the ultrastructure of the respiratory passages epithelium of young children, adolescents as well as older adults who suffered from repeated or chronic respiratory disease using material obtained as a small excision during bronchoscopy. The findings were classified into four categories as follows based on the character of the ultrastructural changes found in the epithelium. In large bronchi, the following was found:

1. A completely unaltered pseudostratified columnar ciliated epithelium;

2. A pseudostratified columnar epithelium with various signs of pathological alteration;

3. An altered pseudostratified columnar epithelium with first ultrastructural signs of the development of squamous metaplasia; and,

4. A developed stratified squamous epithelium (Konradova et al. 1977).

Based on their analysis of these findings, it was the view of these researchers that in the respiratory passages of children and adolescents, even if they suffer from repeated respiratory diseases, the pseudostratiified ciliated epithelium persists; however, it is damaged to different extents (Konradova et al. 1977). According to these researchers, "These patients were classified into the second or at most into the third group. The observation of fully developed squamous metaplasia is reserved to older patients with longer history of chronic respiratory disease" (Konradova et al. 1977, p. 270).

A somewhat later study by Heino, Juntunen-Backman, Leijala, Rapola and Laitinen (1990) investigated the ultrastructural findings in biopsies from the main carina of seven children and adolescents who had experienced a chronic cough for a minimum of 3 months; all of the adolescent subjects also had a history of early lower respiratory illness. The seven subjects in this study experienced their initial lower respiratory illness between birth and the age of 7 years (range, 5 to 11 years) (Heino et al. 1990). According to these researchers, "The cross-sectional area of the epithelium was quantified by point counting for the percentage area of intercellular spaces denoting edema, and the numbers of both inflammatory cells (leukocytes, including eosinophils, and mast cells) and ciliated cells" (Heino et al. 1990, p. 428).

The children and adolescent subjects (not including the single subject who used steroid inhalers) exhibited almost 17- and more than sevenfold increases in the mean area of intercellular spaces as well as the number of inflammatory cells per epithelial area, respectively; the subjects also were shown to have an almost 300% decrease in the mean number of ciliated cells per epithelial area compared with the biopsy specimens from the orifice of the right upper lobe bronchus of two healthy adults (Heino et al. 1990).

In sum, Heino and his associates note that, "In the children, the increase in inflammatory cells (greater than 91% were lymphocytes) was more prominent in the children with two lower respiratory illnesses before the age of 1 year" (p. 429). The findings of the Heino et al. study indicate a close relationship between early lower respiratory illness onset and subsequent epithelial inflammation during chronic cough. The researchers add that it is not possible to rule out allergic mechanisms in the epithelial inflammation because all of the subjects exhibited, either alone or in combination, indications of atopia, positive family history of allergic rhinitis or asthma, and eosinophils or mast cells in the epithelium (Heino et al. 1990).

Finally, according to Lynch, "The relationship between irritant chemical exposure and induction of asthma attacks in asthmatics is well established. Irritant chemical exposure causes bronchial epithelium injury. Persons with asthma are more susceptible to irritant and volatile organic chemicals than nonasthmatics and show greater bronchial hyperresponsiveness to irritant exposures than nonasthmatics" (2000, p. 911).

Remodeling

A number of studies in recent years have examined the role of various factors in the airway remodeling associated with asthma. In this regard, Siddiqui and Martin report that, "Airway remodeling in asthma is a complex process that involves structural changes in virtually all tissues of the airway wall. The histologic changes to the airways consist of epithelial proliferation and goblet cell differentiation, subepithelial fibrosis, airway smooth muscle growth, angiogenesis, matrix protein deposition, gland hyperplasia and hypertrophy, and nerve proliferation" (2008, p. 540).

In addition, cytokines, chemokines, and growth factors from inflammatory cells and structural cells have all been shown to contribute to airway remodeling (Siddiqui & Martin 2008). Not surprisingly, complex interactions among the various signaling pathways involve matrix metalloproteinases that are necessary for the release of the growth factor (Siddiqui & Martin 2008). Based on their research, Siddiqui and Martin conclude that, "The physiologic consequences of remodeling are airway hyperresponsiveness from airway smooth muscle growth and mucus hypersecretion from gland and goblet cell hyperplasia. Airway stiffening is a probable contributor to airway hyperresponsiveness through attenuation of the transmission of potently bronchodilating cyclical stress to the ASM during breathing" (2008, p. 541).

According to Kuramoto, Nishiuma, Kobayashi, Yamamoto, Kono, Funada, Kotani, Sisson, Simon and Nishimura, "The airway remodeling that occurs in asthma is characterized by an excess of extracellular matrix (ECM) deposition in the submucosa, hyperplasia/hypertrophy of smooth muscle, goblet cell metaplasia, and accumulation of fibroblasts/myofibroblasts" (2008, p. 73). In addition, the urokinase-type plasminogen activator (uPA)/plasmin system take part in pericellular proteolysis; this plasminogen activator has the capability of degrading matrix components in a direct fashion as well as being able to activate latent proteinases and growth factors (Kuramoto et al. 2008).

In their study, "Inhalation of urokinase-type plasminogen activator (uPA) reduces airway remodeling in a murine asthma model," these researchers increased plasminogen activator activity in the lung in a mouse ovalbumin asthma model through the administration of exogenous uPA or, in the alternative, through the use of mice that were already genetically deficient in the uPA inhibitor, plasminogen activator inhibitor-1 (PAI-1) in order to determine the role of this system in asthma pathogenesis (Kuramato et al. 2008). According to these researchers, "Following intraperitoneal OVA sensitization, mice inhaled OVA plus uPA (500 IU/mouse) or saline by ultrasonic nebulization for 3 weeks. When studied 24 hours after the final exposure, the groups with upregulated plasmin activity had significantly reduced subepithelial fibrosis within the airway walls and had decreased airway hyperresponsiveness to methacholine (AHR)" (Kuramato et al. 2008, p. 73).

