Vet Res Commun (2012) 36:251–258 DOI 10.1007/s11259-012-9534-x

SHORT COMMUNICATION

Reaginic antibodies from horses with Recurrent Airway Obstruction produce mast cell stimulation G. Moran & H. Folch & C. Henriquez & A. Ortloff & M. Barria

Accepted: 1 August 2012 / Published online: 12 August 2012 # Springer Science+Business Media B.V. 2012

Abstract Reaginic antibodies (IgE and some IgG subclasses) and mast cells play important roles in the induction of type I immediate hypersensitivity reactions. These antibodies bind through their Fc fragment to high affinity receptors (FcεRI) present in the membrane of mast cells and basophils. The cross-linking of the receptor initiates a coordinated sequence of biochemical and morphological events that results in exocytosis of secretory granules containing preformed inflammatory mediators, secretion of newly formed lipid mediators, and secretion of cytokines. Previously, several studies have investigated the role of reaginic antibodies in the pathogenesis of Recurrent Airway Obstruction (RAO). However, whereas the immunological aspects of RAO have been extensively studied, the precise sequence of events involved in the pathogenesis remains not completely understood, and the role of IgE in this disease remains controversial. Therefore, in this study, several bioassays were conducted to determine whether reaginic antibodies from RAO-affected horses have the ability to activate mast cells. These bioassays involved measuring degranulation of rat peritoneal mast cells, activation of NF-κB and morphological changes in basophilic leukemia cells (RBL-2H3) following incubation with horse serum from RAO-affected horses that were sensitive and

G. Moran (*) Department of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, 567, Valdivia, Chile e-mail: [email protected] H. Folch : C. Henriquez : M. Barria Department of Immunology, Faculty of Medicine, Universidad Austral de Chile, 567, Valdivia, Chile A. Ortloff College of Veterinary Medicine, Universidad Catolica de Temuco, Temuco, Chile

insensitive to Aspergillus fumigatus (A. fumigatus) or from unaffected horses. Our results show that reaginic antibodies from horses sensitive to A. fumigatus were able to degranulate rat peritoneal mast cells. In additon, there was an increase in the activity of the transcription factor NF-κB in RBL-2H3 cells, and morphological changes were observed in these cells once cross-linking was produced. These findings were not found in horses not sensitive to A. fumigatus and healthy horses. These bioassays demonstrate the ability of reaginic antibodies to stimulate mast cells and indicate that these antibodies could be involved in the immunological mechanisms leading to RAO. Keyword RAO . Reaginic antibodies . Mast cells . Horses Abbreviations BALF Bronchoalveolar lavage fluid FCS Fetal calves´ serum HBSS Hank´s balanced salt solution NF-κB Nuclear factor- κB PBS Phosphate buffered saline RAO Recurrent airway obstruction

Introduction In general, airway inflammation involves the activation of antigen-specific inflammatory cells, the modulation of transcription factors and the release of inflammatory mediators (Barnes et al. 1998). Recurrent airway obstruction (RAO, “heaves”) is an asthma-like condition that develops in mature horses following stabling and exposure to dusty hay and straw (Robinson 2001). Affected horses respond to this exposure by developing airway bronchoconstriction, neutrophilic inflammation and airway hyper-responsiveness. The disease is

