In vitro bioassay to detect reaginic antibodies from the serum of horses affected with Recurrent Airway Obstruction

Abstract

In horses, Recurrent Airway Obstruction (RAO) is an allergic disease that involves IgE mediated Type I Hypersensitivity responses. The development of this type of allergy involves a series of events that begins with reaginic antibodies, mainly IgE and some IgG subclasses. These reaginic antibodies bind with high affinity, via the Fc portion, to FcepsilonRI receptors on the membrane of mast cells and basophils. Once bound, environmental allergens cross-link the antibodies, which results in mast cell degranulation leading to the production of histamine and other chemical mediators that act together to induce airway inflammation. RAO-affected horses present with coughing, respiratory distress, airway obstruction and poor performance. The aspect of the RAO has been extensively studied, yet the precise sequence of events is still not well understood. Therefore, this study proposes a bioassay for reaginic antibody detection from horse serum of RAO-affected individuals, in order to determine the etiology of disease, which mediate immediate type reactions. The technique involves measuring in vitro calcium mobilization in RBL-2H3 cells following incubation with horse serum from affected or unaffected horses and one of the RAO antigens (Aspergillus fumigatus). The results presented here demonstrate that 30% of RAO-affected horses react positively in this in vitro bioassay, whereas unaffected horses do not. This bioassay may facilitate further research on RAO and other allergic diseases in horses.

Development of an <em>in vitro</em> model system for studying the interaction of <em>Equus</em> <em>caballus</em> IgE with its high-affinity receptor Fc&#949;RI

“…Therefore it is always essential to run mouse IgE positive controls alongside the experiment (Figure 1), and to maintain a large frozen stock of secreting cells. Other research work, such as 20 have indicated that equine RAO is not related to IgE mediation, which is why some discrepancy can be seen with other publishers such as Moran et al 31 when using total equine serum to test to Ca 2+ influx from RAO suffering horses using unspecific reaginic antibodies, for which different antibodies would be responsible for the cellular Ca 2+ influx.…”

Detection of reaginic antibodies against Faenia rectivirgula from the serum of horses affected with Recurrent Airway Obstruction by an in vitro bioassay

“…Ca 2+ mobilization in RBL-2H3 cells Intracellular Ca 2+ mobilization in RBL-2H3 cells was monitored by change in the fluorescence intensity of fura-2AM-loaded cells as previously described (Morán et al 2010). RBL-2H3 cells were adjusted to a concentration of 10 6 cells/ml in RPMI-1640 medium containing 10% FCS plus antibiotics and incubated for 2 hours at 37°C in the presence of 200 μl of serum obtained from RAO-positive and negative horses.…”

“…In horses, type I hypersensitivity has been investigated, suggesting that this reaction might be involved in immune-mediated diseases such as summer eczema (Larsen et al 1988;Ungar-Waron et al 1990), urticaria, and head shaking (Mansfield et al 1998), as well as RAO (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 2010).…”

“…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;Eder et al 2001;Curik et al 2003;Künzle et al 2007;Morán et al 2010). 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 with IgE horses (Wagner 2009). Most recently, Morán et al (2010) proposed a biossay for reaginic antibody detection from horse serum of RAO-affected individuals by using RBL-2H3 cells, which mediate immediate type reactions, in order to determine the etiology of disease. This technique involves measuring in vitro intracellular Ca 2+ mobilization in basophilic leukemia cells (RBL-2H3) following incubation with horse serum from affected individuals and with one of the RAO antigens.…”