In addition, morphometric analysis also found that subepithelial wall thickening of the bronchi (subepithelial area ratio) was diminished as well as collagen and alpha-smooth muscle actin (Kuramato et al. 2008). The results of this study also demonstrated that upregulation of plasmin activity served to elevate the level of hepatocyte growth factor (HGF) activity in bronchoalveolar lavage fluid, while the release of transforming growth factor (TGF)-beta was found to be diminished (Kuramato et al. 2008). Beyond the foregoing findings, Kuramato and his colleagues also determined that the administration of uPA one week following the last ovalbumin inhalation significantly reduced lung hydroxyproline content and airway hyperresponsiveness to methacholine. These findings indicate that enhancing uPA/plasmin activity serves to decrease the airway remodeling in a murine asthma model (Kuramato et al. 2008).

A similar study by Jie, Jin, Cai, Bai, Shen, Yuan, Hu and Holgate (2009) notes that although a disintegrin and metalloprotease domain 33 (ADAM33) has been identified as an asthma susceptibility gene that is associated with small-airway remodeling, the specific role of ADAM33 in the development of allergic airway inflammation remains unclear. To address this gap in the literature, Jie and his associates employed an established murine model of allergen-induced chronic airway inflammation that was sensitized and then challenged through the use of nebulized 2.5% ovalbumin for an 8-week period (i.e., 30 minutes a day, three times a week) (Jie et al. 2009). According to these researchers, "The expression of ADAM33 mRNA detected by real time RT-PCR was significantly enhanced in the lung tissue of mice with OVA challenge, as compared with the group challenged with saline. This OVA-challenged model showed significant Th2-biased airway inflammation as well as airway remodeling with features of sub-epithelial fibrosis and mucus hyper-secretion" (Jie et al. 2009, p. 24).

Moreover, in vitro analyses by Jie and his associates determined that IL-4 and IL-13 were capable of up-regulating the expression of ADAM33 mRNA in human fibroblasts in a concentration- and time- dependent manner significantly compared to normal controls (Jie et al. 2009). The findings of this study were congruent with the notion that Th2 cytokines are capable of up-regulating the expression of ADAM33 mRNA and ADAM33 is capable of functioning in a significant fashion in the development of airway remodeling in allergen-induced chronic airway inflammation (Jie et al. 2009).

A recent study by Labonte, Hassan, Risse, Tsuchiya, Laviolette, Lauzon, and Martin (2009) investigated the effects of repeated allergen challenge on airway smooth muscle structural and molecular remodeling using a rat model of allergic asthma. This study was based on the paucity of timely and relevant studies concerning the effects of remodeling of airway smooth muscle by hyperplasia on airway smooth muscle contractility in vivo (Labonte et al. 2009). The goal of the study by Labonte and his associates was to analyze the relationship between allergen-induced airway smooth muscle hyperplasia and its contractile phenotype.

To this end, brown Norway rats were sensitized using ovalbumin or saline on day 0; thereafter, the rats were either OVA-challenged once on day 14 and sacrificed 24 hours later or ovalbumin-challenged on days 14, 19 and 24 and then sacrificed 2 days or 7 days therafter (Labonte et al. 2009). According to Labonte et al., "Changes in smooth muscle mass, expression of total-myosin, smooth muscle myosin heavy chain fast isoform (SM-B) and myosin light chain kinase (MLCK), tracheal contractions ex-vivo and airway responsiveness to methacholine (MCh) in vivo were assessed" (p. 37).

One day following a single ovalbumin challenge, the number of smooth muscle cells that were found to be positive for proliferating cell nuclear antigen was shown to be greater compared to control animals while the smooth muscle mass, contractile phenotype and tracheal contractility remained unchanged (Labonte et al. 2009). In the group that received three challenges over 2 days, smooth muscle mass and proliferating cell nuclear antigen immunoreactive cells were found to be elevated (3- and 10-fold, respectively p

In addition, Labonte and his associates found lower expression in total-myosin, smooth muscle myosin heavy chain fast isoform and myosin light chain kinase at the mRNA level (p

Finally, repeated insults over time from air pollutants may result in chronic adverse effects in childhood asthma sufferers; in fact, researchers have found that persistent airway inflammation in asthmatics results in airway remodeling, diminished lung growth, and permanent lung function impairment (Delfino, Staimer, Gillen, Tjoa, Sioutas, Fung, George and Kleinman 2006).

Normal epithelial repair

Cytokines. One of the characteristics of asthma is chronic inflammation in the airways and the presence of a predominance of CD[4+] T-helper 2 (Th2) cells; these cells secrete interleukin (IL)-4, IL-5, and IL-13 cytokines (Duramad, Harley, Lipsett, Bradman, Eskenazi, Holland & Tager 2006). Th2 cells contribute to the immunopathogenesis of asthma by recruiting eosinophils and mast cells to the airways and by inducing B-cells to produce immunoglobulin E. antibodies. Conversely, T-helper 1 (Th1) cells that secrete interferon (IFN)-[gamma] are thought to protect against the development of asthma by regulating Th2 cytokine production, although a mixed Th1/Th2 pattern has been reported. Allergic and asthmatic subjects are more likely to have elevated levels of the Th2 cytokines IL-4 and IL-5, and reduced levels of the Th1 cytokines IFN-[gamma] and tumor necrosis factor (TNF)-[beta]; however, increased levels of IFN-[gamma] also have been reported in cases of severe asthma that could involve CD[8.sup.+] T cells (Duramad et al. 2006). Likewise, Spannhake and his associates report that, "Oxidant pollutants can amplify the generation of proinflammatory cytokines by RV16-infected cells and suggest that virus-induced inflammation in upper and lower airways may be exacerbated by concurrent exposure to ambient levels of oxidants commonly encountered the indoor and outdoor environments" (2002, p. 667).

According to Holgate (2008), although asthma is widely regarded as an inflammatory disorder of the conducting airways, the growing body of research concerning this disease indicate that asthma is heterogeneous with regard to immunopathology, clinical phenotypes, response to therapies, and natural history. This researcher reports that, "Once considered purely an allergic disorder dominated by Th2-type lymphocytes, IgE, mast cells, eosinophils, macrophages, and cytokines, the disease also involves local epithelial, mesenchymal, vascular and neurologic events that are involved in directing the Th2 phenotype to the lung and through aberrant injury-repair mechanisms to remodeling of the airway wall" (Holgate 2008, p. 872).

