Michael O Daines

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Scholarly Contributions

Chapters

• Daines, M. O., & Phan, H. (2018). Allergic Rhinitis. In Pharmacotherapy: Principles and Practice. 5th Ed.(pp 967-982). McGraw-Hill.

Journals/Publications

• Daines, M., Zhu, L., Pereira, R., Zhou, X., Bondy, C., Pryor, B. M., Zhou, J., & Chen, Y. (2020). Alternaria induces airway epithelial cytokine expression independent of protease-activated receptor. Respirology (Carlton, Vic.).
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  A novel fungal allergen, Alternaria (Alt), has been previously shown to associate with the pathogenesis of allergic rhinitis and bronchial asthma, particularly in arid and semi-arid regions. Airway epithelial cells are among the first to encounter Alt, and epithelial cytokine production and subsequent airway inflammation are early events in the response to Alt exposure. However, the underlying mechanism is unclear. As protease-activated receptor 2 (PAR2) has been implicated in most of the Alt-induced biological events, we investigated the regulation of airway inflammation and epithelial cytokine expression by PAR2.
• Guilbert, T. W., Bacharier, L. B., Mauger, D. T., Phipatanakul, W., Szefler, S. J., Boehmer, S., Beigelman, A., Fitzpatrick, A. M., Jackson, D. J., Baxi, S. N., Benson, M., Burnham, C. D., Cabana, M. D., Castro, M., Chmiel, J. F., Covar, R., Daines, M., Gaffin, J. M., Gentile, D. A., , Holguin, F., et al. (2019). Challenges in assessing the efficacy of systemic corticosteroids for severe wheezing episodes in preschool children. The Journal of allergy and clinical immunology, 143(5), 1934-1937.e4.
• Hiranrattana, A., Stern, D. A., Guerra, S., Halonen, M., Wright, A. L., Daines, M., Martinez, F. D., & Morgan, W. J. (2018). sensitisation at age 6 years is associated with subsequent airway hyper-responsiveness in non-asthmatics. Thorax, 73(12), 1170-1173.
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  In the non-selected birth cohort Tucson Children's Respiratory Study, early sensitisation to was associated with increased airway hyper-responsiveness (AHR) into adult life among non-asthmatics. The increase in AHR was of a similar magnitude to that seen for sensitised asthmatics and was primarily evident among those who were overweight or obese. In contrast, there was no significant association between early sensitisation to aeroallergens other than and AHR among non-asthmatics. Why this group of sensitised individuals without asthma demonstrated increased AHR of a magnitude similar to asthmatics is unknown and requires further investigation.
• Morris, C. R., Mauger, D. T., Suh, J. H., Phipatanakul, W., Sheehan, W. J., Moy, J. N., Paul, I. M., Szefler, S. J., Jackson, D. J., Fitzpatrick, A. M., & , N. I. (2018). Glutathione and arginine levels: Predictors for acetaminophen-associated asthma exacerbation?. The Journal of allergy and clinical immunology, 142(1), 308-311.e9.
• Yee, M. C., Nichols, H. L., Polley, D., Mahmoud, S., Pal, K., Lee, K., Wilson, E. H., Daines, M. O., Hollenberg, M. D., Boitano, S. A., & DeFea, K. A. (2018). Protease-activated Receptor-2 Signaling through beta-Arrestin-2 Mediates Alternaria Alkaline Serine Protease-induced Airway Inflammation. Am J Physiol Lung Cell Mol Physiol. doi:10.1152/ajplung.00196.2018
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  Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similar to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor, protease-activated receptor-2 (PAR2), to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR2 signaling contributed to asthma symptoms via a PAR2/beta-arrestin signaling axis, to identify the protease activity responsible for PAR2 signaling and determine if protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR2/beta-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR2(-/-) and beta-arrestin-2(-/-) mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type mice, but not PAR2(-/-) or beta-arrestin-2(-/-) mice. Protease was isolated from Alternaria preparations and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria Alkaline Serine Protease (AASP), was sufficient to fully activate PAR2 signaling and induce beta-arrestin-2-dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR2/beta-arrestin signaling axis. Thus, beta-arrestin-biased PAR2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.
