Abstracts from the Conjoint Annual Meeting of L’Association des pneumologues de la province de Québec and le Réseau en santé respiratoire du FRSQ, November 8, 9 and 10, 2007

ENaC expression (mRNA and protein) while pinacidil (K ATP activator) upregulated it. ENac expres- sion could also be regulated after changes in Na + and K + fluxes (with ouabain, valinomycin, monensin). To further study the ENaC regulation mechanisms, we tested the impact of the K + channel modulators on ENaC promoter activity in transfected A549 cells. We found a 2-fold increase in ENaC promoter activity following pinacidil treatment. Regulation of ENaC channel by K + transport is a new avenue and could be of great ben-efit for the resolution of lung pathologies characterized by ion and fluid transport dysfunction. Financial support of NSERC OBJECTIVE: There is considerable genetic variation in asthma suscepti- bility and in response to glucocorticoids, the most commonly used asthma therapeutic. There is also variable asthma susceptibility between Balb/c (hyperresponsive) and C57BL/6 (hyporesponsive) mice. The objective of this study was to determine whether these mouse strains also exhibit vari- ant GC responsiveness. METHODS: Mice were subjected to OVA sensitization followed by intranasal administration of budesonide. Airway responsiveness to metha- choline was measured in anesthetized mechanically ventilated animals. Real-time PCR was used to quantify lung GR and IPO13 (mediates nuclear import of GR) mRNA. Plasma IgE was determined by ELISA. RESULTS: Both Balb/c and C57BL/6 mice had significantly increased respiratory resistance following OVA challenge. Budesonide treatment improved pulmonary function of C57BL/6 but had no effect on Balb/c mice. OVA-induced IgE levels were several fold higher in Balb/c compared to C57BL/6. While budesonide treatment normalized IgE levels in C57BL/6 mice, IgE in Balb/c mice remained unchanged. OVA sensitization lead to significant reduction in IPO13 and GR mRNA levels in both mouse strains. Budesonide treatment restored IPO13 mRNA in C57BL/6 mice to control levels and stimulated GR mRNA to 3-fold over control. In Balb/c mice, budesonide restored GR and IPO13 mRNA to control levels. CONCLUSIONS: Our findings demonstrate a significant difference in steroid responsiveness in C57BL/6 vs. Balb/c mice. The resistance to exogenous steroid observed in the Balb/c mice may reflect a dysregulation of handling of endogenous steroid which in turn may contribute to hyper- responsiveness. BACKGROUND: Interleukin (IL)-13 is a key Th2 mediator in asthma pathogenesis. While the IL-13 receptor (IL-13R), comprised of the IL-4R α and IL-13R α 1 subunits, is expressed by various hemopoietic and non-hemopoietic cells including dendritic cells (DC), airway smooth muscle (ASM) and airway epithelial cells, functional IL-13Rs are not expressed on T cells. We are interested in defining mechanism(s) by which IL-13 regulates T cell function and pathogenesis in experimental asthma. HYPOTHESIS: T cell specific overexpression of IL-13 enhances DC maturation, host T cell recruitment and ASM remodeling in the airways of rats with experimental asthma. METHODS: We have developed methodology to produce high titer recombinant retroviruses with the ability to transduce primary ovalbumin (OVA)-specific Brown Norway (BN) rat CD4+ T cells. We will generate gene-modified CD4+ T cells overexpressing IL-13 and the enhanced green fluorescence protein (EGFP) marker. Following retroviral transduction, gene-modified T cells will be purified magnetically, FACS-sorted and then adoptively transferred into naïve BN rats that will subsequently be challenged with OVA on three occasions. We will assess the effects of T cell- specific IL-13 overproduction on several of the classic pathological features of asthma including (1) airway eosinophilic inflammation, (2) mucus production and (3) ASM remodeling. We will also examine its role in (4) DC maturation in the lungs and draining lymph nodes, as well as (5) host T cell recruitment to the lungs. SIGNIFICANCE: These studies will define the role of IL-13 in the abil- ity of adoptively transferred T cells to induce pathogenesis in experimental asthma. BACKGROUND: Asthma and allergic rhinitis may result from a similar allergen-induced inflammatory process, but to determine possible mechanisms by which the PURPOSE: the of challenges on upper and lower airway inflammation in rhinitic subjects with or without asthma. METHODS: Thirty-six subjects with allergic rhinitis were recruited: 21 mild asthmatics (A) and 15 non-asthmatics (R). Subjects underwent a nasal control challenge with normal saline followed by 4 consecutive daily aller- gen challenges. At each challenge day, increasing allergen dilutions were sprayed into each nostril until a positive response occurred. Induced spu- tum (IS) and nasal lavage (NL) with differential cell counts were obtained 7 hours following the control, the first and the last challenge. RESULTS: No difference between groups was observed in the percentage of IS and NL eosinophils at any points (p>0.05). No change in the per- centage of IS eosinophils was observed after one and 4 days of challenge compared with control day (p>0.05), but some subjects (A=4 (19%) and R=3 (20%)) had an increase ( ≥ 2%) after the first and/or the last challenge. An increase in the percentage of NL eosinophils was observed after 4 days of challenge compared with control challenge (mean±SEM: 16.9±23.0% vs 3.8±9.6%; p<0.05), but not after 1 day (8.8±17.1%; p>0.05). CONCLUSIONS: Repeated nasal allergen challenge is effective to induce upper airway inflammation, but only induces lower airway inflam- mation in about one in 5 subjects. The presence or absence of asthma did not appear to make a difference. in close association with resi-dent cells, such as fibroblasts, raising the possibility of cross talk between these two types of cells. OBJECTIVES: The aim of our study was to evaluate the effect of T cells interaction with bronchial fibroblasts on IL-6 production. METHODS: Bronchial fibroblasts were isolated from mild asthmatics and non atopic healthy controls. T cells were purified from the peripheral blood of healthy and asthmatic subjects. Co-culture of confluent healthy (BNF) or asthmatic bronchial fibroblasts (BAF) with T cells were performed in presence or absence of blocking antibody anti CD18. RESULTS: Direct contact of T cells significantly stimulated IL-6 production by both BNF and BAF. This effect was significantly higher in BAF than BNF (881±113.39%vs 452.70±92.40%; p=0,02). Coculture with anti-CD18 showed a significant inhibition of IL-6 expression in BAF (881±113.39% vs 517.35±162.97%; p=0.03) but, not in BNF (452.70±92.40% vs 429.13±80.93%). ICAM-1 expression on fibroblasts showed a high BAF expression compared to BNF (62.33±5.73% vs 43.27±5.6%; p=0.05). Twenty four and forty eight hours of coculture also leads to activation of T cells from both asthmatic (24h: 8.13±1.74% vs 19.85±4.55%; 48h: 8.58±1.06% vs 34.44±5.76%) and healthy donors (24h: 7.58±0.72% vs 21.39±3.96%; 48h: 7.14±1.17% vs 24.44±4.72%). CONCLUSION: Cellular interaction particularly between T cells and fibroblasts play an important role in the amplification of the inflammatory response in asthma. les deux conditions. En fin d’effort (iso-temps-100%), la chute de la FMV était significativement corrélée avec la différence de ventilation entre l’état fatigué et non-fatigué (r=0.70, P =0.036). Enfin, la diminution de force avait tendance à être corrélée avec la diminution du temps d’endurance (r=0.60 ; P=0.087). CONCLUSION : La fatigue des membres inférieurs est associée à une charge ventilatoire supplémentaire en fin d’effort. Although pressure support ventilation (PSV) promotes inspiratory muscle unloading, it also downregulates respiratory drive and deactivates inspira- tory muscles. During higher intensity exercise, COPD patients exhibit a plateau in their transdiaphragmatic pressure (Pdi) secondary to dynamic hyperinflation and reduced diaphragm neuromechanical coupling, whereas diaphragm electrical activity (EAdi) continues to increase. We hypothesized that inspiratory muscle unloading with PSV would result in a greater diaphragm deactivation in COPD patients compared to healthy individuals. AIM: To compare the effect of PSV on EAdi suppression and mechanical output in such subjects. METHODS: Ten patients (FEV1: 49±13% pred) and nine healthy subjects breathed for six minutes each with 0 and 10 cm H2O of PSV at rest and during 40% and 60% of their maximum exercise workload. RESULTS: PSV increased minute ventilation (VE) in both healthy subjects (P=0.004) and COPD patients (P<0.001); healthy subjects reached a higher VE with PSV than patients during exercise (P=0.03). With increased exercise intensity, COPD patients had larger EAdi and smaller Pdi increases than healthy individuals. PSV application during exercise reduced both EAdi and Pdi in healthy subjects, thus preserving diaphragm neuromechanical coupling, whereas in COPD patients it resulted in greater EAdi deactivation, improving coupling. The larger the EAdi exhibited without PSV, the greater the resulting EAdi suppression that was achieved with PSV (r=0.57, P<0.001). CONCLUSION: PSV during exercise results in greater EAdi downregu-lation and improved neuromechanical coupling in COPD patients com- pared to healthy individuals. Current guidelines recommend treatment of latent