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characterized by pulmonary neutrophilia and excessive mucous production, resulting in reduced dynamic lung compliance and increased pulmonary resistance and pleural pressure excursions (Derksen et al. 1985; Jackson et al. 2004). Moreover, this disease is characterized by episodes of acute airway obstruction (crisis) followed by periods of disease remission (Robinson et al. 1996). Type I hypersensitivity, which is reaginic antibodymediated (mainly of the IgE and certain IgG subclasses) (Ishizaka and Ishizaka 1966) has been suggested to play a role in airway inflammation in RAO-affected horses (Halliwell et al. 1993; Schmallenbach et al. 1998; Eder et al. 2000; Eder et al. 2001; Curik et al. 2003; Künzle et al. 2007; Morán et al. 2010a, b). RAO in horses involves allergen-specific immunoglobulins of the IgE class bound to high-affinity Fcε receptors (FcεR) on the surface of basophils and mast cells present in the subepithelial layer of the airways (van der Haegen et al. 2005). Cross-linking of this receptor initiates a coordinated sequence of biochemical and morphological events that results in exocytosis of secretory granules containing histamine or other pre-formed inflammatory mediators; synthesis and secretion of newly formed lipid mediators, such as prostaglandins and leukotrienes; and synthesis and secretion of cytokines (Kobayashi et al. 1998) that in turn induce airway inflammation. Many mediators are released in RAO-affected horses, and it is clear that these mediators interact with each other. Mediators may act synergistically to enhance the effects of one another, or one mediator may modify the release or action of another mediator (Morán and Folch 2011). In the development of RAO, the activity of IgE antibodies is dependent upon genetic susceptibility, as well as the type, quantity, and time of environmental allergen exposure (Halliwell et al. 1993; Schmallenbach et al. 1998; Eder et al. 2000). Inhalation of mold and fungal antigens induces the inflammatory airway response in susceptible horses within 6 h after exposure (Gerber et al. 2004). Transcription factors are DNA-binding proteins that regulate the expression of inflammatory genes, including enzymes involved in the synthesis of inflammatory mediators, as well as protein and peptide mediators (Barnes et al. 1998). Inflammation associated with hypersensitivity results from the exaggerated expression of inflammatory genes, and a number of researchers have explored the mechanisms implicated in inflammatory gene induction (Barnes and Karin 1997; Barnes and Adcock 1998). Many transcription factors are cell-specific and are crucial for cell differentiation and the regulation of specific cellular processes, including proliferation and enzyme and cytokine expression. In animal models of airway diseases, such as atopic asthma, nuclear factor (NF)κB, activator protein-1 (AP-1), GATA-3, JunB and c-Maf play central roles in the control of airway inflammation (Finotto et al. 2001; Nguyen et al. 2003; Yamashita et al. 2007). Similarly, in horses with RAO exposed to moldy hay, airway

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epithelial cells exhibit increased expression of NF-κB, in particular the p65 homodimers (Bureau et al. 2000a, b; Saunders et al. 2001). Previously, several studies have investigated the role of reaginic antibodies in the pathogenesis of RAO (Halliwell et al. 1993; Schmallenbach et al. 1998; Eder et al. 2000, 2001; Curik et al. 2003; Künzle et al. 2007; Morán et al. 2010a, b). However, the role of IgE-mediated reactions in RAO remains unclear (Marti et al. 2008; Tahon et al. 2009; Wagner 2009). Today, various techniques are available to detect soluble IgE and the sensitization of mast cell or basophils in IgE horses (Wagner 2009). Most recently, Morán et al. (2010a, b) proposed a bioassay for reaginic antibody detection from horse serum of RAO-affected animals using RBL-2H3 cells (basophilic leukemia rat cells), which mediate immediate reactions, to determine the etiology of the disease. This technique involves measuring in vitro intracellular Ca2+ mobilization in RBL-2H3 cells following incubation with horse serum from affected animals and with one of the main RAO antigens. As described above, studies indicated that reaginic antibodies are involved in the immunological mechanisms leading to RAO. However, further investigations of the various relationships between reaginic antibodies and mast cells are required for a better understanding of the immunologic mechanisms involved during the response to RAO. Therefore, we performed several bioassays to determine whether reaginic antibodies from RAO-affected horses sensitive to A. fumigatus have the ability to activate mast cells.

Material and methods Horses and samples Twelve horses with clinical diagnoses of RAO (RAO herd) and eight negative controls (normal horses), ranging from 4 to 20 years old and 420 to 450 kg and of the Chilote and Chilean criollo breeds, were used in this work. The RAOaffected horses were evaluated for sensitivity to Aspergillus fumigatus (A. fumigatus), which was detected by a bioassay based on activation of RBL-2H3 cells, as previously described (Morán et al. 2010a, b). The animals were separated into three groups: control horses (n08), RAO sensitive (n0 6) and insensitive (n06) to A. Fumigatus. This study was conducted by avoiding animal distress and with the approval of the Bioethical Committee of the University. RAO was diagnosed after careful physical examination (including rectal temperature, heart rate, respiratory rate, evaluation of hydration, gut motility, and auscultation of heart and lung), hematology analysis, endoscopic inspection and bronchoalveolar lavage fluid (BALF) examination to establish a clinical score for RAO, as previously described (Robinson et al. 2000). It was also important to rule out

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other lung pathologies or parasitism. RAO-affected horses were demonstrated typical disease symptoms such as coughing, increased respiratory effort, tracheal mucus accumulation and increased neutrophils in the BALF. Prior to performing the bioassays, blood samples were collected by jugular venipuncture into sterile tubes. For preparation of serum, the tubes were incubated for 30 min at 37 °C and centrifuged for 7 min at 650 × g. In addition, all sera in this study were heated to 56 ° C as previously described (Prouvost-Danon et al. 1977). Later, sera were kept frozen at −20 °C until use.