• Yee, M. C., Nichols, H. L., Polley, D., Saifeddine, M., Pal, K., Lee, K., Wilson, E. H., Daines, M. O., Hollenberg, M. D., Boitano, S., & DeFea, K. A. (2018). Protease-activated receptor-2 signaling through β-arrestin-2 mediates Alternaria alkaline serine protease-induced airway inflammation. American journal of physiology. Lung cellular and molecular physiology, 315(6), L1042-L1057.
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  Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR signaling contributed to asthma symptoms via a PAR/β-arrestin signaling axis, identify the protease activity responsible for PAR signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR/β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR and β-arrestin-2 mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR or β-arrestin-2 mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR signaling and induce β-arrestin-2-dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR/β-arrestin signaling axis. Thus, β-arrestin-biased PAR antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.
• Addison, K. J., Morse, J., Robichaud, A., Daines, M. O., & Ledford, J. G. (2017). A Novel System to Test Bronchodilators. Journal of infectious pulmonary diseases, 3(1).
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  The incidence and severity of asthma continue to rise worldwide. β-agonists are the most commonly prescribed therapeutic for asthma management but have less efficacy for some subsets of asthmatic patients and there are concerns surrounding the side effects from their long-term persistent use. The demand to develop novel asthma therapeutics highlights the need for a standardized approach to effectively screen and test potential bronchoprotective compounds using relevant animal models. Here we describe a validated method of testing potential therapeutic compounds for their fast-acting efficacy during the midst of an induced bronchoconstriction in a house dust mite challenged animal model.
• Polley, D. J., Mihara, K., Ramachandran, R., Vliagoftis, H., Renaux, B., Saifeddine, M., Daines, M. O., Boitano, S., & Hollenberg, M. D. (2017). Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 47(7), 946-960.
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  Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized.
• Polley, D., Mihara, K., Ramachandran, R., Vliagoftis, H., Renaux, B., Saifeddine, M., Daines, M. O., Boitano, S., & Hollenberg, H. (2017). Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2 (PAR2). Clinical and Experimental Allergy, Accepted for publication in 2017.
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  BACKGROUND: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES: We aimed to identify, isolate and characterize the trypsin-like proteinases in a German cockroach allergen extract used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50); (2) its amino acid sequence and (3) its ability to activate calcium signaling and/or ERK1/2 phosphorylation via PAR2. METHODS: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signaling. FINDINGS: Each of the three serine proteinase-activity-based probe-labelled enzymes isolated were biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57 to 71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signaling via PAR2. CONCLUSIONS AND RELEVANCE: We have identified three distinct allergen proteinases from the German cockroach that may play different roles for allergen-sensitization in vivo via PAR2 and may represent attractive therapeutic targets for asthma.
• Fitzpatrick, A. M., Jackson, D. J., Mauger, D. T., Boehmer, S. J., Phipatanakul, W., Sheehan, W. J., Moy, J. N., Paul, I. M., Bacharier, L. B., Cabana, M. D., Covar, R., Holguin, F., Lemanske, R. F., Martinez, F. D., Pongracic, J. A., Beigelman, A., Baxi, S. N., Benson, M., Blake, K., , Chmiel, J. F., et al. (2016). Individualized therapy for persistent asthma in young children. The Journal of allergy and clinical immunology, 138(6), 1608-1618.e12.
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  Phenotypic presentations in young children with asthma are varied and might contribute to differential responses to asthma controller medications.
• Sheehan, W. J., Mauger, D. T., Paul, I. M., Moy, J. N., Boehmer, S. J., Szefler, S. J., Fitzpatrick, A. M., Jackson, D. J., Bacharier, L. B., Cabana, M. D., Covar, R., Holguin, F., Lemanske, R. F., Martinez, F. D., Pongracic, J. A., Beigelman, A., Baxi, S. N., Benson, M., Blake, K., , Chmiel, J. F., et al. (2016). Acetaminophen versus Ibuprofen in Young Children with Mild Persistent Asthma. The New England journal of medicine, 375(7), 619-30.
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  Studies have suggested an association between frequent acetaminophen use and asthma-related complications among children, leading some physicians to recommend that acetaminophen be avoided in children with asthma; however, appropriately designed trials evaluating this association in children are lacking.