tuberculosis infection (LTBI) for patients at risk of developing active TB. Physicians must weigh the benefits of TB prevention against the risk of adverse events. Our objective was to determine rates of adverse events, hospitalization and completion associated with LTBI therapy. A historical cohort of all Québec residents dispensed at least 30 days of LTBI therapy between January 1, 1998 and December 31, 2003 was con-structed using the provincial healthcare databases. For each case, two age and sex matched controls were identified. Rates of therapy completion, hospitalization, and adverse events were compared. RESULTS: Over 6 years, 9,393 Québec residents were dispensed at least 30 days of LTBI therapy. Isoniazid (INH) alone was given to 90.4%, Rifampin alone to 6.4% , and rifampin and pyrazinamide (RIF PZA) to 0.4%. Overall completion was 52%, and was lower with older age. Probable drug-related serious adverse events requiring hospitalization occurred in 1.2% of persons taking INH, 2.5% of persons taking RIF, and 0% of persons taking RIF-PZA. Hospitalization rates, hospitalization dura-tion, and serious adverse events were significantly greater among those receiving LTBI therapy vs. controls. Probable drug-induced liver disease requiring hospitalization occurred in 0.3% of patients dispensed INH, 0.1% for RIF. Rates of severe adverse events related to LTBI therapy increased with age. Overall mortality for the six year period was 4.4% among the LTBI cohort and 4.3% among controls. CONCLUSION: Rates of severe adverse events associated with LTBI therapy were low, and similar to previous reports from specialty TB clinics. BACKGROUND: Recognizing the patient perspective is essential in achieving effective tuberculosis (TB) control. TB treatment is free to patients in most DOTS programs, but the patient must bear the costs of care prior to diagnosis, and costs associated with hospitalization, and travel/time with DOT. These costs may deter patients from seeking care for their TB-related symptoms or create a major barrier to continued treat- ment. Thus, patient-related cost information is relevant and necessary in assessing the impact of program design on TB control. PURPOSE: To study the economic impact and burden of care-seeking for pulmonary TB patients in 4 low/middle-income countries, and 3 high-income countries including: Ecuador, Zambia, Brazil, China, Canada, the Netherlands, and the United Kingdom. METHODS: An in-depth questionnaire was adapted from the pilot study and translated into local languages. Locally trained interviewers adminis- tered questionnaires to adults with active bacteriologically confirmed pulmonary TB, who had completed approximately two months of treatment, at government health facilities using the DOTS strategy. Direct (out-of- pocket expenditures) and indirect (time) costs were estimated. Approximately 100 patients were interviewed in each low/middle income country, and 50 patients were interviewed in each high income country. RESULTS: Preliminary analysis revealed that patient expenditures for TB were significant at all sites. When taken as a proportion of the annual gross national income, patients’ expenditures ranged from 10.27% in Canada to 26.10% in Ecuador. The economic burden on patients was the most signif- icant during the diagnosis period. BACKGROUND: Asthma is a chronic respiratory disease that has been previously associated with a disproportionately high level of suicidal ideation (SI), independent of depression. However, the precise mecha- nisms linking asthma to suicidal ideation remain poorly understood. Several asthma medications like theophylline and beta-2 adrenergic- agnonists (which are potent bronchodilators), have been shown to pro-voke intense feelings of anxiety, fear, or panic in some individuals. These drugs may potentially influence rates of SI in asthma patients, but this has yet to be explored. The present study assessed SI in 630 consecutive adult asthma patients presenting to an outpatient clinic (40% men ; mean age=50 yrs). METHODS: All patients underwent a sociodemographic, psychiatric (PRIME-MD), and medical history interview, and completed a battery of questionnaires including the Beck Depression Inventory-II (BDI-II) and Asthma Control Questionnaire (ACQ). Responses e 1 on question 9 of the BDI were used to measure SI. RESULTS: BDI-II results indicated that 12% of asthmatics reported having SI. After controlling for age, sex, smoking status, and major depression, results of general linear model analyses indicated a significant main effect theophylline use (F=3.85, p<.05), but not beta-2 agonist use (F=0.04, p=.8497), on SI. Though asthma control and severity levels were also significantly associated with SI in univariate analyses, these associations were no longer significant after controlling for covariates. CONCLUSIONS: These findings suggest that theophylline use may be associated with a higher risk of SI in adult asthmatics. It is noteworthy that these findings were independent of major depression, which suggests results are not simply the results of depressive symptomatology in asthma patients. Clinically, these findings indicate that physicians should be vigi-lant about the potential psychological side effects of certain asthma med- ications, particularly those that enhance central nervous system activity. BACKGROUND: Pulmonary rehabilitation (PR) programs have been built to meet the various needs of patients suffering from COPD. Knowing to what extent these needs are being met by PR programs is difficult because while health related quality of life (HRQoL) is generally consid-ered as a main outcome of PR, the fulfillment of needs, which some authors view as a central component of HRQOL, is often overlooked in the assessment of PR outcomes. PURPOSE: To develop and pre test an instrument based on a conceptual model of patients needs assessment in PR. METHODS: A list of 120 Items corresponding to our model of needs assessment was submitted to a panel of 14 clinicians involved in PR. Following comments from this panel, questionnaire content was reduced to 98 items. Three successive versions were administered to a total of 15 patients in order to further reduce content to 80 items. The final version corresponds to a specific visual format reflecting the domains of need recognition as well as degree of knowledge, motivation, expectations and goals. Psychometric validation studies of this questionnaire should follow. of 0.14 uM and abrogated it at a concentration of 1uM. Interestingly, increasing concentration of A23187 (up to 2 uM) did not prevent the inhibitory effect of MTK on LTC4 biosynthesis. PTX (25 ng/ml) decreased the A23187-induced LTC4 release by only 30% (p=0.03); such an effect was not sufficient to support a possible inhibition of LTC4 autocrine stimulation by MTK. These data suggest that MTK decreases LTC4 release by a mechanism unrelated to CysLT receptor blockade. To confirm that this inhibition is relevant in vivo, LT release will have to be evaluated ex vivo in blood of asthmatics taking MTK. 5-oxo-ETE is a recently discovered 5-LO product that may have an important role in asthma due to its chemoattractant properties on eosinophils and neutrophils. It is synthesized by the action of the microsomal enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH) on the 5-LO product 5-HETE in the presence of NADP+. Under normal conditions, 5-oxo-ETE synthesis is inhibited by a high intracellular ratio of NADPH to NADP+ but we speculate that as neutrophils undergo aging, the levels of NADP+ increase, which favors the formation of 5-oxo-ETE. To investigate this hypothesis, we incubated neutrophils for 24 h in the presence of 10% FBS. At this time, 5-oxo-ETE synthesis was 15 ± 4 times higher compared to freshly isolated neutrophils (p <0.001). The increase in 5-oxo- ETE synthesis was paralleled with a dramatic increase in intracellular pyridine nucleotides, measured by HPLC. NADP+ (pmol/10 6 cells) rose from 1.2 ± 0.5 in freshly isolated neutrophils to 18 ± 6 after 24 h, whereas NADPH levels remained nearly constant. NAD+ (pmol/10 6 cells) rose from 17 ± 2 to 136 ± 30 over 24 h, suggesting that the increase in NADP+ could be coming from the activation of neutrophil NAD kinase. The increase in 5-oxo-ETE synthesis was partially inhibited by the survival fac- tor GM-CSF (65 ± 6%), LPS (54 ± 7%), and catalase (53 ± 13%), all of which also reduced the ratio of NADP+/NADPH (0.4 ± 0.1, 0.7 ± 0.4, and 0.5 ± 0.3, respectively) compared to vehicle-treated controls (2.0 ± 0.5). We conclude that in aging neutrophils apoptosis-associated oxidative stress increases NADP levels and 5-oxo-ETE synthesis, which could pro- long inflammation due to its chemoattractant effects. RATIONALE: IL-33, a recently described IL-1 cytokine family member, promotes Th2 inflammation but evidence on implications of this cytokine in asthma is lacking. IL-33 is mainly expressed by structural cells including ASMC, but whether pro-inflammatory cytokines and anti-inflammatory drugs modulate its expression in ASMC is unknown. METHODS: Endobronchial biopsies were collected from adults with mild (n = 8), moderate (8), severe (9) asthma and from control subjects (5). Primary ASMC were cultured with or without inflammatory cytokines, Dexamethasone (DEX) or Mithramycin A (MMA). IL-33 expression was investigated by real-time quantitative RT-PCR, immunocytochemistry (ICC) and western