Drew Nahirney

The circular bacteriocin, carnocyclin A, forms anion-selective channels in lipid bilayers.

Bacterial resistance to conventional antibiotics is a major challenge in controlling infectious diseases and has necessitated the development of novel approaches in antimicrobial therapy. One such approach is the use of antimicrobial peptides, such as the bacterially produced bacteriocins. Carnocyclin A (CclA) is a 60-amino acid circular bacteriocin produced by Carnobacterium maltaromaticum UAL307 that exhibits potent activity against many Gram-positive bacteria. Lipid bilayer and single channel recording techniques were applied to study the molecular mechanisms by which CclA interacts with the lipid membrane and exerts its antimicrobial effects. Here we show that CclA can form ion channels with a conductance of 35 pS in 150 mM NaCl solution. This channel displays a linear current-voltage relationship, is anion-selective, and its activation is strongly voltage-dependent. The formation of ion channels by CclA is driven by the presence of a negative membrane potential and may result in dissipation of membrane potential. Carnocyclin As unique functional activities as well as its circular structure make it a potential candidate for developing novel antimicrobial drugs.

Regulation of Cl− secretion by α2-adrenergic receptors in mouse colonic epithelium

Previous studies have shown that α2 adrenoceptor (α2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of α2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that α2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that α2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated Cl− secretion but had no effect on CFTR activation by cAMP‐dependent phosphorylation. Apical administration of an ionophore, nystatin (90 μg ml−1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+–K+‐ATPase current, measured as ouabain‐sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 μm). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin‐activated Isc indicating that activation of α2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that α2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT‐PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by α2AR agonists required activation of pertussis toxin‐sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, α2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.

The role of bestrophin in airway epithelial ion transport

The purpose of this study was to identify Cl− channels in the basolateral membrane of airway epithelial cells at the molecular level. We have focused on a new family of Cl− channels, bestrophins, which have previously been identified in retinal pigment epithelium. RT‐PCR, Western blot and confocal microscopy studies revealed the presence of bestrophin in airway epithelial cells. Decreasing bestrophin expression using siRNA resulted in diminished 36Cl− flux. These studies also showed that bestrophin regulation is similar to that of native basolateral Cl− channels. The data indicate that the presence of a functional bestrophin may contribute to the basolateral cell conductance in airway epithelial cells.

House Dust Mite Interactions with Airway Epithelium: Role in Allergic Airway Inflammation

House dust mite (HDM) allergens are the most prevalent allergens associated with asthma and rhinitis around the world. The mechanisms of allergic sensitization and allergic airway inflammation after exposure to HDM have been studied extensively, but many questions remain unanswered. Airway epithelial cells are the first line of defense against external antigens and are considered an important player in the development of allergic airway inflammation. Both genetic susceptibility to allergic sensitization and HDM composition play decisive roles in the outcome of HDM-epithelium interactions, especially regarding airway epithelial dysfunction and allergic inflammation. Interactions between HDM and the airway epithelium have consequences for both development of allergy and asthma and development of allergic airway inflammation. This review will describe in detail these interactions and will identify issues that require more study.

Cholesterol-dependent regulation of adenosine A2A receptor-mediated anion secretion in colon epithelial cells

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. In this study we have investigated the role of cholesterol in the adenosine-dependent regulation of ion transport in colonic epithelial cells. We observed that methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering molecule, enhanced adenosine A(2A) receptor-activated transepithelial short circuit current (I(sc)), but only from the basolateral side. Cholesterol is a major constituent of membrane microdomains, called lipid rafts that also contain sphingolipids. However, studies with the sphingomyelin-degrading enzyme, sphingomyelinase, and the cholesterol-binding agent, filipin, indicated that the change in the level of cholesterol alone was sufficient to control the adenosine-modulated I(sc). Cholesterol depletion had a major effect on the functional selectivity of A(2A) receptors. Under control conditions, adenosine activated I(sc) more potently than the specific A(2A) agonist, CGS-21680, and the current was inhibited by XE991, an inhibitor of cAMP-dependent K(+) channels. Following cholesterol depletion, CGS-21680 activated I(sc) more potently than adenosine, and the current was inhibited by clotrimazole, an inhibitor of Ca(2+)-activated K(+) (IK1) channels. Co-immunoprecipitation experiments revealed that A(2A) receptors associate with IK1 channels following cholesterol depletion. These results suggest that cholesterol content in colonic epithelia affects adenosine-mediated anion secretion by controlling agonist-selective signaling.