• Sherwood, C. L., Daines, M. O., Price, T. J., Vagner, J., & Boitano, S. (2014). A highly potent agonist to protease-activated receptor-2 reveals apical activation of the airway epithelium resulting in Ca2+-regulated ion conductance. American journal of physiology. Cell physiology, 307(8), C718-26.
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  The airway epithelium provides a barrier that separates inhaled air and its various particulates from the underlying tissues. It provides key physiological functions in both sensing the environment and initiating appropriate innate immune defenses to protect the lung. Protease-activated receptor-2 (PAR2) is expressed both apically and basolaterally throughout the airway epithelium. One consequence of basolateral PAR2 activation is the rapid, Ca(2+)-dependent ion flux that favors secretion in the normally absorptive airway epithelium. However, roles for apically expressed PAR2 activation have not been demonstrated, in part due to the lack of specific, high-potency PAR2 ligands. In the present study, we used the newly developed PAR2 ligand 2at-LIGRLO(PEG3-Pam)-NH2 in combination with well-differentiated, primary cultured airway epithelial cells from wild-type and PAR2 (-/-) mice to examine the physiological role of PAR2 in the conducting airway after apical activation. Using digital imaging microscopy of intracellular Ca(2+) concentration changes, we verified ligand potency on PAR2 in primary cultured airway cells. Examination of airway epithelial tissue in an Ussing chamber showed that apical activation of PAR2 by 2at-LIGRLO(PEG3-Pam)-NH2 resulted in a transient decrease in transepithelial resistance that was due to increased apical ion efflux. We determined pharmacologically that this increase in ion conductance was through Ca(2+)-activated Cl(-) and large-conductance K(+) channels that were blocked with a Ca(2+)-activated Cl(-) channel inhibitor and clotrimazole, respectively. Stimulation of Cl(-) efflux via PAR2 activation at the airway epithelial surface can increase airway surface liquid that would aid in clearing the airway of noxious inhaled agents.
• Boitano, S., Flynn, A. N., Sherwood, C. L., Schulz, S. M., Hoffman, J., Gruzinova, I., & Daines, M. O. (2011). Alternaria alternata serine proteases induce lung inflammation and airway epithelial cell activation via PAR2. American journal of physiology. Lung cellular and molecular physiology, 300(4), L605-14.
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  Allergens are diverse proteins from mammals, birds, arthropods, plants, and fungi. Allergens associated with asthma (asthmagens) share a common protease activity that may directly impact respiratory epithelial biology and lead to symptoms of asthma. Alternaria alternata is a strong asthmagen in semiarid regions. We examined the impact of proteases from A. alternata on lung inflammation in vivo and on cleaving protease-activated receptor-2 (PAR(2)) in vitro. A. alternata filtrate applied to the airway in nonsensitized Balb/c mice induced a protease-dependent lung inflammation. Moreover, A. alternata filtrate applied to human bronchial epithelial cells (16HBE14o-) induced changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), consistent with PAR(2) activation. These effects were blocked by heat inactivation or by serine protease inhibition of A. alternata filtrates, and mimicked by PAR(2) specific ligands SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2), but not the PAR(1)-specific ligand TFLLR-NH(2). Desensitization of PAR(2) in 16HBE14o- cells with 2-furoyl-LIGRLO-NH(2) or trypsin prevented A. alternata-induced [Ca(2+)](i) changes while desensitization of PAR(1), PAR(3), and PAR(4) with thrombin had no effect on A. alternata-induced Ca(2+) responses. Furthermore, the Ca(2+) response to A. alternata filtrates was dependent on PAR(2) expression in stably transfected HeLa cell models. These data demonstrate that A. alternata proteases act through PAR(2) to induce rapid increases in human airway epithelial [Ca(2+)](i) in vitro and cell recruitment in vivo. These responses are likely critical early steps in the development of allergic asthma.
• Muralidhar, A., Borbon, I. A., Esharif, D. M., Ke, W., Manacheril, R., Daines, M., & Erickson, R. P. (2011). Pulmonary function and pathology in hydroxypropyl-beta-cyclodextin-treated and untreated Npc1⁻/⁻ mice. Molecular genetics and metabolism, 103(2), 142-7.