blotting. RESULTS: Higher levels of IL-33 transcripts were detected in biopsies from asthmatic patients (mild, moderate and severe) compared to control subjects (p = 0.932, 0.0186 and 0.002, respectively). ICC staining seemed to correlate with transcript levels. In ASMC, TNF α upregulated IL-33 expression in a time- and dose-dependent manner. MMA reduced the TNF α -induced IL-33 upregulation, whereas DEX did not display significant effect. CONCLUSIONS: IL-33 expression increases with asthma severity in bronchial biopsies. In cultured ASMC, TNF α is a potent inducer of IL-33 expression. IL-33 might consist of an alternative therapeutic target for drugs acting on respiratory disease states which are refractory to corticosteroid therapy, such as severe asthma. IL-33 consists of novel Th2 mediator but its precise role in lung inflammatory diseases remains unclear. Québec BACKGROUND: Obstructive sleep apnea (OSA) is characterized by recurrent episodes of upper airway (UA) during sleep. UA narrowing, increased UA collapsibility, and UA muscle dysfunction may contribute to the occurrence of UA occlusion during sleep. Pro-inflammatory cytokines, such as, TNF and IL-6 are elevated in serum of OSA patients and have been proposed as mediators of muscle weakness. OBJECTIVE: To measure the expression of TNF in uvular tissue of non-apneic snorers and apneic patients. METHODS: Forty patients who underwent an UPPP were enrolled and divided into three groups. The first group was composed of non-apneic snorers (n = 14) and the second group (OSA 1; n = 14) consisted of patients whose body mass index was similar. The third group (OSA 2; n = 12) consisted of OSA patients whose BMI was higher than the one of the other groups. Following UPPP, the resected tissues were divided into two sections (proximal and distal). RESULTS: TNF is highly expressed in the epithelium of the uvula and in the musculus uvulae (MU). Western blotting of whole proximal uvula (n=3) revealed that TNF expression was significantly higher in apneic patients compared to snorers (Snorers: 100.5 ± 2.99% ; OSA 1: 127.07 ± 6.91%; OSA 2: 140.75 ± 10.97%; P=0.01). TNF expression in muscular area was higher in OSA 2 group compared to both OSA 1 and snorer’s groups. This TNF expression was correlated to body mass index (r = 0.8702; p = 0.02) CONCLUSION: Heaviest OSA patients have a higher TNF expression in Musculus Uvulae than less obese OSA and non-apneic snorers. This suggests that in heaviest OSA patients Musculus Uvulae is the main TNF source. mor- phometric analysis (ImagePro). RESULTS: Expression of mRNA for all 3 cytokines was significantly greater in the severe vs. mild group, with mean increases of 310 ± 62% for IL-1alpha, 181 ± 46% for IL-6 and 290 ± 53% for TGF-beta (p < 0.05). There was also significantly greater connective tissue in severe (74.6 ± 8.3 % counts(SD)) compared with mild (60.2 ± 17.4 % counts) OSA (p = 0.007). CONCLUSIONS: Expression of pro-fibrotic cytokine mRNA and con- nective tissue content is significantly increased in severe compared with mild OSA. This may have implications for UA function. Funding: asthma, M±SD age=46±13 yrs, 42% male, completed the asthma control questionnaire (ACQ) and provided demographic information, including height, weight, and waist circumference (WC). Two general linear models were conducted to assess the relationship between adiposity and asthma con- trol. Separate models were run for the main and interaction effects of body mass index (BMI) and sex on ACQ total and its subscales, and WC and sex on ACQ. RESULTS: In general, there was a main effect of sex and an interaction effect of sex and BMI on ACQ total and subscale scores. In addition, there were main effects of sex and interactions of sex and WC on ACQ scores. All analyses used age as a covariate. The main effect of sex revealed that men exhibited worse asthma control than women. The sex X WC interaction suggested that increasing WC was associated with worse control in men, with no effect in women. In contrast, the sex X BMI interaction suggested that increasing BMI was associated with better control in women, with no effect in men. CONCLUSION: Further research is needed in larger samples to establish mechanisms by which central adiposity and total body fat influences asthma control and the variation that exists between sexes. OBJECTIVE: Asthma is the most common chronic disease of children. Abnormalities of the lung predate onset of symptoms. Our objective is to determine how genes that regulate lung development contribute to asthma susceptibility. METHODS: We measured mRNA (real-time PCR) and protein (immunohistochemistry) expression of genes involved in mesenchymal- epithelial signaling and airway muscle maturation. Goblet cells were assessed with PAS stain. Lung function was measured using the flexiVent small animal ventilator. RESULTS: Asthma susceptible (Brown Norway, BN), hyperresponsive (Fisher) and normoresponsive (Lewis) rats showed differences in lung function at postnatal day 14. These rat models also exhibited differential developmental expression in the lung of a ) epidermal and platelet-derived growth factor receptors (associated with lung remodeling in asthma); b) glucocorticoid receptor (associated with steroid responsiveness) c) total and fast myosin (SM-B, associated with airway narrowing). At postnatal day 14 Fisher and BN pups had increased total tracheal myosin. SM-B was upregulated in Fisher and down-regulated in BN pups. Newborn BN rats displayed tracheal goblet cell hyperplasia. CONCLUSIONS: Asthma susceptibility is associated with significant differences in lung function and respiratory gene expression in the newborn period. Increased SM-B/total myosin leads to faster muscle shorten- ing. Increased SM-B/total myosin in developing lung leading to increased smooth muscle contraction may contribute to innate hyperresponsiveness. Neonatal goblet cell hyperplasia, characteristic of asthmatic airways, asso- ciates with the asthma susceptible phenotype of BN rat. Clarification of genetic mechanisms that contribute to asthma-susceptibility will be essen- tial to the design of therapeutic targets for early intervention and primary prevention. compared the effects of the steroid budesonide (Bud) in acute versus repeated allergen challenges and also studied its effects on structure-function relationships. and (1 or 3x at 5-day intervals) with ovalbumin (Ova) (or were administered Bud (or vehicle (Veh)) intranasally 24 or 1h prior to Ova (or Sham) Two days after the final challenge, AHR to methacholine (MCh) and were Lung tissue was for smooth muscle (SM) mass and goblet cells (GCs). in Veh/Ova (p<0.05) is attributable to peripheral airways. In the central air- ways in Veh/Ova, a marked increase in SM mass (p<0.05) and GCs (p<0.05) was observed. Both parameters were not decreased by Bud. Twenty-four hours after a single challenge, there was a marked increase in eosinophils from the bronchoalveolar lavage in Veh/Ova (p<0.02). The increase in total respiratory elastance (Ers) in Veh/Ova was decreased by Bud at MCh (32 and 64mg/mL). DISCUSSION: Repeated allergen challenge in the rat causes AHR in the lung periphery and remodeling in the central airways, reflecting a dissoci- ation of AHR and airway remodeling. While Bud cannot inhibit these effects, it attenuates increases in Ers after a single challenge. These find- ings heed the elucidation of mechanisms by which steroid resistance may play a role after multiple allergen challenges. Supported by CIHR, J.T. Costello Memorial Fund, MUHC and Asthma in the Workplace OBJECTIVE: To determine whether deficiency of lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury. METHODS: An Lgl1 knockout mouse was generated by replacement of exon 2 of the Lgl1 gene with a neomycin cassette. Absence of lgl1 was lethal prior to lung formation. We compared lung morphology, Lgl1 RNA and lgl1 protein, tracheal goblet cells, elastin fibers and lung function in heterozygous Lgl1+/– and wild type mice. RESULTS: At embryonic day 18.5, Lgl1+/– lungs had reduced levels of Lgl1 RNA and lgl1 protein. Postnatal Lgl1+/– lungs (days 1-14) displayed delayed septation, distal airspace enlargement, increased interstitium, gob- let cell hyperplasia, and fragmented elastin fibers. At one month of age, lungs of Lgl1+/+ and Lgl1+/– mice were indistinguishable. CONCLUSIONS: Lgl1 is essential for viability, and is required for developmental processes preceding and during lung formation. Newborn Lgl1+/– mice display features of delayed lung maturation and inflammatory injury. Lgl1 deficiency states may contribute to BPD. (a) the cell both P (SP) and its the receptor (NK-1R), (b) the NK-1R is activated in an autocrine manner in response to FceRI clustering, blocking NK-1R activation with the selective antagonist L-703,606 decreased basophil degranulation. AIM: To determine the role of the tachykinergic system SP/NK-1R in the production of LTC4 in IgE-dependent activation of basophils. METHODS: IgE-sensitized RBL-2H3 cells were treated or not with L-703,606 or glucocorticoids prior to FceRI clustering. The production of LTC4 was measured using the Luminex technology. RESULTS: The kinetic study indicates that LTC4 production occurred as early as 2 min following FceRI clustering, and that most of the LTC4 synthesized is released by basophils. Interestingly, blocking NK-1R activation reduced the synthesis of the LTC4. The glucocorticoids, dexamethasone