Finally, the cells were fixed with 10 % formalin and absolute ethanol. The evaluation of the percentage of degranulated mast cells was done after staining with 0.1 % toluidine blue. Two hundred or more mast cells were counted. The percentage of degranulation was determined as previously described (Astorquiza et al. 1980). All determinations were carried out in duplicate, and the results are shown as the means of four experiments with different pools of mast cells.

Antigenic preparations of A. fumigatus

The activity of NF-κB in the RBL-2H3 cells after stimulation with antigen was determined by the Southwestern immunochemistry as previously described (Mezzano et al. 2001). RBL-2H3 cells were adjusted to a concentration of 106 cells/ ml in RPMI-1640 medium containing 10 % FCS plus antibiotics and separated by glass adherence on covered slides. Later, the cells were incubated with sera from horses and exposed to A. fumigatus protein in a similar manner as described above for peritoneal degranulation mast cells, as well as bioassay controls. Later, the cells were fixed with paraformaldehyde and treated with hydrogen peroxide to eliminate the endogenous peroxidases and to avoid non-specific reactions. Subsequently, the preparations were digested with 0.5 % pepsin (433 U/mg) in 1 M HCl and washed twice with Buffer 1 (10 mM HEPES, 40 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1 mM EDTA, 0.25 % BSA at pH 7.4) and with Buffer 2 (10 mM HEPES, 40 mM NaCl, 1 mM DTT, 10 mM EDTA, 0.25 % BSA at pH 7.4). Then, the cell preparations were dehydrated in alcohol and subsequently incubated with Buffer 1 containing 0.5 μg/ ml poli (di-dc), and the double strain preparations were washed with Buffer 1 Plus (0.03 % Tween 20, 10 mM maleic acid, 0.15 %mM NaCl, pH 7.4), then incubated with blocking buffer (0.1 X saline sodium citrato, 0.1 % SDS 1:10 in washing buffer). Later, the cells were incubated with streptavidinperoxidase (1:250). Finally, the peroxidase substrate aminoethylcarbazole (AEC) was added to reveal the reaction. The reaction was stopped by incubation with Hank’s balanced salt solution. Finally, the cells were mounted in glycerol.

The culture of A. fumigatus was carried out in two steps. First, the fungus was cultured in peptone-agar for 7 days at 37 °C and then in malt broth for an additional 7 days. The resulting fungus was used for soluble protein extraction. To extract the proteins from A. fumigatus cultures, the fungus was blended and subsequently sonicated (Ultrasonic Homogenizer, series 4710 Cole Parmer, USA). The resulting suspension was centrifuged at 12,500 × g for 10 min, and the supernatants containing the soluble proteins were kept at −20 °C until use. RBL-2H3 cell line Rat basophilic leukemia cells (RBL-2H3) were grown in RPMI-1640 medium (Sigma, USA) supplemented with 10 % fetal calf serum (FSC) plus antibiotics. The cells were maintained at 37 °C in a 5 % CO2 humidified chamber. RBL-2H3 cells were used just before they reached confluency in culture. Mast cell degranulation test Fresh mast cells were obtained from 3-month-old Sprague– Dawley rats by intraperitoneal lavage with 10 ml of heparinized Hank’s balanced salt solution. Cells were pooled and washed twice and resuspended at the required concentration in RPMI-1640 medium containing 10 % FCS. For degranulation tests, rat peritoneal mast cells were separated by glass adherence on covered slides and were adjusted to a concentration of 106 cells/ml in RPMI-1640 medium containing 10 % FCS plus antibiotics as previously described (Astorquiza and Fernández 1999). Later, the cells were incubated for 2 h at 37 °C in the presence of 200 μl of serum obtained from horses affected by RAO that were positive and negative to A. fumigatus and control horses. Cells incubated without horse serum were used as the negative control. Each group received 77 μg of soluble protein obtained from A. fumigatus via incubation at 37 °C for 15 min. Hank’s solution was used as the incubation control. In addition, to establish a positive control characterized by total degranulation, the cells were induced with 0.5 μM digitonin (Sigma Chemical Co, USA).

