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1.  IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells 
PLoS ONE  2014;9(8):e103263.
The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.
doi:10.1371/journal.pone.0103263
PMCID: PMC4139276  PMID: 25141009
2.  The bitter taste receptor T2R38 is an independent risk factor for chronic rhinosinusitis requiring sinus surgery 
Background
The bitter taste receptor T2R38 was recently described to play a role in upper airway innate mucosal defense. When activated by bacterial quorum-sensing molecules, T2R38 stimulates the ciliated epithelial cells to produce nitric oxide (NO), resulting in bactericidal activity and an increase in mucociliary clearance (MCC). Polymorphisms within the T2R38 gene (TAS2R38) confer variability in activation of the receptor yielding dramatic differences in upper airway defensive responses (NO production and accelerated MCC) to microbial stimulation based on genotype. Our objective was to determine whether the non protective TAS2R38 polymorphisms, which render the receptor inactive, correlate with medically recalcitrant chronic rhinosinusitis (CRS) necessitating surgical intervention in the context of known risk factors, and thus identify whether the TAS2R38 genotype is an independent risk factor for patients undergoing functional endoscopic sinus surgery (FESS).
Methods
CRS patients undergoing primary FESS were prospectively genotyped for TAS2R38. Chi-square analysis was performed on the genotype distribution with respect to other risk factors, including allergies, asthma, nasal polyposis, aspirin sensitivity, diabetes, and smoking exposure.
Results
Seventy primary FESS patients were genotyped demonstrating a statistically significant skewing from the expected distribution of the general population (p < 0.0383). CRS patients with a particular polymorphism seemed less likely to have allergies, asthma, nasal polyposis, aspirin sensitivity, and diabetes, but this did not demonstrate statistical significance.
Conclusion
Our investigation suggests that TAS2R38 genotype is an independent risk factor for patients failing medical therapy, necessitating surgical intervention.
doi:10.1002/alr.21253
PMCID: PMC4082560  PMID: 24302675
innate immunity; antimicrobial; nitric oxide; mucociliary clearance; endoscopic sinus surgery; genetics
3.  Bitter and sweet taste receptors regulate human upper respiratory innate immunity 
The Journal of Clinical Investigation  2014;124(3):1393-1405.
Bitter taste receptors (T2Rs) in the human airway detect harmful compounds, including secreted bacterial products. Here, using human primary sinonasal air-liquid interface cultures and tissue explants, we determined that activation of a subset of airway T2Rs expressed in nasal solitary chemosensory cells activates a calcium wave that propagates through gap junctions to the surrounding respiratory epithelial cells. The T2R-dependent calcium wave stimulated robust secretion of antimicrobial peptides into the mucus that was capable of killing a variety of respiratory pathogens. Furthermore, sweet taste receptor (T1R2/3) activation suppressed T2R-mediated antimicrobial peptide secretion, suggesting that T1R2/3-mediated inhibition of T2Rs prevents full antimicrobial peptide release during times of relative health. In contrast, during acute bacterial infection, T1R2/3 is likely deactivated in response to bacterial consumption of airway surface liquid glucose, alleviating T2R inhibition and resulting in antimicrobial peptide secretion. We found that patients with chronic rhinosinusitis have elevated glucose concentrations in their nasal secretions, and other reports have shown that patients with hyperglycemia likewise have elevated nasal glucose levels. These data suggest that increased glucose in respiratory secretions in pathologic states, such as chronic rhinosinusitis or hyperglycemia, promotes tonic activation of T1R2/3 and suppresses T2R-mediated innate defense. Furthermore, targeting T1R2/3-dependent suppression of T2Rs may have therapeutic potential for upper respiratory tract infections.
doi:10.1172/JCI72094
PMCID: PMC3934184  PMID: 24531552
4.  GABA and Glutamate are not colocalized in mossy fiber terminals of developing rodent hippocampus 
Brain research  2012;1474:40-49.