Using a "soil" and "seed" analogy, Holgate notes that the structural cells provide the requisite basis for the inflammatory response that takes place in the lungs and also serve to maintain a chronic phenotype; on this foundation are superimposed acute and subacute episodes that typically are caused by various environmental factors such as exposure to allergens, microorganisms, pollutants or caused by inadequate antiinflammatory treatment (Holgate 2008). Taken together, Holgate suggests that, "Greater consideration of additional immunologic and inflammatory pathways are revealing new ways of intervening in the prevention and treatment of the disease. Thus increased focus on environmental factors beyond allergic exposure (such as virus infection, air pollution, and diet) are identifying targets in structural as well as immune and inflammatory cells at which to direct new interventions" (2008, p. 873).

Tissue factor. Tissue factor has a particular relevance for asthma patients. In this regard, Kemona-Chetnik, Bodzenta-Lukaszyk, Kucharewicz and Rogalewska (2005) report that, "Allergic asthma is characterized by bronchial inflammation and repair processes at the same time, that cause increased airway obstruction. Recent evidences suggest that monocytes and macrophages may play important role in allergic inflammation" (p. 98). When proinflammatory factors are activated, they are able to express tissue factor as well as tissue factor pathway inhibitor (Kemona-Chetnik et al. 2005), which is discussed further below. The goal of the study by Kemona-Chetnik and his associates was to assess tissue factor as well as tissue factor pathway inhibitor in the plasma of bronchial asthma patients.

To this end, the Kemona-Chetnik study analyzed 17 patients suffering from asthma with established Dermatophagoides pteronyssinus allergy; 15 subjects who were healthy served as a negative control for the study (Kemonoa-Chetnik et al. 2005). During the course of the study, specific bronchial challenge with Dermatophagoides pteronyssinus extracts was performed among the asthma patients, with blood specimens being obtained prior to the allergen challenge (A0), during the early asthmatic reaction (EAR), A0 during the late asthmatic reaction (LAR), A2, and 24 hours following the administration of the first allergen dose (A3) (Kemona-Chetnik et al. 2005).

The concentrations of TF and TFPI were measured by the enzyme-linked immunosorbent assay (ELISA) method. The concentration of tissue factor in the patients with asthma was found to be substantially higher compared to the healthy subjects in the negative control group (Kemona-Chetnik 2005). According to these researchers, "In patients that developed only early asthmatic reaction, the concentration of tissue factor increased at A1, then decreased at time when late asthmatic reaction should be developed (A2) and it was comparable with beginning values 24 hours after starting the challenge" (Kemona-Chetnik et al. 2005, p. 99). Patients who developed early asthmatic reaction as well as late asthmatic reaction, the mean tissue factor concentration was shown to be elevated during early asthmatic reaction (a,) and declining thereafter during late asthmatic reaction (A2) (Kemona-Chetnik et al. 2005).

Twenty-four hours following the initiation of the challenge, the tissue factor concentration was found to be elevated compared to the beginning values (A3); in addition, the concentration of tissue factor pathway inhibitor among those patients who experienced early asthmatic reaction only was found to be substantially higher than the values in the health negative control group (Kemona-Chetnik et al. 2005).

The concentration of tissue factor, though, decreased during early asthmatic reaction (A1) and became elevated 24 hours following the initiation of the challenge (A3) (Kemona-Chetnik et al. 2005). Although the mean concentration of tissue factor pathway inhibitor in the subjects who developed early and late asthmatic reaction was not found to be significantly higher than the values in the healthy negative control group, the mean concentration of tissue factor pathway inhibitor did increase during early and late asthmatic reaction with the highest values being experienced 24 hours following the initiation of the challenge (Kemona-Chetnik et al. 2005). In sum, the researchers conclude that, "Coagulation system seems to be activated during allergic inflammation after allergen challenge. Increased levels of tissue factor and tissue factor pathway inhibitor in asthma patients may be connected with chronic bronchial inflammation and remodeling of bronchial wall" (Kemona-Chetnik et al. 2005, p. 99).

In addition, the results of a study by Karoly, Li, Dailey, Hyseni and Huangthat (2007) found that tissue factor, coagulation factor II receptor-like 2, interleukin 6, and interleukin 8 were up-regulated; in addition, these researchers demonstrated that the release of IL-6 and IL-8 induced by ultrafine particulates was dependent in part on the activation of the tissue factor pathway.

Tissue factor pathway inhibitor. Tissue factor pathway inhibitor (TFPI) is a naturally occurring anticoagulant protein that serves to inhibit the growth of tumors and the formation of new blood vessels in preclinical models (EntreMed and Affymax form tissue factor pathway inhibitor peptides collaboration 2004). Researchers have found that TFPI possesses a high level of activity against endothelial cells, the foundation of blood vessels, causing specific antiangiogenic properties. According to this report, "Coagulation is an important component of the metastatic spread of tumors, and by inhibiting both coagulation and angiogenesis this new finding uncovers the dual role of TFPI as a potential agent for cancer treatment" (Entremed reports on tissue factor pathway inhibitor as an antiangiogenic agent 2001, p. 4).

The smaller molecular weight of low molecular weight heparins alters their anticoagulant profile compared to unfractionated heparin; however, both of these substances function by facilitating antithrombin activity (Adler 2004). Because low molecular weight heparin contains a significantly smaller quantity of the large polymers required for thrombin inactivation, their primary anticoagulant effect takes place through an inhibition of factor Xa (Adler 2004). Consequently, these agents have minimal impact on the partial thromboplastin time, the clinical, antithrombotic effect of these agents, though, appears to involve more than the factor Xa inhibition, and may partially depend on the capability of low molecular weight heparin to increase the release of tissue factor pathway inhibitor (Adler 2004).