NF-κB activity determination in RBL-2H3 cells

Electron microscopy To visualize the morphological changes in RBL-2H3 cells after antigen exposure (performed in a manner similar to the method described above for peritoneal degranulation mast cells) by electron microscopy, treated RBL-2H3 cells and controls were initially fixed in 4 % buffered formalin. Thereafter, the cells were transferred to half-strength modified Karnovsky's fixative before two washes with 0.2 M sodium cacodylate and postfixation in 2 % osmium tetroxide reduced with 2.5 % potassium ferrocyanide. After fixation, the cells were washed in 0.2 M sodium cacodylate and

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The results from the quantitation of the percentage of degranulated peritoneal mast cells were assessed by ANOVA. When significant, the means were compared using Tukey's Multiple Comparison Test. The results are reported as the means ± SDs. A value of P<0.05 was considered significant.

fumigtus contained increased activity of the transcription factor NF-κB when they were challenged with this fungus (Fig. 3a). The increased activity of this transcription factor was not observed in RAO-affected horses that were insensitive to A. fumigatus and in healthy animals (Fig. 3b). In addition, when we analyzed the morphological changes in RBL-2H3 cells using the protocol described above, in cells sensitized with sera from horses with RAO that were positive for A. fumigatus and challenged with Aspergillus proteins, we observed a loss of electrodense granules and abundant prolongations of the plasma membrane after the exocytosis of the granules, in contrast with the intact the cells exposed to sera from horses insensitive to A. fumigatus or healthy animals exposed to the antigen Fig. 4.

Results

Discussion

To determine whether reaginic antibodies specific for A. fumigatus in horses are capable of stimulating mast cells and can be used as in vitro bioassays to determine the antigen responsible for inducing RAO in a given individual animal, sera from 12 horses with an RAO diagnosis (7 positive and 5 negative for A. fumigatus) and 8 sera samples obtained from clinically normal controls were used to sensitize peritoneal rat mast cells and RBL-2H3 cells, as described above. When the population of peritoneal mast cells was sensitized with sera from RAO-affected horses positive for A. fumigatus, the soluble proteins from this fungus induced a clear increase in the degranulation percentages of these cells (Fig. 1a). In contrast, degranulation of the peritoneal mast cells was not observed when these cells were sensitized with sera from RAO-affected horses negative for A. fumigatus and control horse sera (Fig. 1b). For the percentage of degranulation specified (Fig. 2), the percentage of RAO-affected horses sensitized to A. fumigatus was statistically significant (P<0.001) compared with RAOaffected horses not sensitized to A. fumigatus and other control groups. Similarly, RBL-2H3 cells sensitized with sera from horses affected with RAO and sensitive to A.

It is well known that mast cells are key effector cells of the Th2- and IgE-associated immune response; that is, in an appropriately sensitized host, an encounter with a specific antigen will induce mast cells to undergo activation dependent on IgE and the Fcε-receptor I (FcεRI) and/or certain classes of IgG and FcγRII-depend activation. The mast cell activation contributes to the local or systemic immunological response (Galli et al. 2005). Mast cell activation in horses has been hypothesized to contribute to host resistance to certain parasites (Suter and Fey 1981; Dowdall et al. 2004; Hill et al. 2007) or to drive the pathology of allergic disorders, such as anaphylaxis (Wagner 2009) and RAO (Halliwell et al. 1993; Schmallenbach et al. 1998; Eder et al. 2000, 2001; Curik et al. 2003; van der Haegen et al. 2005; Künzle et al. 2007; Morán et al. 2010a, b). In this work, as in our previous study, we used the generic name of reaginic antibodies to encompass all immunoglobulin molecules that can sensitize peritoneal rat mast cells and RBL-2H3 cells (Morán et al. 2010a, b). IgE is greatly responsible for mediating hypersensitivity; however, other subclasses of IgG have been proposed to mediate mast cell degranulation in humans (Okayama et al. 2001, 2003),

dehydrated using a graded ethanol series before infiltration and embedding in Spurr's epoxy resin. Thick sections were cut, mounted on 150 mesh copper grids, stained with 6 % methanolic uranyl acetate and Reynold's lead citrate, and examined using a LEO 905E (Zeiss) transmission electron microscope at 60 V accelerating voltage. Data analysis