It has been hypothesized that, in the developing rodent hippocampus, mossy fiber terminals release GABA together with glutamate. Here, we used transgenic glutamic acid decarboxylase-67 (GAD67)-GFP expressing mice and multi-label immunohistochemistry to address whether glutamatergic and GABAergic markers are colocalized. We demonstrate that in the dentate gyrus, interneurons positive for GABA/GAD are sparsely distributed along the edge of the hilus, in a different pattern than the densely packed granule cells. Co-staining for synaptophysin and vesicular glutamate transporter1 (VGLUT1) in postnatal day 14 brain sections from both mice and rats identified mossy fiber terminals as a group of large (2 – 5μm in diameter) VGLUT1-positive excitatory presynaptic terminals in the stratum lucidum of area CA3a/b. Furthermore, co-staining for synaptophysin and vesicular GABA transporter (VGAT) revealed a group of small-sized (~0.5μm in diameter) inhibitory presynaptic terminals in the same area where identified mossy fiber terminals were present. The two types of terminals appeared to be mutually exclusive, and showed no colocalization. Thus, our results do not support the hypothesis that GABA is released as a neurotransmitter from mossy fiber terminals during development.
doi:10.1016/j.brainres.2012.07.042
PMCID: PMC3695404  PMID: 22842523
granule cell; vesicular glutamate transporter; vesicular GABA transporter; immunofluorescence; synaptic button
5.  Surfactants in the management of rhinopathologies 
Background:
Surfactants are a class of amphiphilic surface active compounds that show several unique physical properties at liquid–liquid or liquid–solid surface interfaces including the ability to increase the solubility of substances, lower the surface tension of a liquid, and decrease friction between two mediums. Because of these unique physical properties several in vitro, ex vivo, and human trials have examined the role of surfactants as stand-alone or adjunct therapy in recalcitrant chronic rhinosinusitis (CRS).
Methods:
A review of the literature was performed.
Results:
The data from three different surfactants have been examined in this review: citric acid zwitterionic surfactant (CAZS; Medtronic ENT, Jacksonville FL), Johnson's Baby Shampoo (Johnson & Johnson, New Brunswick NJ), and SinuSurf (NeilMed Pharmaceuticals, Santa Rosa, CA). Dilute surfactant therapy shows in vitro antimicrobial effects with modest inhibition of bacterial biofilm formation. In patients with CRS, surfactants may improve symptoms, most likely through its mucolytic effects. In addition, surfactants have several distinct potential benefits including their ability to improve an irrigant's penetration of the nonoperated sinus and their synergistic effects with antibiotics. However, surfactants potential for nasal irritation and possible transient ciliotoxicity may limit their use.
Conclusion:
Recent data suggest a possible therapeutic role of surfactants in treating rhinopathologies associated with mucostasis. Further investigation, including a standardization of surfactant formulations, is warranted to further elucidate the potential benefits and drawbacks of this therapy.
doi:10.2500/ajra.2013.27.3873
PMCID: PMC3901440  PMID: 23710951
Biofilms; cilia; contamination; mucociliary clearance; nasal irrigation; sinusitis; surfactants; surface tension
6.  Heterogeneity of Gene Expression in Murine Squamous Cell Carcinoma Development—The Same Tumor by Different Means 
PLoS ONE  2013;8(3):e57748.
Transformation is a complex process, involving many changes in the cell. In this work, we investigated the transcriptional changes that arose during the development of squamous cell carcinoma (SCC) in mice. Using microarray analysis, we looked at gene expression during different stages in cancer progression in 31 mice. By analyzing tumor progression in each mouse separately, we were able to define the global changes that were common to all 31 mice, as well as significant changes that occurred in fewer individuals. We found that different genes can contribute to the tumorigenic process in different mice, and that there are many ways to acquire the malignant properties defined by Hanahan and Weinberg as “hallmarks of cancer”. Eventually, however, all these changes lead to a very similar cancerous phenotype. The finding that gene expression is strongly heterogeneous in tumors that were induced by a standardized protocol in closely related mice underscores the need for molecular characterization of human tumors and personalized therapy.
doi:10.1371/journal.pone.0057748
PMCID: PMC3601100  PMID: 23526950
7.  T2R38 taste receptor polymorphisms underlie susceptibility to upper respiratory infection 
The Journal of Clinical Investigation  2012;122(11):4145-4159.