Finally, a related study by White, Witt, Pan, Mueske, Kleppe, Holroyd, Champion and Simari (2009) notes that, "Pulmonary hypertension is a commonly recognized complication of chronic respiratory disease. Enhanced vasoconstriction, pulmonary vascular remodeling and in situ thrombosis contribute to the increased pulmonary vascular resistance observed in pulmonary hypertension associated with hypoxic lung disease" (p. 37). According to these researchers, the tissue factor pathway functions by regulating the deposition of fibrin deposition in response to acute and chronic vascular injury. In this study, White and his associates speculated that an inhibition of the tissue factor pathway would cause a reduction in the pathophysiologic parameters that are generally associated with hypoxiainduced pulmonary hypertension. To this end, White et al. used a chronic hypoxia-induced murine model of pulmonary hypertension through the use of mice that overexpress tissue factor pathway inhibitor through the smooth muscle specific promoter SM22 (TFPI[SM22]). According to White and his colleagues, "TFPI (SM22) mice have increased pulmonary TFPI expression compared to wild type (WT) mice. In WT mice, exposure to chronic hypoxia (28 days at 10% O2) resulted in increased systolic right ventricular and mean pulmonary arterial pressures, changes that were significantly reduced in TFPI (SM22) mice. Chronic hypoxia also resulted in significant pulmonary vascular muscularization in WT mice which was significantly reduced in TFPI (SM22) mice" (p. 37). Based on the pleiotropic effects of tissue factor pathway inhibitor, autocrine and paracrine mechanisms for these hemodynamic effects were taken into account in their analysis (White et al. 2009).

The results of this study showed that the TFPI (SM22) mice experienced less deposition of pulmonary fibrin than wild type mice at 3 days after being exposed to hypoxia consistent with antithrombotic effects of tissue factor pathway inhibitor (White et al. 2009). In addition, the TFPI (SM22) mice were found to have a substantial decrease in the number of proliferating (proliferating cell nuclear antigen positive) pulmonary vascular smooth muscle cells vs. The wild type mice congruent with in vitro findings (White et al. 2009). In sum, these researchers conclude that, "These findings demonstrate that overexpression of tissue factor pathway inhibitor results in improved hemodynamic performance and reduced pulmonary vascular remodeling in a murine model of hypoxia-induced pulmonary hypertension. This improvement is in part due to autocrine and paracrine effects of tissue factor pathway inhibitor overexpression" (White et al. 2009, p. 37).

Coagulation factors. According to a study by Kannan, Misra, Dvonch and Krishnakumar concerning the biologically plausible mechanistic pathways by which exposure to particulate matter may result in adverse perinatal outcomes, "Systemic alterations in rheologic factors, including blood coagulability and whole blood viscosity as a result of exposure to particulate matter, represent one of the potential mechanisms of particulate matter toxicity" (2006, p. 1636). Responses to exposure to particular matter could potentially elevate the presence of the proteins of the clotting cascade which represent a possibility for coagulation (Kannan et al. 2006). The results of a cross-sectional study performed by Pekkanen, Brunner, Anderson, Tiittanen and Atkinson (2000) identified mixed results concerning the relationship between particulate matter less than 10 microns in aerodynamic diameter and plasma fibrinogen; this relationship was shown to be significant during warm seasons only though. According to Kannan and her colleagues, "Other measurable biomarkers include factors VII-IX, fibrin D-dimer, and von Willebrand factor. Particulate matter exposures may also lead to changes in hemoglobin, platelets, and white blood cells, which may potentially contribute to the association between particulate matter and adverse fetal growth" (2006, p. 1637).

In addition, for women, high levels of oral estrogen in contraceptive pills can serve to increase the production of coagulation factors in the liver; this increased production of coagulation factors may result in the deterioration of coronary arteries (Orth-Gomer, Chesney & Wenger 1998). According to these researchers, "The current doses of postmenopausal estrogen-replacement therapy are much lower than those previously used in contraceptive pills. Consequently, dose-dependent procoagulant activity by alkylated estrogens has been proposed. It has also been reported that estrogens induce an increase in fibrinolytic activity by increasing levels of tissue plasminogen, and decreasing the levels of plasminogen activator-I (Orth-Gomer et al. 1998, p. 190).

The results of a comparative study of postmenopausal women using untreated or estrogen treated subjects determined that the levels of procoagulative factors such as fibrinogen, factor VII and PAI-1 were significantly higher among those women who were untreated women; the results of this study (Scarabin et al. 1993) suggest an increased coagulation and decreased fibrinolysis in the women who were not using estrogen treatment; likewise, a positive anticoagulative effect on coagulation factors was identified in the group of women who were using estrogen therapy (Scarabin et al. 1993). According to Orth-Gomer and her associates, "The overall balance between coagulation and fibrinolysis induced by estrogens favors a reduction in thrombosis. Even though some new studies showed a slight increase in thrombolembolic events, others could not show an association" (1998, p. 190).

Finally, the goal of a study by Karwat, Kumor and Chazan (2005) entitled, "Coagulation parameters associated with inhaled glucocorticosteroid therapy in stable asthma" was to determine if there are alterations in laboratory analyses that could provide the opportunity for researchers to identify coagulation disturbances associated with inhaled glucocorticoid therapy.

To achieve this goal, Karwat and his associates used a study group that was comprised of 26 patients diagnosed with stable asthma (n=5 male, 21 female) without risk factors of venous thrombosis, mean age 45 +/- 14 years. Of these 26 subjects, 15 were treated with 500-1000 mg beclomethasone on a daily regimen and 11 patients were administered a daily dose of 1000-2000 microg of beclomethasone (Karwat et al. 2005). The laboratory investigations included the following:

1. Hemoglobin concentration (Hb);

2. Hematocrit (Ht);

3. Platelet count (PLT);

4. Red blood cell count (E);

5. White blood cell count (L);

6. Percentage of eosinophils (Eos%);

7. Fibrinogen (Fib);

8. D-dimer concentration;

9. Activated partial thromboplastin time (APTT);

10. Prothrombin time (PT);

11. Prothrombin index (Quick); and,

12. INR and von Willebrand factor (vWF) (Karwat et al. 2005).

The results of this study found an increased concentration of D-dimers in four of the patients (or 15%), while the other investigated coagulation parameters were shown to be within the normal range. According to Karwat and his colleagues, "We found a significant correlation between the dose of inhaled glucocorticosteroid and the PT, prothrombin index and INR.

The results revealed no evidence of increased fibrinolytic activity in subjects with stable bronchial asthma treated with inhaled glucocorticosteroids" (2005, p. 528).