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  Lung dysfunction is an important part of the pathology of the neurodegenerative disorder, Niemann-Pick C1 (NPC1). We have studied the pulmonary disease in the Npc1(NIH/NIH) mouse model. On histology, we find large numbers of alveolar foamy macrophages but no alveolar proteinosis. Lung weight as percent of body weight was markedly increased; using the flexiVent small animal ventilator (SCIREQ, Inc.), we find inspiratory capacity, elastance and hysterisivity to be increased while resistance was not changed. Cholesterol measurements show a doubling of lung cholesterol levels. Collagen is also increased. Treatment of Npc1(-/-) mice with hydroxypropyl-β-cyclodextrin (HPBCD), despite efficacious effects in brain and liver, results in little difference from age-matched controls (using a CNS-expressed transgene to extend the life expectancy of the Npc1(-/-) mice) for these variables.
• Sivaprasad, U., Askew, D. J., Ericksen, M. B., Gibson, A. M., Stier, M. T., Brandt, E. B., Bass, S. A., Daines, M. O., Chakir, J., Stringer, K. F., Wert, S. E., Whitsett, J. A., Le Cras, T. D., Wills-Karp, M., Silverman, G. A., & Khurana Hershey, G. K. (2011). A nonredundant role for mouse Serpinb3a in the induction of mucus production in asthma. The Journal of allergy and clinical immunology, 127(1), 254-61, 261.e1-6.
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  Asthma is a major public health burden worldwide. Studies from our group and others have demonstrated that SERPINB3 and SERPINB4 are induced in patients with asthma; however, their mechanistic role in asthma has yet to be determined.
• Chen, W., Tabata, Y., Gibson, A. M., Daines, M. O., Warrier, M. R., Wills-Karp, M., & Hershey, G. K. (2008). Matrix metalloproteinase 8 contributes to solubilization of IL-13 receptor alpha2 in vivo. The Journal of allergy and clinical immunology, 122(3), 625-32.
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  IL-13 receptor alpha2 (IL-13R alpha 2) is a high-affinity receptor for IL-13, a central mediator of allergic asthma. It acts predominantly as a decoy receptor but can also contribute to IL-13 responses under certain conditions. IL-13R alpha 2 exists in soluble and membrane forms, which can both bind IL-13 and modulate its activity. Yet the proteolytic processes that contribute to the generation of soluble IL-13R alpha 2 are largely unknown.
• Marsh, R. A., Lucky, A. W., Walsh, T. J., Pacheco, M. C., Rinaldi, M. G., Mailler-Savage, E., Puel, A., Casanova, J. L., Bleesing, J. J., Filippi, M. D., Williams, D. A., Daines, M. O., & Filipovich, A. H. (2008). Cutaneous infection with Metarhizium anisopliae in a patient with hypohidrotic ectodermal dysplasia and immune deficiency. The Pediatric infectious disease journal, 27(3), 283-4.
• Daines, M. O., Chen, W., Tabata, Y., Walker, B. A., Gibson, A. M., Masino, J. A., Warrier, M. R., Daines, C. L., Wenzel, S. E., & Hershey, G. K. (2007). Allergen-dependent solubilization of IL-13 receptor alpha2 reveals a novel mechanism to regulate allergy. The Journal of allergy and clinical immunology, 119(2), 375-83.
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  Allergic sensitization affects half of western populations and often precedes the development of allergic disorders including asthma. Despite the critical role of allergens in the pathogenesis of these disorders, little is known about how allergens modulate the immune response. IL-13 receptor alpha2 (IL-13Ralpha2) is a decoy receptor for IL-13.
• Daines, M. O., Tabata, Y., Walker, B. A., Chen, W., Warrier, M. R., Basu, S., & Hershey, G. K. (2006). Level of expression of IL-13R alpha 2 impacts receptor distribution and IL-13 signaling. Journal of immunology (Baltimore, Md. : 1950), 176(12), 7495-501.