Innate and adaptive defense mechanisms protect the respiratory system from attack by microbes. Here, we present evidence that the bitter taste receptor T2R38 regulates the mucosal innate defense of the human upper airway. Utilizing immunofluorescent and live cell imaging techniques in polarized primary human sinonasal cells, we demonstrate that T2R38 is expressed in human upper respiratory epithelium and is activated in response to acyl-homoserine lactone quorum-sensing molecules secreted by Pseudomonas aeruginosa and other gram-negative bacteria. Receptor activation regulates calcium-dependent NO production, resulting in stimulation of mucociliary clearance and direct antibacterial effects. Moreover, common polymorphisms of the TAS2R38 gene were linked to significant differences in the ability of upper respiratory cells to clear and kill bacteria. Lastly, TAS2R38 genotype correlated with human sinonasal gram-negative bacterial infection. These data suggest that T2R38 is an upper airway sentinel in innate defense and that genetic variation contributes to individual differences in susceptibility to respiratory infection.
doi:10.1172/JCI64240
PMCID: PMC3484455  PMID: 23041624
8.  Pharmacologic Activation of the Innate Immune System to Prevent Respiratory Viral Infections 
Drugs that can rapidly inhibit respiratory infection from influenza or other respiratory pathogens are needed. One approach is to engage primary innate immune defenses against viral infection, such as activating the IFN pathway. In this study, we report that a small, cell-permeable compound called 5,6-di-methylxanthenone-4-acetic acid (DMXAA) can induce protection against vesicular stomatitis virus in vitro and H1N1 influenza A virus in vitro and in vivo through innate immune activation. Using the mouse C10 bronchial epithelial cell line and primary cultures of nasal epithelial cells, we demonstrate DMXAA activates the IFN regulatory factor-3 pathway leading to production of IFN-β and subsequent high-level induction of IFN-β–dependent proteins, such as myxovirus resistance 1 (Mx1) and 2′,5′-oligoadenylate synthetase 1 (OAS1). Mice treated with DMXAA intranasally elevate mRNA/protein expression of Mx1 and OAS1 in the nasal mucosa, trachea, and lung. When challenged intranasally with a lethal dose of H1N1 influenza A virus, DMXAA reduced viral titers in the lungs and protected 80% of mice from death, even when given at 24 hours before infection. These data show that agents, like DMXAA, that can directly activate innate immune pathways, such as the IFN regulatory factor-3/IFN-β system, in respiratory epithelial cells can be used to protect from influenza pneumonia and potentially in other respiratory viral infections. Development of this approach in humans could be valuable for protecting health care professionals and “first responders” in the early stages of viral pandemics or bioterror attacks.
doi:10.1165/rcmb.2010-0288OC
PMCID: PMC3265219  PMID: 21148741
innate immunity; interferon; influenza; pneumonia; bronchial epithelium
9.  Mouse Dendritic Cells Pulsed with Capsular Polysaccharide Induce Resistance to Lethal Pneumococcal Challenge: Roles of T Cells and B Cells 
PLoS ONE  2012;7(6):e39193.