Fig. 1 Rat peritoneal mast cell degranulation test. a) A representative image of positive degranulation obtained from mast cells sensitized with sera from RAO-affected horses sensitive to A. fumigatus. b) A representative image of negative degranulation obtained from mast cells sensitized with sera from RAOaffected horses insensitive to A. fumigatus or with sera from healthy horses; Scale: 20 μm

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% degranulation

80

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Controls horses RAO sensitive to A.fumigatus RAO non-sensive to A. fumigatus

**

60 40 20 0

Fig. 2 Percentage of rat peritoneal mast cells that degranulated in response to sensitized sera from control horses and RAO-affected horses that were sensitive or insensitive to A. fumigatus (** P<0.01)

mice (Latour et al. 1992; Katz and Lobell 1995) and horses (Hellberg et al. 2006). In our experiments, it is unknown which immunoglobulin isotype is mediating the mast cell activation. However, the fact that the sensitizing capacity of the horse serum disappeared after being heated to 56 °C for 30 min is consistent with the thermosensitivity of the IgE molecule (Prouvost-Danon et al. 1977), and this characteristic is generally absent in the IgG subclasses. Based on these facts, we hypothesize that IgE immunoglobulins play important roles in these in vitro bioassays. The technique of mast cell degranulation has been extensively used to study IgE-mediated anaphylaxis (Astorquiza et al. 1980; Astorquiza and Fernández 1999). Using this method, we showed that reaginic antibodies of RAO-affected horses, in combination with specific antigen, have the ability to stimulate rat peritoneal mast cells, as we observed a clear degranulation of these cells. These results suggest that type I hypersensitivity plays a role in the development of RAO. However, although the immunological aspects of RAO have been extensively studied, the precise pathogenic mechanisms

remain not well understood. Moreover, the involvement of an IgE-mediated mechanism in the pathogenesis of RAOaffected horses remains unclear and is still controversial (Marti et al. 2008; Wagner 2009). Wagner (2009) suggested that more recent studies do not confirm IgE-mediated pathogenesis of RAO. The same author also argued that RAOaffected horses display chronic inflammatory disease, with some indication for the involvement of a delayed type of hypersensitivity mechanism. However, these results suggest that reaginic antibodies, which mediate immediate hypersensitivity, could be involved in the immunological mechanisms leading to the development of RAO. In addition, as mentioned previously, several studies have investigated the role of reaginic antibodies in the pathogenesis of RAO (Halliwell et al. 1993; Schmallenbach et al. 1998; Eder et al. 2000, 2001; Curik et al. 2003; Künzle et al. 2007; Morán et al. 2010a, b), further demonstrating the significant role of these antibodies in the development of this disease. RBL-2H3 cells have been widely used by many groups for an extremely broad range of applications, from degranulation studies and investigation of mast cell stabilizers to identifying the physical–chemical properties of the FcεR and its interaction with the cytoskeleton (Kulczycki et al. 1974; Fewtrell and Metzger 1980; Maeyama et al. 1986; Robertson et al. 1986; Ikawati et al. 2001). RBL-2H3 cells present the unquestionable advantage of easy cultivation; hence, a large number of homogeneous cells can be easily obtained (Tkaczyk and Gilfillan 2001). These cells respond with degranulation following cross-linking of their IgE-bound FcεRI by multivalent allergens, and they release of a range of preformed and newly synthesized mediators that evoke a potent immune allergic response. Successful studies examining human FcεRI-IgE binding were performed in RBL-2H3 cells (Tkaczyk and Gilfillan 2001). In addition, Morán et al. (2010a, b) were able to demonstrate the ability of these cells to bind horse reaginic

Fig. 3 NF-κB activity determination in RBL-2H3 cells by Southwestern blot analysis. a) A representative blot showing positive activation of NF-κB in RBL-2H3 cells exposed to sera from RAOaffected horses sensitive to A. fumigatus. b) A representative blot showing negative activation of NF-κB in RBL-2H3 cells exposed to sera from RAO-affected horses not sensitive to A. fumigatus or to sera from healthy horses; Scale: 20 μm