Tissue-type plasminogen activator (t-PA). As noted above, coagulation involves a number of mechanisms and different molecules. In this regard, Mukamal and Rimm report that, "Coagulation is a highly complex and dynamic process that involves numerous molecules. Thus, some proteins that circulate in the blood (e.g., fibrinogen and factor VII) tend to promote thrombosis, as part of the clotting cascade. Conversely, other molecules that act only locally (e.g., tissue-type plasminogen activator [t-PA]) can help dissolve newly formed thrombi, a process called fibrinolysis" (2001, p. 255). In addition, the process of coagulation can involve yet other cells and molecules which can function as part of the complex process of blood clot formation and dissolution (Mukamal & Rimm 2001).

As an example, Mukamal and Rimm note that, "Fibrinogen and other coagulation-promoting molecules activate platelets -- a type of blood cell that helps to seal off injured blood vessels -- and encourage platelet aggregation at the site of the plaque rupture. Again, this aggregation can enhance the blockage of the affected blood vessel. Meanwhile, other substances, such as plasminogen-activator-inhibitor 1 (PAI-1), temper the fibrinolytic process" (2001, p. 256). In the majority of instances, the final phase in the blockage of a coronary vessel and the beginning of a myocardial infarction is the formation of a new thrombus when an atherosclerotic plaque ruptures; therefore, the processes of atherosclerosis, inflammation, and thrombosis all serve to interact to produce clinical coronary heart disease (Mukamal & Rimm 2001).

According to Wilansky and Willerson, "In addition to atherosclerotic plaque fissuring or ulceration, other reasons for endothelial injury include flow shear stress, hypertension, immune complex deposition and complement activation, infection, and mechanical injury to the endothelium as it occurs with coronary artery angioplasty and after heart transplantation" (2002, p. 68). In addition, endothelial injury diminishes endothelium-derived relaxing factor (EDRF), or nitric oxide; prostaglandin I 2 (PGI 2); as well as tissue-type plasminogen activator (t-PA) (Wilanksy & Willerson 2002). Fibrin-selective thrombolytic agents such as tissue-type plasminogen activator posses a significantly higher affinity for fibrin-bound plasminogen compared to circulating plasminogen. In this regard, Wilanksy and Willerson note that, "They are 1000 times less potent than their nonselective counterparts in activating circulating plasminogen. The purported advantage of these agents is concentration of their activity at the site of interest without requiring systemic thrombolysis" (2002, p. 207).

Plasminogen-activator-inhibitor (PAI-1). According to Ma, Paek and Oh, "Plasminogen activator inhibitor (PAI)-1 is a major inhibitor of the fibrinolytic system. PAI-1 levels are markedly increased in asthmatic airways, and mast cells (MCs), a pivotal cell type in the pathogenesis of asthma, are one of the main sources of PAI-1 production" (2009, p. 37). The results of studies in recent years indicate that PAI-1 may contribute to the onset of asthma through regulation of airway remodelling, airway hyperresponsiveness, and allergic inflammation (Ma et al. 2009). In this regard, Ma and his associates report that, "The single guanosine nucleotide deletion/insertion polymorphism (4G/5G) at -675 bp of the PAI-1 gene is the major genetic determinant of PAI-1 expression. Plasma PAI-1 level is higher in people with the 4G/4G genotype than in those with the 5G/5G genotype" (2009, p. 37). In addition, the recent research has indicated a strong correlation between the 4G/5G polymorphism and the risk and the severity of developing asthma (Ma et al. 2009).

In addition, these researchers note that the respective levels of plasma IgE and PAI-1 and the severity of airway hyperresponsiveness are increased among asthmatic patients with the 4G/4G genotype compared to those patients with the 5G/5G genotype (Ma et al. 2009). Moreover, the PAI-1 promoter corresponding to the 4G allele results in increased transcription activity compared to the PAI-1 promoter with the 5G allele in stimulated mast cells (Ma et al. 2009). In sum, these researchers conclude that, "The molecular mechanism for the 4G allele-mediated higher PAI-1 expression is associated with greater binding of upstream stimulatory factor-1 to the E-box adjacent to the 4G site (E-4G) than to the E-5G. In summary, PAI-1 may play an important role in the pathogenesis of asthma. Further studies evaluating the mechanisms of PAI-1 action and regulation may lead to the development of a novel prognostic factor and therapeutic target for the treatment and prevention of asthma and other PAI-1-associated diseases" (Ma et al. 2009, p. 38).

The results of a recent study by Cosan, Kurt, Degirmenci, Kucukarabaci, Metintas, Kucuk, Gunes and Colak (2009) concerning plasminogen activator inhibitor type-1 gene 4G/5G polymorphism in Turkish adult patients with asthma are noteworthy. In this regard, Cosan and his associates analyzed asthmatic patients in the Turkish population to identify the frequency of 4G/5G polymorphism genotypes of plasminogen activator inhibitor type-1 gene with the goal of determining the function of this polymorphism in asthma development. To achieve this goal, Cosan and his colleagues obtained genomic DNA from 165 persons (98 patients with asthma; 67 healthy patients for a control group); the DNA so obtained was multiplied with polymerase chain reaction through the use of 4G and 5G allele-specific primers. Polymerase chain reaction products were analyzed using a charged coupled device camera with an exposure of 2% agarose gel electrophoresis and the results evaluated using a chi-square test (Cosan et al. 2009).

The results of this analysis determined that there were no statistically significant differences between the 98 patients with asthma and the 67 patients in the control group with regard to genotype distribution; the 4G allele frequency was shown to be 48% and 5G allele was 52% in the asthmatic patients, while there was precise 50-50% found in the control group (Cosan et al. 2009). The results of this study show that 4G/5G polymorphism genotypes of plasminogen activator inhibitor type-1 gene do not play a role in the development of asthma, at least in the segment of the Turkish population analyzed by these researchers (Cosan et al. 2009).