and fluticasone, also reduced FceRI clustering-induced LTC4 synthesis. Very interestingly, the combination of glucocorticoids and the NK-1R antagonist has further inhibited the production of LTC4 which suggests a synergic interplay between these two inhibitory pathways. CONCLUSION: Our study provides the first evidence that the tachykin- ergic system plays a role in the production of LTC4 in response to FceRI clustering. The combination of a NK-1R antagonist with a glucocorticoid may be an interesting medication for the treatment of allergic diseases. Supported by the CIHR In male, HVR higher in pups exposed to neonatal IH than in normoxic pups vs. of baseline value, respectively). In female, HVR was similar between neonatal IH and normoxic pups vs. of baseline value, respectively). a male-specific enhancement of HVR in rat pups. This study suggests that neonatal apnea contribute to respiratory disorder breathing in sex specific manner in infant. BACKGROUND: C57BL/6 mice are susceptible to Pseudomonas aeruginosa lung infection and extensively used to explore the pathogenesis of infection with this microorganism. Time course of this infection has been studied in details using the conventional invasive method of infection and partially studied using the less invasive non-surgical method of infection, METHODS: Female C57BL/6 mice were infected with 10 6 or 5 x 10 5 CFUs/mice of P. aeruginosa in agar beads. Mortality, daily body weight, total and differential leukocyte count in broncheoalveolar lavage fluid (BALF) were assessed at day 3 and 7 post-infection. Bacterial burden in lungs was tested at 6 hrs, 24 hrs, 3 and 7 days. RESULTS: There was a considerable mortality by day 3 post-infection (32.3 %, 12 out of 33) when mice were infected with 10 6 CFUs mean-while all the mice survived the infection when the dose was decreased to 5 x 105 CFUs. Body weight loss peaked (- 12%) at day 2 or 3 (in both doses) and baseline body weight was regained by day 7 post-infection. time point (6-12 hours post-infection). 3) We plan to study whether an intermediate dose (7-8 x 105 CFUs) will minimize mortality while prominent inflammation will be still induced. BACKGROUND: A growing body of evidence suggests that exogenous nitric oxide (NO) may have beneficial roles in airway diseases such as asthma and COPD. TPI 1020 is a novel respiratory anti-inflammatory drug which incorporates an NO-donating moiety and displays broncho-dilating activities in the lungs. PURPOSE: The aim of this study was to compare TPI 1020 to budesonide in animal models of neutrophil-mediated lung inflammation and broncho-constriction. METHODS: Mice were treated intra-tracheally with dry powder formula-tions of TPI 1020 or budesonide at equimolar concentrations, prior to challenge with lipopolysaccharide (LPS). Neutrophil recruitment and inflammatory mediators were measured in lung lavages. The broncho-dilating activity of TPI 1020 was assessed in guinea pigs challenged with histamine. RESULTS: In mice, TPI 1020 (8.0 mg/kg) was found to inhibit the LPS-induced neutrophil recruitment by 33%. In addition, TPI 1020 signifi- cantly decreased the secretion of TNFa (83%, p<0.001), MCP-1 (49%, p<0.05) and pro-MMP-9 (84%, p<0.05) in the lung while it had no effect on KC levels. Budesonide (5.6 mg/kg) had a weaker effect on neutrophil recruitment (19%), secretion of TNFa (71%, p<0.001), and pro-MMP-9 (54%), and had no effect on MCP-1 and KC secretion. In guinea pigs, TPI 1020 had a greater protective effect (90%) than budesonide (30%) against broncho-constriction induced by histamine. CONCLUSION: Together, these data indicate that TPI 1020 exerts broader anti-inflammatory and broncho-dilating effects as compared to budesonide, and demonstrates the potential for unique anti-inflammatory effects on prognostic indicators that are believed to be relevant in COPD. BACKGROUND: Cystic Fibrosis (CF) is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation is the deletion of phenylalanine 508 (F508del) that causes mild misfolding of CFTR and its retention in the endoplasmic reticulum (ER) leading to a drastic decrease in cAMP-stimulated Cl- permeability at the cell surface. Most evidence suggests that even partial correction (6-15%) of the ER-retained F508del-CFTR could provide therapeutic ben- efit for CF patient. PURPOSE: To identify small molecule correctors of abnormal F508del- CFTR processing, we developed and used a robust cell-based assay using F508del-CFTR with three hemagglutinin (3HA) tags inserted into the fourth external loop. METHODS: Baby Hamster Kidney (BHK) cells expressing 3HA tagged F508del-CFTR were incubated with test compounds at 10μM for 24 hours at 37°C. One of the hits we identified was the phosphodiesterase inhibitor sildenafil which has previously been reported to increase CFTR activity in nasal epithelia. A computional search for similar structures yielded ana- logues of sildenafil carrying the distinctive sulfonyl-piperazine group. We evaluated these analogues and found that one, called KM60, was surpris- ingly potent. It partially restored F508del trafficking and increased maturation significantly when BHK cells were treated with 10nM for 24 h or 10μM for 2 h. Partial correction was confirmed by the appearance of mature CFTR in Western blots and by halide flux, patch-clamp and short- circuit current measurements in unpolarized BHK cells, monolayers of human airway epithelial cells, and intestines isolated from The matrix metalloprotease (MMPs) are an enzyme family, which have an important role in the degradation of extracellular matrix. Amongst MMPs, MMP-9 has been implicated in airway remodeling in asthma. Our previous findings indicated that MMP-9 is not expressed by HBSMC but it appears to bind to these cells as a function of cell confluency. Recent studies have reported that MMP-9 is a ligand for the CD44 receptor. However, little is known about the expression and the role of CD44 in HBSMC, and there is no data on its possible interaction with MMP-9. AIMS: To determine (a) the expression of CD44 in HBSMC as a function of cell confluency, (b) its functionality and (c) its potential interaction with MMP-9. METHODOLOGY: CD44 expression was determined by Western blot (WB), and cytometry. RESULTS: RT-PCR analysis showed that CD44 is expressed in HBSMC, at least, as the v6 and v8 isoforms. WB analysis confirmed the expression of CD44. Flow cytometric analysis indicated that, similarly to cell-bound MMP-9, CD44 expression decreased as a function of cell confluency. These results were confirmed by semi-quantitative WB analysis. The functionality of CD44 was demonstrated through its internalization in the presence of hyaluronan, one of its orthosteric ligands. CONCLUSION: Our study indicates that HBSMC express a functional CD44 receptor. Furthermore, changes in its level of expression suggest a role for CD44 in cell growth. However, whether MMP-9 is an orthosteric ligand for CD44 and regulates HBSMC function remain to be determined. RATIONALE: Bronchopulmonary dysplasia (BPD), characterized by arrested lung growth and alveolarization, is a leading cause of morbidity in premature infants. Glucocorticoid (GC), retinoic acid (RA) and trans- forming growth factor- 1 (TGF-B1) regulate lung development and are implicated in the pathology of BPD. Late-Gestation-Lung 1 (LGL1) is reduced in O2 toxicity models of BPD and normalizes during recovery. LGL1 transgenic mice show advanced lung maturation. Analysis of the LGL1 promoter identified binding sites for GC receptor, RA, vit D and SMAD 3/4 (mediates TGF-B1 signaling). We hypothesized that GC, RA, vit D and TGF-B1 regulate LGL1 expression during late lung development. METHODS: Fibroblasts isolated from fetal and neonatal rat lung were treated with 10-7 M GC, 10-5 M RA, 10-8M vit D, 25 ng/ml TGF- B1or combinations of these for up to 72 hr (n>4). LGL1 mRNA was quantified by real-time RT-PCR. Embryonic day (E) 20 and postnatal day (PN) 7 rat lung fibroblasts transfected with an LGL1 promoter-driven luciferase expression construct were similarly treated and assayed for luciferase activity (n=4). RESULTS: GC treatment for 24 hr stimulated LGL1 mRNA from E18 until PN14 (range 7-115 fold), with maximal increase observed from PN1 to PN4. RA treatment for 48-72 hr, but not 24 hr, led to a reduction in LGL1 mRNA (5 fold). TGF-B1 and vit D caused a decrease in LGL1 mRNA. Luciferase assays confirmed these results. CONCLUSION: GC and RA have opposing effects on lung maturation and repair in the face of O2 injury. TGF-B1 influences lung growth and inflammation. Our findings suggest that these agents modulate the expression of LGL1 during lung maturation and in neonatal lung. Severe lesions of airway epithelia are observed in cystic fibrosis (CF) patients. Epithelial repair then involve cell migration and proliferation processes. Their regulatory mechanisms, controlled partially by growth fac-tors (EGF) secreted by epithelial cells or fibroblasts, are not well defined. A model of mechanical wound of non-CF (NuLi) and CF (CuFi) bronchial cell monolayers was employed to study their (WT) CFTR tagged with green fluorescent protein (GFP) can traffic to the apical membrane of polarized CF airway epithelial cell (CFBE41o-) monolayers whereas the GFP-tagged mutant protein has a distribution consistent with the ER. Apical expression of WT-CFTR is correlated with an increase in the trans-epithelial resistance (TER) (518 +/- 69 ohm•cm2 vs control: 381 +/- 44 ohm•cm2, n=6, p<0.05). Expression of GFP- dF508-CFTR does not affect TER (353 +/- 69 ohm•cm2, n=6, p=1). In non-CF polarized CALU-3 cells, GFP-CFTR, but not GFP-dF508-CFTR, is trafficked to the apical membrane, but TER is not altered (1800 +/- 378.9 ohms•cm2 vs control: 1243 +/- 243 ohms•cm2, n=4, p=0.25). This shows that the barrier function of CF epithelial cells is enhanced by expression of WT CFTR, and that CFTR apical targeting is important for cell polarization. Defective trafficking of dF508-CFTR may impair the barrier function of airway epithelium and its ability regener-ate in CF. BACKGROUND: While the importance of phosphodiesterases (PDE) in the regulation of cell function is now widely accepted as a pathway to potential respiratory drug candidates, data from large, long-term clinical trials with the first selective PDE4 oral drugs have significantly tempered expectations due to serious dose limiting side effects. Topigen has devel- oped TPI 1100, a combination of two antisense oligonucleotides (AON) targeting PDE subtypes 4B, 4D and 7A, which are major regulators of cAMP metabolism in lung tissue and immune cells. PURPOSE: The aim of this study was to investigate the potential of an antisense therapy directed against specific PDE isoforms for reducing lung inflammation. METHODS: TPI 1100 was transfected in lung epithelial cells (A549 or NHBE) and PDE gene expression was measured by RT-PCR. Cells were stimulated with IL-1 β following treatment with TPI 1100 and the quan-tification of IL-8, MCP-1 and GM-CSF protein levels were performed by ELISA. The effect of PDE inhibition on lung inflammation was assessed in a sub-acute cigarette smoke model in mice. RESULTS: Transfected in lung epithelial cells, TPI 1100 nearly abolished PDE mRNA expression and reduced by at least 50% the secretion level of inflammatory markers (IL-8, MCP-1 and GM-CSF) following stimulation with IL-1 β . In mice, TPI 1100 reduced target gene expression in lung lavage cells and blocked neutrophil recruitment (68%; p<0.05) induced by sub-acute exposure to cigarette smoke. CONCLUSION: Delivered directly to the lungs, TPI 1100 represents a novel RNA-antagonist therapy for COPD without the systemic side effects widely associated with known small molecule inhibitors of PDE. BACKGROUND: Sleep apnea (SA) is highly prevalent among dialysis patients and contributes to impaired quality of life and adverse clinical outcomes. Increasing dialysis beyond conventional thrice-weekly sched- ules can improve SA, but is impractical. Hemodiafiltration (HDF) is an adjunct to Conventional Hemodialysis (CHD) which can increase dialysis efficiency during conventional schedules. The aim of the present study was to compare SA severity during 3 months of CHD vs. HDF while main-taining a conventional 3 day per week schedule. METHODS: 15 clinically stable CHD patients identified with SA on an overnight polysomnograpm (apnea-hypopnea hypopnea index (AHI) > 15 events/h) have been randomized to two 3-month periods of CHD and HDF, in random order. Follow-up complete in-home PSG has been performed at the end of each 3-month intervention period. Full dialysis details and body weight are recorded at each dialysis session, and Creatinine clearance and B2 microglobulin are periodically assessed. 24h blood pressure monitoring, Epworth score, generic and sleep-related quality of life indices are determined at baseline and the end of each intervention period. RESULTS: 5 men and 1 woman (mean age 59 SD 11 ) of the 15 enrolled have completed the protocol. The mean AHI with CHD is 46.3 SD 14.9 compared to 39.9 SD 15 events/h on HDF (p = .56). CONCLUSIONS: Our pilot data thus far indicates that the study is design is feasible; however data collection on the remaining recruits will have to be completed before further conclusions can be drawn. Upper airway stabilizing muscles play a crucial role in the maintenance of UA patency. Transcranial magnetic stimulation allows the investigation of respiratory muscles’ corticomotor activation process. This technique has also been used to evaluate the genioglossus corticomotor response. The aims of this study were to characterize the response of different upper air- way stabilizing muscles to focal cortical stimulation of the genioglossus. METHODS: Alae nasi, genioglossus, levator palatini, palatoglossus and diaphragm motor evoked potential responses to transcranial magnetic stimulation were recorded during expiration, tidal inspiration and deep inspiration in 9 normal awake subjects. RESULTS: A concomitant response of the four studied upper airway mus- cles was observed in the majority of cortical stimuli. The response of these muscles was independent of the diaphragmatic one that was only occa- sionally observed. Significant positive relationships were found between alae nasi, levator palatini and palatoglossus motor evoked potential laten- cies and amplitudes and the corresponding values of the genioglossus. CONCLUSION: Transcranial magnetic stimulation applied in the genioglossus area induces a concomitant motor response of upper airway stabilizing muscles with consistent changes in their motor responses during inspiratory maneuvers. BACKGROUND: A proportion of subjects with work-related respiratory symptoms (WRS) may be misclassified as asthmatic subjects. We sought to assess the proportion of confirmed diagnoses of WRA in a cohort of work- ers with respiratory symptoms suggestive of WRA and to identify the types of occupations associated with WRA and WRS. METHODS: Prospective observational study of workers referred for symptoms suggestive of WRA over a one-year period. Detailed medical and occupational questionnaires were asked. Pulmonary function tests were performed in order to confirm the diagnosis of asthma. Specific inhalation challenges to occupational agents were performed in order to distinguish occupational asthma (OA) from work-exacerbated asthma (WEA). RESULTS: One hundred and twenty workers were investigated: 51 had WRA (OA: 33; WEA: more frequently done in swimmers. Further studies are needed to understand why respiratory symptoms do not correlate with these challenges. cockroaches, tobacco smoke) and outdoor (e.g., urban pollution) allergens, and poorer access to health care, which may increase risk for asthma exacerbations. Though the SES- asthma link has been well established in children, less is known about this association in adult asthmatics. This study assessed associations between adult SES and five measures of asthma morbidity in 782 adult asthmatics (n=467 women): asthma control; pulmonary function (%FEV1); asthma- related health service use in the past year (emergency department, ED vis-its and hospitalizations); asthma self-efficacy, and asthma-related quality of life. All patients underwent a sociodemographic and medical history inter-view and pulmonary function testing on the day of their asthma clinic visit, and completed a battery of questionnaires (Asthma Control Questionnaire, ACQ; Asthma Quality of Life Questionnaire, AQLQ; Asthma Self-Efficacy Scale, ASES). General Linear Model’s assessed associations between SES (measured using educational level: M=12.9 yrs; range 2-35 yrs) and each morbidity measure, controlling for age, sex and asthma severity. Results indicated that lower SES was significantly associ- ated with worse asthma control (F=11.63, p<.001) greater health service use (hospitalizations) (F=5.96, p<.05), and worse asthma self-efficacy (F=12.04, p<.01), independent of covariates. SES was not related to worse pulmonary function or quality of life. Results suggest that SES (measured according to education level), is associated with several indices of worse asthma morbidity in adult asthmatics. Results are consistent with previous studies linking lower SES to worse asthma in children. Future studies should examine the mechanisms by which low SES adversely impacts asthma among adults. CONCLUSION: Results suggest that smoking influences AQL when controlling for age and sex. However, the relationship between smoking and AQL seems to be mediated by asthma control. Further studies are needed to explore the mechanisms and impact of these relationships. For example, further examination of the relationship using packages/year and an assessment of the longitudinal relationship is warranted. > 60% et trois, une éosinophilie > 2%. Le score de qualité de vie était de 4.3 (ET= 1.5) sur une échelle de 0 (pire) à 7 (meilleur). Le score général du questionnaire psychologique utilisé était de 25 (ET = 21), onze sujets ayant un score > 25 (légèrement anomal). En conclusion, notre étude montre que dans cet échantillon de 30 sujets ayant eu un SIrB significatif (18 ayant dû consulter dans les jours qui ont suivi l’événement) et revus en moyenne 12.6 ans après l’événement accidentel : 1) tous sont encore symptomatiques; 2) seulement 4 n’ont plus d’hyperex-citabilité bronchique; 3) l’expectoration induite ne montre pas d’inflammation; 4) il y a une atteinte franche de la qualité de vie et de l’état psychologique. et altération des fonctions pulmonaires. La signification neutrophilie demeure Inhaled corticosteroids (ICS) play an important role in the management of asthma symptoms by reducing airway inflammation, one of the hallmark physiological characteristics of this respiratory illness. Several studies have shown that asthma is related to body weight and weight gain