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Fig. 4 a) A representative electron microscopy image of RBL-2H3 cells exposed to sera from RAO-affected horses sensitive to A. fumigatus. b) A representative image of cells exposed to sera from RAOaffected horses insensitive to A. fumigatus or to sera from healthy horses. Note the loss of electrodense granules and abundant prolongations of the plasma membrane (arrow) after the exocytosis of the granules in contrast with the intact cells exposed to sera from nonantigen-sensitive and control horses; Scale: 3 μm

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antibodies. These cells are activated once their Fc receptors are occupied and crosslinked. Ca2+ mobilization occurs as a consequence of this activation (Kim et al. 1997; Ali et al. 2001; Ching et al. 2001). Using this method, Morán et al. (2010b) demonstrated that out of 20 cases of RAO-positive horses arriving at a hospital facility, 15 % of the cases were caused by Faenia rectivirgula. In similar studies, using this in vitro bioassay, Morán et al. (2010a) demonstrated that 30 % of thirty RAO-affected horses tested positive for A. fumigatus. Moreover, the bioassay described above enabled us to form groups of animals in this study (Morán et al. 2010a, b). The NF-κB family is composed of five structurally related DNA-binding proteins, p50, p52, p65/RelA, c-Rel/Rel, and RelB (Baldwin 1996; Chen et al. 1996). The most common form of NF-κB is a heterodimer composed of the p50 and p65 subunits. It was reported that the DNA-binding activity of NFκB plays an important role in the regulation of the rapid and transient transcriptional activation of the inflammatory genes via the FcεRI in RBL-2H3 cells (Pelletier et al. 1998). In this study, we showed that the IgE antibodies of RAO-affected horses that were sensitive to A. fumigatus produced a clear increase in the activity of NF-κB. This result demonstrates the ability of antibodies from horses to bind and stimulate RBL2H3 cells in the presence of specific antigen, and these results are similar to those reported in other studies (Morán et al. 2010a, b). In addition, in RAO-affected horses exposed to moldy hay, airway epithelial cells exhibit increased expression of NF-κB, and in particular the p65 homodimers (Bureau et al. 2000a, b; Saunders et al. 2001). Other studies reported two cases of horses with RAO with a high percentage of eosinophils in the BALF; in both horses, immunohistochemistry studies confirmed high p65 homodimer activity in the bronchial cells (Ortloff et al. 2011). Other studies indicated that the

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effect of exposure to moldy hay in RAO-affected horses is at least partly mediated by an increase in AP-1 binding activity in the airways. Prolonged allergen exposure results in the upregulation of cyclic AMP response element binding protein (CREB) and the downregulation of AP-1 (Couetil et al. 2006). These findings suggest that the transcription factors NF-κB, AP-1 and CREB play an important role in the modulation of airway inflammation in horses with RAO. As mentioned above, the cross-linking of IgE-receptor complexes has been shown to provide a necessary and sufficient signal to trigger the process of cellular degranulation. These events lead to morphological changes in RBL-2H3 cells when activated (Robertson et al. 1986). In this study, we showed that reaginic antibodies in RAO-affected horses sensitive to A. fumigatus were able to produce obvious morphological changes in RBL-2H3 cells, while these changes were not observed in the other groups of animals. These findings further demonstrate the role of reaginic antibodies from RAOaffected horses in mediating hypersensitivity. In summary, these results suggest that the bioassays utilized in this study have the ability to detect serum reaginic antibodies specific for a particular antigen. Our results indicate that reaginic antibodies could be involved in the immunological mechanisms leading to the development of RAO. Moreover, these studies may provide the impetus for further studies focusing on the delineation of how the signaling pathways of other activating or synergizing receptors on mast cells are integrated in the regulation of cellular activation in RAO-affected horses. Further investigations of the various aspects of reaginic antibodies and mast cells (receptors and/or pathways) are required for a better understanding of the mechanisms involved during the immune response to RAO and might provide a basis for the development of new

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strategies for the treatment of this disease and other allergy diseases in equines. Acknowledgments This work was supported by FONDECYT Nº 11100196 (Conicyt- Chilean Government). Conflict of interest statement None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper.

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