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  IL-13, a critical cytokine for allergic inflammation, exerts its effects through a complex receptor system including IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2. IL-4Ralpha and IL-13Ralpha1 form a heterodimeric signaling receptor for IL-13. In contrast, IL-13Ralpha2 binds IL-13 with high affinity but does not signal. IL-13Ralpha2 exists on the cell surface, intracellularly, and in soluble form, but no information is available regarding the relative distributions of IL-13Ralpha2 among these compartments, whether the compartments communicate, and how the relative expression levels impact IL-13 responses. Herein, we investigated the distribution of IL-13Ralpha2 in transfected and primary cells, and we evaluated how the total level of IL-13Ralpha2 expression impacted its distribution. Our results demonstrate that the distribution of IL-13Ralpha2 is independent of the overall level of expression. The majority of the IL-13Ralpha2 protein existed in intracellular pools. Surface IL-13Ralpha2 was continually released into the medium in a soluble form, yet surface expression remained constant supporting receptor trafficking to the cell surface. IL-13Ralpha2 inhibited IL-13 signaling proportionally to its level of expression, and this inhibition could be overcome with high concentrations of IL-13.
• Tabata, Y., Chen, W., Warrier, M. R., Gibson, A. M., Daines, M. O., & Hershey, G. K. (2006). Allergy-driven alternative splicing of IL-13 receptor alpha2 yields distinct membrane and soluble forms. Journal of immunology (Baltimore, Md. : 1950), 177(11), 7905-12.
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  IL-13 is a key mediator of allergic inflammation. Its diverse functions are mediated by a complex receptor system including IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2. IL-4Ralpha and IL-13Ralpha1 form a high-affinity signaling heterodimer. IL-13Ralpha2 binds IL-13 with high affinity and has been found to exist in membrane and soluble forms. Soluble IL-13Ralpha2 has been postulated as a critical endogenous modulator of IL-13 responses. However, the mechanism of generation for the soluble form remains unclear. We present the initial study that a mechanism for generation of the soluble form is alternative splicing and that alternative splicing yields a distinct form of soluble IL-13Ralpha2. We found that several mouse organs expressed two IL-13Ralpha2 transcripts, the 1152-bp transcript encoding the full-length protein and the 1020-bp transcript lacking exon10, which encodes the transmembrane region. Deletion of exon 10 (DeltaEx10) caused a frameshift resulting in a different amino acid sequence from position 327 to position 339 and early termination. Constructs encoding both splice variants were transfected into WEHI-274.1 cells. Transfectants expressing the full-length transcript had IL-13Ralpha2 on the cell surface but produced minimal soluble IL-13Ralpha2 in the supernatants. In contrast, transfectants expressing the DeltaEx10 transcript displayed no membrane IL-13Ralpha2 but secreted high levels of soluble IL-13Ralpha2 capable of inhibiting IL-13 signaling. Both variants bound IL-13, but the DeltaEx10 variant displayed approximately 2-fold increase in IL-13 binding activity. Expression of the two IL-13Ralpha2 transcripts was differentially regulated in vivo in an experimental allergic asthma model. Thus, alternatively spliced variants of IL-13Ralpha2 may have a distinct biologic function in vivo.
• Boesch, R. P., Daines, M., Kaul, A., Cotton, R., & Amin, R. (2005). Lymphoproliferative disorder of the airway of an adolescent without immunodeficiency. International journal of pediatric otorhinolaryngology, 69(11), 1591-4.
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  Epstein-Barr virus (EBV) is a ubiquitous viral pathogen in humans that has a unique ability to immortalize B-cells. In immunosuppressed individuals, EBV can produce non-neoplastic lymphoproliferative disorders involving various organs. We describe a case report of EBV-associated lymphoproliferative disorder in an immunocompetent 14-year-old male. The case provides a description of EBV-associated lymphoproliferation affecting the upper and lower respiratory tract. The massive submucosal infiltration of B-cells in the lingual tonsils, trachea, and bronchi produced near-complete airway obstruction resulting in tracheotomy. Neither surgical reduction of lingual tonsils nor treatment with steroids was of benefit. An extensive evaluation for immunodeficiency and neoplasia was normal. Treatment with rituximab, an anti-CD20 antibody, resulted in near-complete resolution of the infiltrative process, sufficient to allow decannulation. Rituximab is a treatment option for the rare occurrence of non-neoplastic, EBV-associated, lymphoproliferative disorders.
• Guajardo, J. R., Schleifer, K. W., Daines, M. O., Ruddy, R. M., Aronow, B. J., Wills-Karp, M., & Hershey, G. K. (2005). Altered gene expression profiles in nasal respiratory epithelium reflect stable versus acute childhood asthma. The Journal of allergy and clinical immunology, 115(2), 243-51.
