Atopic asthma is a complex disease process that has a significant social, personal and economic burden across all ages. Leukotriene-receptors are involved in the cascade of inflammation that may result in symptoms of atopy and asthma. Two leukotriene receptors have been identified in the lung. The cysteinyl leukotriene receptor 1 and cysteinyl leukotriene receptor 2 genes (i.e., CYSLTR1 and CYSLTR2) have been sequenced, and a number of single nucleotide polymorphisms (SNPs) within these genes have been identified.The purpose of this study was to: (1) Determine the relationship between CYSLTR1 genotypes, CYSLTR2 genotype, atopy, elevated IgE level, and eosinophilia, (2) Determine the relationship between CYSLTR1 genotypes, CYSLTR2 genotype, asthma, and atopic asthma, and (3) Determine the degree of interaction between CYSLTR2 genetic variation and gender in atopic asthma.Nested within two sub-studies of the Tucson Epidemiological Study of Airway Obstructive Disease (TESAOD) study, a prospective longitudinal cohort, 853 individuals were entered into this study. Study criteria included Non-Hispanic white adults, who consented to genetic testing in the two sub-studies. Tagging SNPs of the CYSLTR1 and CYSLTR2 genes were genotyped. Serum IgE status and eosinophilia were obtained from existing dataset. Questionnaires collected in the parent study were used to obtain demographic and clinical data.SNP rs321006 in the CYSLTR1 gene was associated with atopy among Non-Hispanic white women. Assuming a recessive model, among female Non-Hispanic white adults, the odds of having rs321073 CC genotype was 5.82 times higher among those with atopic asthma than those without atopic asthma. No gene by gender interaction was found between SNP of interest in CYSLTR2 and atopic asthma. Genetic association of SNPs rs321006 with atopy and rs321073 with atopic asthma are novel findings to date.Implications for nurses, clinicians, and scientists include better understanding of associations of these genetic variations with asthma, atopy, and atopic asthma that can generate further inquiry into other mechanisms of atopic asthma. These novel genetic associations with atopy and atopic asthma may have the potential for personalized medicine that might afford patients with appropriate treatment based on their genotype.

Cysteinyl leukotriene receptor 2, a G-protein coupled receptor known to be expressed and functional on human platelets. However, it seems that upon ligand activation the cysteinyl leukotriene receptor 2 activates a variety of signaling pathways in multiple cell types among different species. Previously, a former laboratory member Vrinda Kulkarni found cysteinyl leukotriene receptor 2 to be expressed on the surface of adult zebrafish thrombocytes. In this work I studied the characteristics of aggregation in adult zebrafish thrombocytes with the knockdown of cysteinyl leukotriene receptor 2. I used a newly developed knockdown method to study the function of cysteinyl leukotriene receptor 2. Knockdown of the cysteinyl leukotriene was confirmed using RT-PCR results showed p=.001, reduced sell surface level of expression of the cysteinyl leukotriene receptor 2 results showed that p=.002. I found that the knockdown of cysteinyl leukotriene receptor 2 results in prothrombotic thrombocytes by using flow cytometry p=.0001.

Montelukast (ML) is primarily an antagonist of type 1 cysteinyl leukotriene receptors (CysLT1R), an activity which underpins its therapeutic efficacy in bronchial asthma. However, ML has also been reported to be useful in the treatment of acute and chronic inflammatory disorders of both infective and non-infective origin in which CysLTs are unlikely to be the predominant mediators of harmful inflammatory responses. These include conditions such as chronic obstructive pulmonary disease and cystic fibrosis in which the neutrophil is believed to be the primary offender, suggesting that ML may possess neutrophil-targeted, CysLT1R-independent mechanisms of anti-inflammatory activity. Accordingly, the laboratory research presented in this thesis was designed with the primary objectives of characterizing possible CysLT1R-dependent and – independent neutrophil-targeted anti-inflammatory activities of ML in vitro, and consisted of 3 phases. These were investigation of: i) the effects of the CysLTs, LTC4 and LTD4 (in the absence and presence of ML) on mobilization of intracellular Ca2+ stores, generation of reactive oxygen species (ROS) and release of primary and secondary granule proteinases; ii) the effects of ML on a series of pro-inflammatory activities of neutrophils following activation of the cells with the chemoattractants FMLP and platelet-activating factor (PAF); and iii) the interactive, anti-inflammatory effects on neutrophils of ML in combination with the long-acting beta-2 agonist, formoterol. In addition to the aforementioned activities, measurement of the production and expression of CR3, as well as generation of inositol triphosphate (IP3), cyclic AMP, and activities of the enzymes cAMP- and cGMP-phosphodiesterases (PDEs) in isolated neutrophil cytosol and membrane fractions, were also included. The following assays were used: i) chemiluminescence procedures for the detection of ROS; ii) a colourimetric procedure for the detection of elastase; iii) ELISA procedures for the detection of the matrix metalloproteinases (MMPs) 8- and -9, LTB4, and cyclic AMP; iv) fura-2-based spectrofluorimetry and a radiometric procedure for monitoring cytosolic Ca2+ fluxes; v) flow cytometry for CR3; and vi) radioassays for IP3 and activity of cAMP- and cGMP-PDEs. Exposure of neutrophils to LTD4, but not LTC4, activated a very modest and transient increase in cytosolic Ca2+, but failed to initiate the generation of ROS or release of elastase or MMP-8. However, brief pre-treatment with either LTC4 or LTD4 sensitized the cells for increased production of ROS and release of granule proteinases following activation with FMLP, which was partially attenuated by inclusion of ML. In the second part of the study, pre-treatment of neutrophils with ML, at therapeutically relevant concentrations, resulted in dose-related inhibition of the FMLP- or PAF-activated generation of ROS and LTB4, as well as the release of elastase, with the former being unaffected by an inhibitor of 5-lipoxygenase (MK886), compatible with a CysLT1R-independent mechanism of anti-inflammatory activity. From a mechanistic perspective, these interactions of ML with neutrophils were associated with accelerated clearance of Ca2+ from the cytosol of the cells which could not be attributed to inhibition of production of IP3, but was, however, associated with increased levels of cAMP, apparently as a consequence of non- specific inhibition of cyclic nucleotide phosphodiesterases. In the third part of the study, combining ML with formoterol caused (in most cases) additive inhibitory effects on the generation of ROS and LTB4, release of granule proteinases, as well as expression of CR3, which again were associated with elevations in cAMP and interference with Ca2+ mobilization. In conclusion, ML appears to attenuate neutrophil activation by CysLT1R-dependent and –independent mechanisms. In the case of the former by interfering with the modest sensitizing (priming) interactions of LTC4 and LTD4 with neutrophils, and in the latter by inhibition of PDEs, leading a to sustained elevation in cAMP, resulting in rapid clearance of Ca2+ from the cytosol and decreased uptake of the cation from the extracellular milieu. / Thesis (PhD)--University of Pretoria, 2011. / Immunology / Unrestricted