Epidermal growth factor. According to Loewenstein, the members of the growth factor (GF) family are characterized by a distinctive looped polypeptide chain, described by this author as "a stretch 30 to 40 amino acids long with three pairs of disulfide-bonded cysteines -- a sort of family insignia that is displayed in various repeats" (1999, p. 254). This distinctive looped polypeptide chain can be seen in Figure 2 below. As an example, Loewenstein notes that the precursor of the epidermal growth factor, proEGF, possesses nine repeats but proTGF? just has one. Other growth factors such as FGF and TGF? are cleavage products of these membrane-anchored precursor molecules; these growth factors function as messengers in hormonal communication; following their cleavage (Figure 2, see arrows), these growth factors are then secreted into the extracellular medium and ultimately attach themselves to specific receptors on target cells to which they communicate their growth-stimulating message (Loewenstein 1999). According to this researcher, "This is what was known to biochemists and so was the growth-stimulation effect on cells of the diffusible cleavage products. These products reach the responding cells via the intercellular medium or the blood; so they were appropriately classified as hormones (hence, the name growth factors)" Loewenstein 1999, p. 255).

Figure 2. The EGF insignia. Three representatives of the EGF family displaying, in various repeats, the clan motif -- a stretch of polypeptide chain puckered by disulfide bonding between three pairs of cysteines (the points of contact between the loops represent the three disulfide bonds). The arrows indicate where the membraneanchored molecule can be enzymatically cleaved off, an option exercised when the insignia information is used for hormonal, instead of syncretic, communication (the cleavage points in Delta are unknown). (M) Membrane; (O) outside medium; (I) cell interior. ProTGF?, proEGF, and Delta are all membrane glycoproteins; the first two are instrumental in controlling cellular growth in vertebrates and the last, in guiding nervous-system development in some invertebrates.

Source: Loewenstein 1999, p. 254

According to Flake, Anderson and Dixon, "Growth factors with mitogenic activity, such as transforming growth factor, basic fibroblast growth factor, epidermal growth factor, and insulin-like growth factor-I, are elevated in fibroids and may be the effectors of estrogen and progesterone promotion" (2003, p. 1037). To date, a number of growth factors and their receptors have been identified in both myometrium and leiomyomas; the growth factors that have been the focus of the most research include transforming growth factor (TGF)-[beta], bFGF, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF) (Flake et al. 2003).

The epidermal growth factor is mitogenic for the cells of myometrium as well as leiomyomas in tissue cultures; moreover, and potentially a unique aspect of EGF, is the growth factor's seemingly upregulation in fibroids by progesterone (Maruo, Centonze Bernasconi, Fazzuoli, Berta & Giordano 2000). The concentration of EGF mRNA in leiomyomas is comparable to that found in the myometrium during the follicular phase; however, it is substantially elevated in leiomyomas during the luteal phase, while the concentration in the myometrium remains relatively unchanged otherwise (Flake et al. 2003). In addition, since the mitotic activity of leiomyomas is at its peak during the luteal phase of the cycle, Flake and his colleagues suggest it is reasonable to conclude that this indicates that the production of EGF may be one way by which progesterone functions to stimulate mitotic activity in fibroids (Flake et al. 2003).

The mRNA for the EGF receptor has been detected in both myometrial and leiomyoma cells; while the levels of EGF receptors are not substantially higher in leiomyomas compared to those found in the myometrium and do not appear to be altered during the menstrual cycle, Flake and his associates point out that there is a significant decrease in the EGF-receptor levels in the leiomyomas but not in the myometrium of women who have been administered GnRH agonists before surgery (Flake et al. 2003).

Although more research is required in this area, the findings to date indicate that the EGF receptors in fibroids are more sensitive to regulation via the ovarian sex steroids compared to those found in the myometrium (Flake et al. 2003). Perhaps even more significantly, since the decrease of EGF receptor levels is related to reduction in the size of the fibroids as a consequence of the GnRH-agonist therapy, these findings also indicate that the impact of sex steroids on fibroid growth may be altered, at least partially, by EGF (Flake et al. 2003). In sum, these researchers note that, "It is of interest that in cultures of leiomyoma cells, estradiol augmented the expression of the EGF receptor, whereas progesterone increased the expression of EGF, suggesting to the authors that estradiol and progesterone may act in combination to stimulate proliferation in fibroids through the induction of EGF and its receptor" (Flake et al. 2003, p. 1038).

A study by Li, Stonehuerner, Devlin and Huang (2005) concerning the effects of zinc and vanadium on human bronchial epithelial cells notes that zinc is present everywhere in the natural environment, including ambient air. When humans are exposed to inordinately high levels of zinc, though, it represents a health threat. For instance, industrial workers who perform welding and smelting operations and inhale large concentrations of zinc oxide or zinc chloride can experience respiratory epithelial cell damage, inflammation, and acute injury (Li et al. 2005). According to these researchers, "Treatment of lung epithelial cells in vitro with zinc compounds enhanced inflammatory signaling and produced cytotoxicity and cell death" (Li et al. 2005, p. 1747).

While vanadium and zinc are members of completely different elemental classes in the periodic table, the two elements have a number of biologic properties in common. In this regard, Li and his associates note that, "Both metals are potent enhancers for phosphorylation of signaling proteins, including mitogen-activated protein kinase and epidermal growth factor receptors, and both increase Ras activity and interleukin-8 (IL8) release. Many of these effects may be attributed to the capability of these metals to inhibit protein tyrosine phosphatase activity. Both vanadium and zinc also inhibit metabolic activity of the cells" (2005, p. 1747). It is also possible for both zinc and vanadium to exist simultaneously in the ambient environment following their release from different emission sources (Li et al. 2005).

A timely study by Kassel, Schulte and Toews (2009) concerning the modulation of epidermal growth factor receptor binding to human airway smooth muscle cells by glucocorticoids and beta-2 adrenergic receptor agonists notes that, "Epidermal growth factor receptors are increased in airway smooth muscle in asthma, which may contribute to both their hyperproliferation and hypercontractility. Lysophosphatidic acid is a candidate pathologic agent in asthma and other airway diseases, and LPA up-regulates EGFRs in human airway smooth muscle cells" (p. 37). The goal of this study was to determine whether therapeutic glucocorticoids and/or beta-2 adrenergic receptor (beta2AR) agonists also affect epidermal growth factor receptors binding in human airway smooth muscle cells (Kassel et al. 2009). According to these researchers, "Exposure to glucocorticoids for 24-hour induced a two-fold increase in epidermal growth factor receptors binding similar to that with lysophosphatidic acid; fluticasone was markedly more potent than dexamethasone. The increase in epidermal growth factor receptors binding by glucocorticoids required 24-hour exposure, consistent with transcription-mediated effects" (2009, p. 37).