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  Asthma is the most common chronic disease of childhood and has a strong genetic component.
• Chen, W., Daines, M. O., & Hershey, G. K. (2004). Methylation of STAT6 modulates STAT6 phosphorylation, nuclear translocation, and DNA-binding activity. Journal of immunology (Baltimore, Md. : 1950), 172(11), 6744-50.
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  Signal transducer and activator of transcription 6 is a transcription factor important for the development of Th2 cells and regulation of gene expression by IL-4 and IL-13. It has been reported that STAT1 activity is regulated by methylation of a conserved arginine residue in the N-terminal domain. Methylation of STAT6 has not yet been explored. We observed methylation of STAT6 in cells transfected with wild-type STAT6, but not in cells transfected with Arg(27)Ala mutant, confirming that STAT6 is methylated on Arg(27). Transfectants expressing mutant Arg(27)Ala STAT6 displayed markedly diminished IL-4-dependent STAT6 phosphorylation and nuclear translocation, and no STAT6 DNA-binding activity compared with wild-type STAT6 transfectants. To confirm this, the experiments were repeated using inhibitors of methylation. In the presence of methylation inhibitors, STAT6 methylation was diminished, as was phosphorylation of STAT6 and STAT6 DNA-binding activity. Thus, methylation is a critical regulator of STAT6 activity, necessary for optimal STAT6 phosphorylation, nuclear translocation, and DNA-binding activity. Furthermore, methylation of STAT6 has distinct effects from those reported with STAT1.
• Chen, W., Daines, M. O., & Khurana Hershey, G. K. (2004). Turning off signal transducer and activator of transcription (STAT): the negative regulation of STAT signaling. The Journal of allergy and clinical immunology, 114(3), 476-89; quiz 490.
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  Signal transducer and activator of transcription (STAT) proteins are a group of transcription factors that transmit signals from the extracellular milieu of cells to the nucleus. They are crucial for the signaling of many cytokines that are mediators of allergic inflammation. Considerable information is known about the activation of STATs and their role in gene transcription; comparably much less is known about how STAT signaling is regulated. Because STATs are critical for the induction of many genes crucial for the allergic cascade and immune host defense, understanding the regulation of these molecules will provide novel insights into allergic and immunodeficiency disorders and will likely identify new targets for therapeutic interventions. This review will summarize the current understanding of the regulation of STAT signaling, emphasizing recent observations.
• Daines, M. O., Andrews, R. P., Chen, W., El-Zayaty, S. A., & Hershey, G. K. (2003). DNA binding activity of cytoplasmic phosphorylated Stat6 is masked by an interaction with a detergent-sensitive factor. The Journal of biological chemistry, 278(33), 30971-4.
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  Signal transducer and activator of transcription (Stat) 6 is vital to interleukin (IL)-4 and IL-13 responses and the generation of Th2 immunity. We investigated the cellular location of phosphorylated Stat6 and Stat6 DNA binding activity in A201.1 murine B cells and primary splenocytes. Phosphorylated Stat6 was present in cytoplasmic and nuclear extracts from IL-4-treated cells. Confocal microscopy confirmed the presence of phosphorylated Stat6 in the cytoplasm of IL-4-treated cells. In contrast, Stat6 DNA binding activity was present in nuclear extracts, but not in cytoplasmic extracts. Thus, cytoplasmic extracts from IL-4-stimulated cells were devoid of Stat6 DNA binding activity despite the presence of phosphorylated Stat6. Addition of cytoplasmic extracts to nuclear extracts did not inhibit Stat6 DNA binding present in the nuclear extracts. Detergent treatment restored Stat6 DNA binding activity in cytoplasmic extracts of IL-4-stimulated cells. Thus, DNA binding activity of cytoplasmic phosphorylated Stat6 is masked by a factor dissociable by detergent treatment.

Presentations

• Kiela, P. R., West, J. J., Murray, M., Galindo, K., Mayate-Ortiz, L., Laubitz, D., Daines, M. O., & Rice, S. A. (2019, May). Assessing the Causes, Epidemiology and Under-diagnosis of Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) in Arizonans. 4th Annual ABRC Research Conference, Phoenix, AZ May 2, 2019. Phoenix, AZ: Arizona Biomedical Research Commission.