Mice are exceedingly sensitive to intra-peritoneal (IP) challenge with some virulent pneumococci (LD50 = 1 bacterium). To investigate how peripheral contact with bacterial capsular polysaccharide (PS) antigen can induce resistance, we pulsed bone marrow dendritic cells (BMDC) of C57BL/6 mice with type 4 or type 3 PS, injected the BMDC intra-foot pad (IFP) and challenged the mice IP with supra-lethal doses of pneumococci. We examined the responses of T cells and B cells in the draining popliteal lymph node and measured the effects on the bacteria in the peritoneum and blood. We now report that: 1) The PS co-localized with MHC molecules on the BMDC surface; 2) PS-specific T and B cell proliferation and IFNγ secretion was detected in the draining popliteal lymph nodes on day 4; 3) Type-specific resistance to lethal IP challenge was manifested only after day 5; 4) Type-specific IgM and IgG antibodies were detected in the sera of only some of the mice, but B cells were essential for resistance; 5) Control mice vaccinated with a single injection of soluble PS did not develop a response in the draining popliteal lymph node and were not protected; 6) Mice injected with unpulsed BMDC also did not resist challenge: In unprotected mice, pneumococci entered the blood shortly after IP inoculation and multiplied exponentially in both blood and peritoneum killing the mice within 20 hours. Mice vaccinated with PS-pulsed BMDC trapped the bacteria in the peritoneum. The trapped bacteria proliferated exponentially IP, but died suddenly at 18–20 hours. Thus, a single injection of PS antigen associated with intact BMDC is a more effective vaccine than the soluble PS alone. This model system provides a platform for studying novel aspects of PS-targeted vaccination.
doi:10.1371/journal.pone.0039193
PMCID: PMC3377650  PMID: 22723962
10.  Acquired cilia dysfunction in chronic rhinosinusitis 
Background:
Cilia are complex and powerful cellular structures of the respiratory mucosa that play a critical role in airway defense. Respiratory epithelium is lined with cilia that perform an integrated and coordinated mechanism called mucociliary clearance. Mucociliary clearance is the process by which cilia transport the mucus blanket overlying respiratory mucosa to the gastrointestinal tract for ingestion. It is the primary means by which the airway clears pathogens, allergens, debris, and toxins. The complex structure and regulatory mechanisms that dictate the form and function of normal cilia are not entirely understood, but it is clear that ciliary dysfunction results in impaired respiratory defense.
Methods:
A literature review of the current knowledge of cilia dysfunction in chronic rhinosinsusitis was conducted.
Results:
Ciliary dysfunction may be primary, the result of genetic mutations resulting in abnormal cilia structure, or, more commonly, secondary, the result of environmental, infectious, or inflammatory stimuli that disrupt normal motility or coordination. Patients with chronic rhinosinusitis (CRS) have been found to have impaired mucociliary clearance. Many biochemical, environmental, and mechanical stimuli have been shown to influence ciliary beat frequency, and common microbial pathogens of respiratory mucosa such as Pseudomonas aeruginosa and Haemophilus influenzae have developed toxins that appear to interrupt normal mucociliary function. Furthermore, inflammatory mediators known to be present in patients with CRS appear to impair secondarily mucociliary clearance.
Conclusion:
The goal of this article is to summarize the recent developments in the understanding of cilia dysfunction and mucociliary clearance in CRS.
doi:10.2500/ajra.2012.26.3716
PMCID: PMC3906518  PMID: 22391065
Calcium; chronic rhinosinusitis; cilia; cytokines; dynein; mucostasis; Pseudomonas
11.  Regulation of Virulence Gene Expression Resulting from Streptococcus pneumoniae and Nontypeable Haemophilus influenzae Interactions in Chronic Disease 
PLoS ONE  2011;6(12):e28523.
Chronic rhinosinusitis (CRS) is a common inflammatory disease of the sinonasal cavity mediated, in part, by polymicrobial communities of bacteria. Recent molecular studies have confirmed the importance of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) in CRS. Here, we hypothesize that interaction between S. pneumoniae and NTHi mixed-species communities cause a change in bacterial virulence gene expression. We examined CRS as a model human disease to validate these polymicrobial interactions. Clinical strains of S. pneumoniae and NTHi were grown in mono- and co-culture in a standard biofilm assay. Reverse transcriptase real-time PCR (RTqPCR) was used to measure gene expression of key virulence factors. To validate these results, we investigated the presence of the bacterial RNA transcripts in excised human tissue from patients with CRS. Consequences of physical or chemical interactions between microbes were also investigated. Transcription of NTHi type IV pili was only expressed in co-culture in vitro, and expression could be detected ex vivo in diseased tissue. S. pneumoniae pyruvate oxidase was up-regulated in co-culture, while pneumolysin and pneumococcal adherence factor A were down-regulated. These results were confirmed in excised human CRS tissue. Gene expression was differentially regulated by physical contact and secreted factors. Overall, these data suggest that interactions between H. influenzae and S. pneumoniae involve physical and chemical mechanisms that influence virulence gene expression of mixed-species biofilm communities present in chronically diseased human tissue. These results extend previous studies of population-level virulence and provide novel insight into the importance of S. pneumoniae and NTHi in CRS.