and asthma exacerbation, and that this relationship might be sex specific. Corticosteroids have been associated with weight gain. However, little is known about the effect of ICS use on weight gain among patients with asthma. We hypothesized that the use of ICS will trigger weight gain among patients with asthma, and specifically, women with asthma will have greater weight gain compared to men. In total 169 adult patients with asthma provided details of their medical history (including ICS use) and demographic information (including weight and height). All participants provided the same information at one year. General linear models revealed a main effect of ICS dose (F=5.23, P=.024), sex (F=3.87, P=.051), as well as the interaction between ICS dose and sex (F=4.69, P=.032) on BMI change, controlling for age, smoking status, level of education and baseline BMI. Results indicate that a greater ICS dose was associated with increased weight gain at 1 year and women had greater weight gain than men. Further analysis of the interaction indicated that women had greater weight gain with increased ICS dose; whist men had decreased weight gain with increased ICS dose. This finding could possibly be due to hormonal sex differences interacting with ICS usage, yet more studies are needed to examine this relationship. Prognostic model among apprentices exposed to laboratory animal (LA) allergens will support career counseling and decision making in medical surveillance. OBJECTIVES: Develop prognostic models for incidence of sensitization to LA allergens and symptoms at work. METHODS: The models were derived from cohort data of Canadian animal health technology apprentices. The potential predictors at baseline were used to predict health endpoints at the 32-month visit. Four multivariable logistic regression models were developed for each endpoint: (1) question- naire, (2) questionnaire and skin reactivity to common allergens (atopy), (3) questionnaire and bronchial responsiveness (BR) to methacholine, and (4) questionnaire, atopy, and BR model. The models’ validity was assessed inter-nally by bootstrapping procedure. The models’ calibration was evaluated with the Hosmer-Lemeshow (HL) test; the discrimination was determined by the area under the receiver operating characteristic curve (ROC area). RESULTS: The final questionnaire model for LA sensitization and symp- toms at work consisted of symptoms indicative for asthma and symptoms in contact with pets. For LA sensitization, only the ROC area of Model 4 (0.78) was significantly higher than Model 1 (0.72). highest ferent both endpoints, the predictive All good calibration (H-L p-value > 0.10) and internal validity. CONCLUSIONS: questionnaire models were recommended for sur-veillance for occurrence of sensitization to LA allergens and symptoms at work. completed, compared to 339 of 420 (81%) who completed 4RIF (P<.0001). Grade 3-4 SAE occurred in 15 (4.1%) on 9INH, compared to 5 (1.8%) taking 4RIF (p=.02). Grade 3-4 hepatitis occurred in 15 of 427 (4.1%) randomized to 9INH, compared to 3 of 420 (0.8%) taking 4RIF (P=.002). Grade 1-2 AE judged probably! related to study drugs by the DSMB were not different between the two arms. CONCLUSIONS: Compared to 9INH, 4RIF had significantly better completion rate, and significantly lower rate of grade 3-4 SAE particularly hepatotoxicity. These findings justify a large scale trial to assess efficacy. The emergence of multi-drug resistant (MDR) tuberculosis (TB) poses a major challenge to the control of world-wide. In this present study, we analyzed the association between prevalence of initial MDR and outcomes of initial treatment and re-treatment. 250 cases annually 2003 were selected for analysis. Prevalence of initial MDR was taken from global were into four cate- gories on of initial MDR: 1-2%, 2-3 and T-tests. WHO standard treatment regimens, failure rates with initial therapy and retreatment were significantly higher in countries with the highest MDR prevalence (p<0.0001). CONCLUSION: Prevalence of MDR in new cases, is highly associated with rates of failure of initial therapy and retreatment. Current recommended standardized treatment regimens should be re-evaluated in coun- tries with high prevalence of MDR-TB. RATIONALE: This study tried to validate the relationship of the GOLD staging system with respiratory symptoms presentation in a clientele at risk of COPD. METHODS: A program for early detection of COPD was introduced in 11 family medicine practice clinics. Patients selected for screening did not have a previous diagnosis of COPD or asthma and were not under regular respiratory medication. Post-BD spirometry was performed on patients over the age of 40, having a minimum of 10 pack-years smoking history and at least one respiratory symptom (cough, cough with sputum, shortness of breath of at least 2 in the MRC scale, wheezing or frequent respiratory infections). COPD was classified based on the GOLD Standards (2001). RESULTS: We screened 240 eligible subjects (mean age 55.7 years, smoking history 33.7 pack-years). After performing spirometry, we discovered 31 new cases of COPD (GOLD 0=191, GOLD I=18, GOLD II=31). We found a trend indicating higher prevalence of COPD among subjects presenting 2 or more respiratory symptoms as compared to those with only 1 symptom (15% vs. 8%, p=0.18). No statistically significant differences were seen among COPD stages in terms of number of respiratory symptoms present, type of symptoms or severity of shortness of breath. CONCLUSIONS: The recent GOLD update (2006) has eliminated an important COPD stage, “0: at risk”. The incidence of chronic respiratory symptoms in subjects negative to COPD represents an unmet need for treatment. As well, spirometry remains absolutely necessary to establish a diagnosis of COPD; symptom presentation is not a reliable indicator of the of


GAPDH : UN NOUVEAU PARTENAIRE DU RÉCEPTEUR DE LA FRACTALKINE
A Chamberland, S Gagnon, V Legendre-Guillemin, C Laprise Université du Québec à Chicoutimi, Chicoutimi, Québec CONTEXTE: Des résultats issus des puces à ADN et d'une étude d'association déjà réalisée, ont permis de cibler le récepteur de la fractalkine (CX3CR1) comme biomarqueur dans l'asthme. Ainsi, des analyses fonctionnelles devraient permettre de documenter le rôle de CX3CR1 dans la pathophysiologie, en particulier la composante inflammatoire, de l'asthme. OBJECTIFS: 1) Cloner et produire des protéines de fusion GST incluant respectivement chaque domaine intracellulaire de CX3CR1. 2) Identifier les partenaires intracellulaires de liaison pour chaque domaine de CX3CR1. MÉTHODE: Les quatre domaines intracellulaires de CX3CR1 ont été clonés dans le vecteur pGEX4T1 puis l'expression des protéines de fusion GST a été vérifiée sur gel SDS-PAGE. Les partenaires de liaison aux différents domaines intracellulaires de CX3CR1 ont été identifiés par "pulldown" à partir de lysat de foie adulte de rat. RÉSULTATS: Jusqu'à présent, des protéines de fusion GST ont été obtenues pour trois des quatre domaines intracellulaires de CX3CR1. Puis, un candidat spécifique au deuxième domaine intracellulaire de CX3CR1 a été identifié par spectrométrie de masse. La protéine identifiée est la glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cette interaction spécifique a été confirmée par western blot. PERSPECTIVES: La suite de l'étude fonctionnelle consistera à caractériser l'implication de la GAPDH dans la fonction de CX3CR1 au niveau des lymphocytes T et plus particulièrement dans le CONTEXTE de l'asthme utilisant des lymphocytes CD8 isolés à partir de prélèvements sanguins chez des sujets asthmatiques. Immuno-modulatory compounds are increasingly studied for their therapeutic potential in asthma. However, the mechanisms by which they induce favourable clinical outcomes are incompletely understood. Using a Balb/c murine model of allergic asthma, we have found that mice sensitized to a birch tree pollen extract (BPEx) and then subsequently treated with a mixture of BPEx and a mucosal adjuvant, ProtollinTM, had partial inhibition of airway hyperresponsiveness, eosinophilia and serum IgE, measured 48 hours following the final BPEx allergen challenge. We are seeking to clarify the mechanisms by which the intranasal co-administration of Protollin and BPEx (Pro/BPEx), subsequent to allergen sensitization, but prior to allergen challenge, inhibits features of experimental allergic asthma. We wished to specifically examine whether Protollin, which partially consists of toll-like receptor 2 and 4 agonists, suppresses birch polleninduced allergic asthma by skewing the immune response away from a type 2 towards a type 1 profile (interferon (IFN)-γ) and/or by inducing regulatory T cells and anti-inflammatory cytokines, such as interleukin (IL)-10. Balb/c mice were sensitized to BPEx and intranasally "immunized" with a mixture of Pro/BPEx or BPEx alone on Days 7, 10 and 13. Using splenocyte cell culture techniques we assessed the systemic effects of Pro/BPEx immunization. Splenocytes harvested and cultured for 96 hrs with BPEx, one day after the final immunization (Day 14), expressed the cytokines IFN-γ, IL-4, IL-13 and IL-10, as well as the T helper (Th)2 cell-and Th1 cell-differentiating transcription factors, GATA-3 and T-bet, respectively. Splenocytes from mice pre-treated in vivo with Pro/BPEx, as opposed to BPEx alone, had significantly lower expression of GATA-3 and T-bet, although there was no difference in Th1 or Th2 cytokine expression. IL-10 mRNA and protein expression were higher in the splenocytes and supernatants, respectively, of Pro/BPEx-treated mice. However, IL-13 and IFN-γ protein levels were also elevated in the supernatants of Pro/BPEx-treated mice compared to BPEx-treated mice. Thus, the inhibitory effect of Protollin in this model may involve elements related to regulatory T cell activation, as well as, Th1 biasing. Supported by CIHR grant #10381