While the increase in epidermal growth factor receptors binding was blocked by the protein synthesis inhibitor cycloheximide for lysophosphatidic acid, fluticasone, and dexamethasone, Kassel and his colleagues also note that just lysophosphatidic acid caused a significant increase in epidermal growth factor receptor protein expression identified through immunoblotting. In addition, the beta2AR agonists isoproterenol, albuterol and salmeterol were found to cause a decrease in epidermal growth factor receptor binding in contrast to the increased binding caused by the glucocorticoids (Kassel et al. 2009). Moreover, beta2AR agonist effects were shown to be multiphasic, with an initial decrease being identified at 2-4-hour that was reversed by hour 6 and a second slightly greater decrease by hours 18-24 (Kassel et al. 2009). In the cells that were pretreated with glucocorticoids, Kassel and his colleagues note that the decrease in epidermal growth factor receptor binding by follow-up beta2AR treatment did not result in statistically significant changes; likewise, glucocorticoid up-regulation of epidermal growth factor receptor prevented additional increases by; ysophosphatidic acid as well. Comparable decreases by beta2AR agonists as well as increases by glucocorticoids were identified in HFL-1 lung fibroblasts (Kassel et al. 2009). In sum, these researchers conclude that, "These complex and opposing effects of clinically relevant glucocorticoids and beta2AR agonists on airway mesenchymal cell in epidermal growth factor receptor likely contribute to their overall therapeutic profile in the diseased airway" (Kassel et al. 2005, p. 37).

Finally, in their study, "Actions of Ethanol on Epidermal Growth Factor Receptor Activated Luteinizing Hormone Secretion," Hiney, Dearth, Srivastava, Rettori and Dees (2003) report that the epidermal growth factor receptor (EGF-R) is one of the members of the ErbB family of tyrosine kinase receptors that are activated by EGF and the transforming growth factor. According to these researchers, "Recent evidence has shown that these receptors are located on the glia and tanycytes lining the third ventricle in close contact with the luteinizing hormone-releasing hormone (LHRH) terminals and play a pivotal role in the cell to cell interactive mechanism by which glial cells regulate LHRH neuronal function" (Hiney et al. 2003, p. 809). In addition, Hiney and his associates note that EGF-R mRNA and protein levels in the medial basal hypothalamus are elevated during puberty; consequently, to the extent that this receptor is changed is the extent to which the onset of puberty is delayed (Hiney et al. 2003).

Hepatocyte growth factor. The protein, hepatocyte growth factor, serves to assist in the maintenance of cell life and skin barrier function without a concomitant increase in the risk of cancer after damage have been experienced due to ultraviolet light exposure (Tschachler 2003). According to Okunishi, Sasaki, Okasora, Nakagome, Imamura, Harada, Matsumoto, Tanaka, Yamamoto, Tabata and Dohi, "Hepatocyte growth factor (HGF) has an important role in many biological events such as angiogenesis and cell proliferation, as well as anti-fibrotic and anti-apoptotic effects" (2009, p. 91). In their study, "Intratracheal delivery of hepatocyte growth factor directly attenuates allergic airway inflammation in mice," these researchers determined that HGF serves to suppress antigen-induced immune responses in the airway through a suppression of dendritic cell functions using a HGF-producing plasmid vector (Okunishi et al. 2009).

In this study, Okunishi and his colleagues investigated whether delivery of the HGF protein in the lung suppresses allergic airway inflammation in a mouse model. In this regard, the researchers report that, "Generally, HGF is rapidly cleared from organs. So, to achieve the efficient delivery of HGF, we prepared a slow-releasing form by mounting recombinant human (rh) HGF protein in biodegradable gelatin hydrogels. BALB/c mice were immunized and then challenged with ovalbumin to induce eosinophilic airway inflammation" (p. 91).

One of the more significant -- and promising -- findings of this study was that a single intratracheal delivery of a very small amount of gelatin-coupled rhHGF (0.3 microg) prior to the inhalation of ovalbumin was found to significantly suppress eosinophilic airway inflammation; moreover,, cytokine production in thoracic lymph nodes and the antigen-presenting capacity of lung CD11c+ cells were also found to be diminished (Okunishi et al. 2009). By contrast, delivery of 1.0 microgram of rhHGF failed to demonstrate any significantly suppressive effect (Okunishi et al. 2009). The results of this study indicate that the controlled release of rhHGF protein can suppress antigen-induced allergic immune responses in the lung indicating that HGF representa a potential new therapeutic option for asthma (Okunishi et al. 2009).

In addition, researchers at the Centre de Recherches et d'Investigations Epidermiques et Sensorielles in Paris have found that hepatocyte growth factor helps maintain the skin's barrier function. One of the lead researchers in the project, Tschachler, reports that, "After cells have been exposed to a potentially toxic dose of ultraviolet light, they release IL-1 and TNF-alpha, which increases production of hepatocyte growth factor. In turn, hepatocyte growth factor seems to ensure that the cells remain alive and keep their place in the skin barrier, but they lose the capacity to further divide. This minimizes the risk for cancer development" (Tschachler 2003, 14). In addition, human saliva contains regulatory peptides such as transforming growth factor a2 and hepatocyte growth factor, as well as antimicrobials such as thiocyanate, and lysozymes (Colon & Colon 1999).