doi:10.1371/journal.pone.0028523
PMCID: PMC3230614  PMID: 22162775
12.  Tobacco Smoke Mediated Induction of Sinonasal Microbial Biofilms 
PLoS ONE  2011;6(1):e15700.
Cigarette smokers and those exposed to second hand smoke are more susceptible to life threatening infection than non-smokers. While much is known about the devastating effect tobacco exposure has on the human body, less is known about the effect of tobacco smoke on the commensal and commonly found pathogenic bacteria of the human respiratory tract, or human respiratory tract microbiome. Chronic rhinosinusitis (CRS) is a common medical complaint, affecting 16% of the US population with an estimated aggregated cost of $6 billion annually. Epidemiologic studies demonstrate a correlation between tobacco smoke exposure and rhinosinusitis. Although a common cause of CRS has not been defined, bacterial presence within the nasal and paranasal sinuses is assumed to be contributory. Here we demonstrate that repetitive tobacco smoke exposure induces biofilm formation in a diverse set of bacteria isolated from the sinonasal cavities of patients with CRS. Additionally, bacteria isolated from patients with tobacco smoke exposure demonstrate robust in vitro biofilm formation when challenged with tobacco smoke compared to those isolated from smoke naïve patients. Lastly, bacteria from smoke exposed patients can revert to a non-biofilm phenotype when grown in the absence of tobacco smoke. These observations support the hypothesis that tobacco exposure induces sinonasal biofilm formation, thereby contributing to the conversion of a transient and medically treatable infection to a persistent and therapeutically recalcitrant condition.
doi:10.1371/journal.pone.0015700
PMCID: PMC3017060  PMID: 21253587
13.  Secondhand smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial cells 
Respiratory Research  2009;10(1):120.
Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure.
doi:10.1186/1465-9921-10-120
PMCID: PMC2792224  PMID: 19943936
14.  Erythropoietin Signaling Promotes Invasiveness of Human Head and Neck Squamous Cell Carcinoma1 
Neoplasia (New York, N.Y.)  2005;7(5):537-543.
Abstract
Erythropoietin (Epo) is used for managing anemia in cancer patients. However, recent studies have raised concerns for this practice. We investigated the expression and function of Epo and the erythropoietin receptor (EpoR) in tumor biopsies and cell lines from human head and neck cancer. Epo responsiveness of the cell lines was assessed by Epoetin-α-induced tyrosine phosphorylation of the Janus kinase 2 (JAK2) protein kinase. Transmigration assays across Matrigel-coated filters were used to examine the effects of Epoetin-α on cell invasiveness. In 32 biopsies, we observed a significant association between disease progression and expression of Epo and its receptor, EpoR. Expression was highest in malignant cells, particularly within hypoxic and infiltrating tumor regions. Although both Epo and EpoR were expressed in human head and neck carcinoma cell lines, only EpoR was upregulated by hypoxia. Epoetin-α treatment induced prominent JAK2 phosphorylation and enhanced cell invasion. Inhibition of JAK2 phosphorylation reduced both basal and Epo-induced invasiveness. Our findings support a role for autocrine or paracrine Epo signaling in the malignant progression and local invasiveness of head and neck cancer. This mechanism may also be activated by recombinant Epo therapy and could potentially produce detrimental effects in rhEpo-treated cancer patients.
PMCID: PMC1501166  PMID: 15967106
Erythropoietin; HIF; cancer; invasion; hypoxia

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