REGULATION OF ALVEOLAR ENAC CHANNEL EXPRESSION FOLLOWING CHANGES IN KVLQT1 AND KATP K + CHANNEL ACTIVITY
JC Bourret 1,2 , O Bardou 2 , A Privé 2 , A Dagenais 2 , Y Berthiaume 1,2 , E Brochiero 1,2 1 UdeM, Département de Médecine, Montréal, Québec; 2 CRCHUM Hôtel-Dieu, Montréal, Québec Normal respiratory function and resorption of lung oedemas are known to rely on Na + transport via ENaC channel through alveolar epithelium. We have recently shown that K ATP and KvLQT1 K + channel activity could control Na + and Cltransport (via ENaC and CFTR channels) as well as fluid clearance across alveolar ATII cells monolayers. We then explored the hypothesis that K + channel activity could have an impact on ENaC expression. We observed that K ATP and KvLQT1 inhibitors (respectively glibenclamide and clofilium) downregulated ENaC expression (mRNA and protein) while pinacidil (K ATP activator) upregulated it. ENac expression could also be regulated after changes in Na + and K + fluxes (with ouabain, valinomycin, monensin). To further study the ENaC regulation mechanisms, we tested the impact of the K + channel modulators on ENaC promoter activity in transfected A549 cells. We found a 2-fold increase in ENaC promoter activity following pinacidil treatment. Regulation of ENaC channel by K + transport is a new avenue and could be of great benefit for the resolution of lung pathologies characterized by ion and fluid transport dysfunction. Financial support of NSERC METHODS: Mice were subjected to OVA sensitization followed by intranasal administration of budesonide. Airway responsiveness to methacholine was measured in anesthetized mechanically ventilated animals. Real-time PCR was used to quantify lung GR and IPO13 (mediates nuclear import of GR) mRNA. Plasma IgE was determined by ELISA. RESULTS: Both Balb/c and C57BL/6 mice had significantly increased respiratory resistance following OVA challenge. Budesonide treatment improved pulmonary function of C57BL/6 but had no effect on Balb/c mice. OVA-induced IgE levels were several fold higher in Balb/c compared to C57BL/6. While budesonide treatment normalized IgE levels in C57BL/6 mice, IgE in Balb/c mice remained unchanged. OVA sensitization lead to significant reduction in IPO13 and GR mRNA levels in both mouse strains. Budesonide treatment restored IPO13 mRNA in C57BL/6 mice to control levels and stimulated GR mRNA to 3-fold over control. In Balb/c mice, budesonide restored GR and IPO13 mRNA to control levels. CONCLUSIONS: Our findings demonstrate a significant difference in steroid responsiveness in C57BL/6 vs. Balb/c mice. The resistance to exogenous steroid observed in the Balb/c mice may reflect a dysregulation of handling of endogenous steroid which in turn may contribute to hyperresponsiveness.

MODULATION OF ASTHMA PATHOGENESIS, DENDRITIC CELL MATURATION AND HOST T CELL RECRUITMENT BY GENE-MODIFIED T CELLS OVEREXPRESSING IL-13
E Nakada, M Kinyanjui, E Fixman Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec BACKGROUND: Interleukin (IL)-13 is a key Th2 mediator in asthma pathogenesis. While the IL-13 receptor (IL-13R), comprised of the IL-4Rα and IL-13Rα1 subunits, is expressed by various hemopoietic and nonhemopoietic cells including dendritic cells (DC), airway smooth muscle (ASM) and airway epithelial cells, functional IL-13Rs are not expressed on T cells. We are interested in defining mechanism(s) by which IL-13 regulates T cell function and pathogenesis in experimental asthma. HYPOTHESIS: T cell specific overexpression of IL-13 enhances DC maturation, host T cell recruitment and ASM remodeling in the airways of rats with experimental asthma. METHODS: We have developed methodology to produce high titer recombinant retroviruses with the ability to transduce primary ovalbumin (OVA)-specific Brown Norway (BN) rat CD4+ T cells. We will generate gene-modified CD4+ T cells overexpressing IL-13 and the enhanced green fluorescence protein (EGFP) marker. Following retroviral transduction, gene-modified T cells will be purified magnetically, FACS-sorted and then adoptively transferred into naïve BN rats that will subsequently be challenged with OVA on three occasions. We will assess the effects of T cellspecific IL-13 overproduction on several of the classic pathological features of asthma including (1) airway eosinophilic inflammation, (2) mucus production and (3) ASM remodeling. We will also examine its role in (4) DC maturation in the lungs and draining lymph nodes, as well as (5) host T cell recruitment to the lungs. SIGNIFICANCE: These studies will define the role of IL-13 in the ability of adoptively transferred T cells to induce pathogenesis in experimental asthma.

CHANGES IN UPPER AND LOWER AIRWAY INFLAMMATION FOLLOWING REPEATED NASAL ALLERGEN CHALLENGES IN RHINITIC SUBJECTS WITH OR WITHOUT ASTHMA
MC Rousseau 1 , ME Boulay 1 , L Goronfolah 2 , M Lin 2 , P Ferrie 2 , JA Denburg 2 , P Keith 2 , LP Boulet 1 1 Centre de recherche de l'Hôpital Laval, Institut universitaire de cardiologie et de pneumologie de l'Université Laval, Québec; 2 McMaster University, Health Sciences, Hamilton, Ontario BACKGROUND: Asthma and allergic rhinitis may result from a similar allergen-induced inflammatory process, but studies are needed to determine possible mechanisms by which one influences the other.

PURPOSE:
To determine the influence of repeated nasal allergen challenges on upper and lower airway inflammation in rhinitic subjects with or without asthma. METHODS: Thirty-six subjects with allergic rhinitis were recruited: 21 mild asthmatics (A) and 15 non-asthmatics (R). Subjects underwent a nasal control challenge with normal saline followed by 4 consecutive daily allergen challenges. At each challenge day, increasing allergen dilutions were sprayed into each nostril until a positive response occurred. Induced sputum (IS) and nasal lavage (NL) with differential cell counts were obtained 7 hours following the control, the first and the last challenge. RESULTS: No difference between groups was observed in the percentage of IS and NL eosinophils at any points (p>0.05). No change in the percentage of IS eosinophils was observed after one and 4 days of challenge compared with control day (p>0.05), but some subjects (A=4 (19%) and R=3 (20%)) had an increase (≥2%) after the first and/or the last challenge. An increase in the percentage of NL eosinophils was observed after 4 days of challenge compared with control challenge (mean±SEM: 16.9±23.0% vs 3.8±9.6%; p<0.05), but not after 1 day (8.8±17.1%; p>0.05). CONCLUSIONS: Repeated nasal allergen challenge is effective to induce upper airway inflammation, but only induces lower airway inflammation in about one in 5 subjects. The presence or absence of asthma did not appear to make a difference. Asthma is a chronic inflammatory disease of the airways characterized by an infiltration of activated inflammatory cells like T lymphocytes into bronchial mucosal tissues. These T cells are in close association with resident cells, such as fibroblasts, raising the possibility of cross talk between these two types of cells. OBJECTIVES: The aim of our study was to evaluate the effect of T cells interaction with bronchial fibroblasts on IL-6 production. METHODS: Bronchial fibroblasts were isolated from mild asthmatics and non atopic healthy controls. T cells were purified from the peripheral blood of healthy and asthmatic subjects. Co-culture of confluent healthy (BNF) or asthmatic bronchial fibroblasts (BAF) with T cells were performed in presence or absence of blocking antibody anti CD18. RESULTS: Direct contact of T cells significantly stimulated IL-6 production by both BNF and BAF. This effect was significantly higher in BAF than BNF (881±113.39%vs 452.70±92.40%; p=0,02  'âge médian était de 3,8 ans (3 jours-17 ans). Les principales indications de VI étaient : 37,3% une défaillance respiratoire, 18,1% en post-opératoire de chirurgie cardiaque,14,2% un état de choc. La médiane de la durée de séjour aux SI était de 9 jours (1-92), la médiane de la durée de VI était de 1,5 jours (0-77 jours). En analyse univariée, les facteurs de risque associés à une VI prolongée étaient un poids < 10 kg, une admission pour polytraumatisme (p = 0,016), défaillance respiratoire (p<0,001) ou syndrome infectieux (p = 0,001), un score de PRISM ≥ 10 (p<0,001), un score de PELOD à l'admission ≥ 10 (p = 0,001), la présence d'« Acute Lung Injury » (p = 0,006), une pression moyenne des voies aériennes ≥ 13 cmH2O au 2ème jour de VI (p<0,001) et une sédation continue au 2ème jour de VI (p<0,001). Aux SI pédiatriques, 30% des patients sont ventilés plus de 4 jours. La gravité des patients à l'admission, le jeune âge et la défaillance respiratoire sont associés à une VI prolongée. Les résultats de cette étude sont préliminaires. L'analyse sur un an permettra de définir précocement la population à risque de VI prolongée et d'identifier les facteurs de risque permettant d'améliorer leur prise en charge.