Elevated levels of circulating CD4(+) CRTh2(+) T cells characterize severe asthma.

Chemoattractant receptor‐homologous molecule expressed on Th2 cells (CRTh2) is a receptor for PGD2 and expressed by T cells, eosinophils, basophils, and ILC2 cells. CRTh2 expression by CD4+ T cells identifies the Th2 subset, and these cells have been characterized as allergen‐specific central memory Th2 cells. Recently, activation of the PGD2‐CRTh2 pathway in the lungs was associated with severe asthma.

Expression of polycystins and fibrocystin on primary cilia of lung cells.

Mutations in polycystin-1, polycystin-2, or fibrocystin account for autosomal dominant or recessive polycystic kidney disease. Renal cystogenesis is linked to abnormal localization and function of these cystoproteins in renal primary cilia. They are also expressed in extrarenal tissues in which their functions are unclear. Here we found that human type-II alveolar epithelial A549, airway submucosal Calu-3 cells, and rat bronchioles contain primary or multiple cilia in which we detected these cystoproteins. At sub-confluency, polycystin-1 was expressed on plasma membrane, while polycystin-2 was localized to the ER of resting cells. Both polycystins were detected on the spindle and mid-body of mitotic cells, while fibrocystin was on centrosome throughout cell cycle. Polycystins and fibrocystin may participate in regulating mucociliary sensing and transport within pulmonary airways.

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma.

Background Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied. Methods We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry. Results Subjects with severe asthma had higher PAR-2 expression on CD14++CD16+ monocytes (intermediate monocytes) and also higher percentage of CD14++CD16+PAR-2+ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14++CD16+PAR-2+ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14++CD16+ PAR-2+ cells in peripheral blood compared to subjects without exacerbations. Conclusions PAR-2 expression is increased on CD14++CD16+ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14++CD16+ monocytes may play a role in the pathogenesis of severe asthma.

Proteinase-activated receptor-2 blockade inhibits changes seen in a chronic murine asthma model

Proteinase‐Activated Receptor‐2 (PAR2) is a G protein‐coupled receptor activated by serine proteinases. We have shown that PAR2 activation in the airways is involved in the development of allergic inflammation and airway hyperresponsiveness (AHR) in acute murine models. We hypothesized that functional inhibition of PAR2 prevents allergic inflammation, AHR and airway remodeling in chronic allergic airway inflammation models.

Proteinase-activated receptor-2 (PAR-2) expression on inflammatory cells in severe asthma

Background PAR-2, a G-coupled receptor activated by serine proteinases, is widely expressed in the human body and is involved in inflammation. We have shown that PAR-2 activation in the airways plays a pathogenetic role in mouse models of asthma. PAR-2 expression is increased in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells of asthmatic individuals has not been. Severe asthma has different phenotypic characteristics from mild-moderate disease. In this study we compared PAR-2 expression on immune cells between subjects with mild/moderate and severe asthma. Methods A total of 36 asthma subjects (24 mild/moderate; 12 severe by ATS guidelines) were recruited at the University of Alberta Hospital and peripheral blood obtained. PAR-2 expression was analyzed by flow cytometry and qRT-PCR in whole blood. Results There were no differences in the % of eosinophils, neutrophils or CD4+ T cells expressing PAR-2 between severe and mild/moderate asthmatics. CD14 high monocytes were classified as classical (CD14++CD16-) or intermediate (CD14++CD16+). No difference in total numbers of either monocyte sub-population was noted between the two asthma groups. More intermediate monocytes from patients with severe asthma (33.6 ±5.1%) expressed PAR-2 compared to patients with mild/moderate asthma (22.4±4.0%, p=0.039), but there was no difference between asthma phenotypes in the percent of classical monocytes expressing PAR-2 (11.7±2.8% vs. 12.5±2.9%). PAR-2 mRNA expression was not different between severe and mild/moderate asthmatics, however, PAR-2 mRNA correlated with total dose of inhaled steroids and inversely correlated with % predicted FEV1. Conclusions PAR-2 expression is increased on intermediate monocytes in subjects with severe asthma. Intermediate monocytes are pro-inflammatory and their numbers are increased in inflammatory diseases. Our data suggest that PAR-2-mediated activation of intermediate monocytes may play ar ole in the pathogenesis of severe asthma, although the effects of PAR-2-mediated activation of these cells are not known.