Yet another study by Ito, Takeda, Fujita, Kamada, Kato, Chiba, Yamaguchi, Ueki, Kayaba, Kanehiro and Chihara (2008) concerning the manner in which hepatocyte growth factor suppresses production of reactive oxygen species and the release of eosinophil-derived neurotoxin from human eosinophils holds some promise for identifying new approaches to the treatment of asthma. According to Ito and his associates, "Reactive oxygen species and eosinophilic granule proteins such as eosinophil-derived neurotoxin are known to damage bronchial tissue and cause airway hyperresponsiveness in asthma. Hepatocyte growth factor regulates various biological activities and is known to be a multifunctional factor" (2008, p. 331). In a previous parallel study, Ito and his colleagues determined that hepatocyte growth factor suppressed allergic airway inflammation and airway hyperresponsiveness in a murine model of asthma; to date, though, there has been a dearth of studies concerning the precise mechanism of the anti-allergic effect of that hepatocyte growth factor in asthma. In their study, Ito et al. examined the potential of recombinant that hepatocyte growth factor to regulate the production of reactive oxygen species as well as the release of eosinophil-derived neurotoxin from human eosinophils (Ito et al. 2008).

To achieve this goal, Ito and his associates obtained eosinophils from subjects with mild eosinophilia through the use of a modified CD16-negative selection; thereafter, these researchers analyzed the expression of CD69, an activation marker of eosinophils, on eosinophils, through the use of flow cytometry. In addition, reactive oxygen species production from eosinophils was examined through the use of luminol-dependent chemiluminescence, and eosinophil-derived neurotoxin release was gauged with the use of an enzyme-linked immunosorbent assay (Ito et al. 2008).

The results of this study showed that treatment with hepatocyte growth factor suppressed interleukin-5-induced upregulation of CD69 expression, the production of reactive oxygen species and eosinophil-derived neurotoxin release in human eosinophils. Based on these results, Ito and his associates indicate that in asthma, hepatocyte growth factor diminishes allergic airway inflammation and airway hyperresponsiveness through at least the suppression of the production of reactive oxygen and eosinophil-derived neurotoxin release from eosinophils (Ito et al. 2008).

Nitric oxide. As noted above, airway inflammation is a specific characteristic of asthma. According to Delfino and his associates, "The fractional concentration of nitric oxide in exhaled air (F[ENO]) is a noninvasive biomarker of airway inflammation and is higher in subjects with poorly controlled asthma. Therefore, exhaled air is potentially useful in epidemiologic field research to evaluate the impacts of air pollution on the inflammatory state of airways in children with asthma" (2006, p. 1736). Nitric oxide (NO) is produced endogenously in the airways from L-arginine by NO synthetase; there are two constituent isoforms and an inducible isoform involved in airway inflammation:

1. Inflammatory cytokines (e.g., tumor necrosis factor-[alpha]) increase the expression of inducible NO synthetase in airway epithelium. There is extensive evidence that F[ENO] is elevated in patients with untreated asthma and decreases with corticosteroid treatment; and,

2. There is also evidence that F[ENO] is correlated with eosinophilic inflammation in asthmatic patients, particularly in those sufferers who are atopic. For instance, after withdrawal of inhaled steroids, F[ENO] serves to accurately predict loss of asthma control (positive predictive value 80-90%) just as well as either eosinophils from induced sputum or bronchial hyperresponsiveness (Delfino et al. 2006, p. 1737).

According to Hill, Snider, Galbraith, Forst and Robbins, tumor necrosis factor-alpha (TNF-alpha) is released in inflammatory lung conditions, thereby elevating airway nitric oxide concentrations through the cytokine-mediated induction of nitric oxide synthase (NOS). These researchers report that, "Cardiopulmonary bypass creates an inflammatory state, characterized by the release of TNF-alpha that may result in lung injury following cardiopulmonary bypass" (1995, p. 1791). In their study, Hill and his associates measured plasma levels of TNF-alpha and interleukin-6 (IL-6) together with airway nitric oxide concentrations during cardiopulmonary bypass procedures and the impact of methylprednisolone on the respective levels of these inflammatory compounds.

To this end, 20 adult males who were previously scheduled for coronary artery bypass grafting (CABG) were anesthetized and assigned to randomized groups who were administered methylprednisolone at 1 gm intravenously 5 minutes prior to their CPB (Group S) and a group that was not administered methylprednisolone (Group N); the subjects' plasma levels of TNF-alpha and IL-6 were measured using an enzyme-linked immunosorbent assay (ELISA) and the airway nitric oxide concentration through the use of chemiluminescence (Hill et al. 1995). According to these researchers, "TNF-alpha was significantly (p < 0.05) increased at 30 min after the termination of CPB, while IL-6 was significantly (p < 0.05) increased at 50 min into CPB and 30 min after the end of CPB in Group N. As compared with controls in the same group and with Group S. At the same time intervals" (Hill et al. 1995, p. 1791).

A group of 10 subjects who were having their infrarenal aortic aneurysms repaired served as a control group for the study for their plasma levels of TNF-alpha; the control group did not experience any significant changes in their TNF-alpha concentrations at any point during the repair of their aneurysms. In Group N, airway nitric oxide concentrations were shown to be significantly increased (p < 0.01) versus Group S. At 5, 20, 35, and 50 minutes of the CPB procedure (Hill et al. 1991).

A study by Gruchalla, Sampson, Matsui, David, Gergen, Calatroni, Brown, Liu, Bloomberg, Chmiel, Kumar, Lamm, Smartt, Sorkness, Steinbach, Stone, Szefler and Busse emphasizes the high prevalence of asthma among inner-city adolescents and notes that more attention must be focused on evaluating those factors that can predict future asthma control when guidelines-based management is used. To this end, Gruchalla and his associates evaluated the function of fraction of exhaled nitric oxide in parts per billion, markers of allergic sensitization, airway inflammation, and measures of asthma severity in determining future risk of asthma symptoms and exacerbations in adolescents and young adults participating in the Asthma Control Evaluation study (2009).

The population for this ambitious study consisted of 546 inner-city residents, aged 12 through 20 years, who were diagnosed with persistent asthma; these subjects received an extensive evaluation upon admission to the study in order to identify predictors of future symptoms and exacerbations over the following 46 weeks; during the duration of the study, guidelines-based, optimal asthma management was provided to the subjects with baseline measurements including fraction of exhaled nitric oxide in parts per billion, total IgE, allergen-specific IgE, allergen skin test reactivity, asthma symptoms, lung function, peripheral blood eosinophils, and, for a subset, airway hyperresponsiveness and sputum eosinophils (Gruchalla et al. 2009).