THE VERSATILITY OF PROTON MAGNETIC RESONANCE IMAGING (MRI) IN PULMONARY RESEARCH
H Karmouty-Quintana 1,2 , N Beckmann 2 , C Page 1 , JR Fozard 2 1 King's College London, United Kingdom; 2 Novartis Pharma AG, Basel, Switzerland MRI has only recently been applied in the area of pulmonary pharmacology. Scans are performed in spontaneously breathing rats deriving anatomical and functional read outs of models of airways disease non-invasively with the aim to profile drugs. Examples of distinct pulmonary changes in animal models are described. Edema: Challenge with the mast cell secretagogue, compound 48/80 has been shown to cause a marked edematous MRI signal 24-48 hrs after treatment that correlates with increased protein concentration in bronchoalveolar lavage (BAL) fluid. The observations are qualitatively similar to rats sensitized and challenged with ovalbumin (OA) and imaged 24 h after challenge. Mucus: Treatment with capsaicin results in a qualitatively distinct signal to that of OA or 48/80 that correlates with increased mucin concentration present in BAL fluid. Pleural Inflammation: Edema associated to the pleural space has been detected at early time points (6 h) following OA challenge. Emphysema: The induction of experimental emphysema by elastase leading to air trapping results in modulations in the signal from the lung parenchyma that can be detected by MRI. The non-invasive nature of MRI and the versatility of this tool provide the basis for testing of compounds for respiratory diseases. It also opens the opportunity for the clinical assessment of distinct lung pathologies noninvasively by MRI.

16
A POPULATION-BASED STUDY OF COMPLICATIONS OF LATENT TUBERCULOSIS TREATMENT B Smith, M-A Sepas-Khah, D Menzies, K Schwartzman, G Bartlett McGill University, Montreal, Quebec Current guidelines recommend treatment of latent tuberculosis infection (LTBI) for patients at risk of developing active TB. Physicians must weigh the benefits of TB prevention against the risk of adverse events. Our objective was to determine rates of adverse events, hospitalization and completion associated with LTBI therapy. A historical cohort of all Québec residents dispensed at least 30 days of LTBI therapy between January 1, 1998 and December 31, 2003 was constructed using the provincial healthcare databases. For each case, two age and sex matched controls were identified. Rates of therapy completion, hospitalization, and adverse events were compared. RESULTS: Over 6 years, 9,393 Québec residents were dispensed at least 30 days of LTBI therapy. Isoniazid (INH) alone was given to 90.4%, Rifampin alone to 6.4% , and rifampin and pyrazinamide (RIF PZA) to 0.4%. Overall completion was 52%, and was lower with older age. Probable drug-related serious adverse events requiring hospitalization occurred in 1.2% of persons taking INH, 2.5% of persons taking RIF, and 0% of persons taking RIF-PZA. Hospitalization rates, hospitalization duration, and serious adverse events were significantly greater among those receiving LTBI therapy vs. controls. Probable drug-induced liver disease requiring hospitalization occurred in 0.3% of patients dispensed INH, 0.1% for RIF. Rates of severe adverse events related to LTBI therapy increased with age. Overall mortality for the six year period was 4.4% among the LTBI cohort and 4.3% among controls. CONCLUSION: Rates of severe adverse events associated with LTBI therapy were low, and similar to previous reports from specialty TB clinics. Recognizing the patient perspective is essential in achieving effective tuberculosis (TB) control. TB treatment is free to patients in most DOTS programs, but the patient must bear the costs of care prior to diagnosis, and costs associated with hospitalization, and travel/time with DOT. These costs may deter patients from seeking care for their TB-related symptoms or create a major barrier to continued treatment. Thus, patient-related cost information is relevant and necessary in assessing the impact of program design on TB control. PURPOSE: To study the economic impact and burden of care-seeking for pulmonary TB patients in 4 low/middle-income countries, and 3 highincome countries including: Ecuador, Zambia, Brazil, China, Canada, the Netherlands, and the United Kingdom. METHODS: An in-depth questionnaire was adapted from the pilot study and translated into local languages. Locally trained interviewers administered questionnaires to adults with active bacteriologically confirmed pulmonary TB, who had completed approximately two months of treatment, at government health facilities using the DOTS strategy. Direct (out-ofpocket expenditures) and indirect (time) costs were estimated. Approximately 100 patients were interviewed in each low/middle income country, and 50 patients were interviewed in each high income country. RESULTS: Preliminary analysis revealed that patient expenditures for TB were significant at all sites. When taken as a proportion of the annual gross national income, patients' expenditures ranged from 10.27% in Canada to 26.10% in Ecuador. The economic burden on patients was the most significant during the diagnosis period. Asthma is a chronic respiratory disease that has been previously associated with a disproportionately high level of suicidal ideation (SI), independent of depression. However, the precise mechanisms linking asthma to suicidal ideation remain poorly understood. Several asthma medications like theophylline and beta-2 adrenergicagnonists (which are potent bronchodilators), have been shown to provoke intense feelings of anxiety, fear, or panic in some individuals. These drugs may potentially influence rates of SI in asthma patients, but this has yet to be explored. The present study assessed SI in 630 consecutive adult asthma patients presenting to an outpatient clinic (40% men ; mean age=50 yrs). METHODS: All patients underwent a sociodemographic, psychiatric (PRIME-MD), and medical history interview, and completed a battery of questionnaires including the Beck Depression Inventory-II (BDI-II) and Asthma Control Questionnaire (ACQ). Responses e 1 on question 9 of the BDI were used to measure SI. RESULTS: BDI-II results indicated that 12% of asthmatics reported having SI. After controlling for age, sex, smoking status, and major depression, results of general linear model analyses indicated a significant main effect theophylline use (F=3.85, p<.05), but not beta-2 agonist use (F=0.04, p=.8497), on SI. Though asthma control and severity levels were also significantly associated with SI in univariate analyses, these associations were no longer significant after controlling for covariates. CONCLUSIONS: These findings suggest that theophylline use may be associated with a higher risk of SI in adult asthmatics. It is noteworthy that these findings were independent of major depression, which suggests results are not simply the results of depressive symptomatology in asthma patients. Clinically, these findings indicate that physicians should be vigilant about the potential psychological side effects of certain asthma medications, particularly those that enhance central nervous system activity.

CONSTRUCT, ITEM DEVELOPMENT AND FORMAT OF A NEW PATIENTS' NEEDS ASSESSMENT INSTRUMENT FOR PULMONARY REHABILITATION
JL Sully 1,2 , L Demers 1 , MA Baltzan 2,3 , N Wolkove 2 1 Université de Montréal; 2 Centre hospitalier Mont-Sinaï; 3 Université McGill, Montreal, Quebec BACKGROUND: Pulmonary rehabilitation (PR) programs have been built to meet the various needs of patients suffering from COPD. Knowing to what extent these needs are being met by PR programs is difficult because while health related quality of life (HRQoL) is generally considered as a main outcome of PR, the fulfillment of needs, which some authors view as a central component of HRQOL, is often overlooked in the assessment of PR outcomes. PURPOSE: To develop and pre test an instrument based on a conceptual model of patients needs assessment in PR. METHODS: A list of 120 Items corresponding to our model of needs assessment was submitted to a panel of 14 clinicians involved in PR. Following comments from this panel, questionnaire content was reduced to 98 items. Three successive versions were administered to a total of 15 patients in order to further reduce content to 80 items. The final version corresponds to a specific visual format reflecting the domains of need recognition as well as degree of knowledge, motivation, expectations and goals. Psychometric validation studies of this questionnaire should follow.

MONTELUKAST DOWNREGULATES THE RELEASE OF CYSTEINYL-LEUKOTRIENES BY BLOOD EOSINOPHILS THROUGH A MECHANISM UNRELATED TO CYSLT RECEPTOR BLOCKADE
F Chouinard, A Langlois, N Flamand, C Ferland, M Laviolette Unité de recherche en pneumologie, Centre de recherche de l'Hôpital Laval, Québec, Québec Cysteinyl leukotrienes (CysLT) (LTC4, LTD4 and LTE4) play major roles in asthma pathogenesis. Asthma is characterized by leukocyte infiltration in the bronchial mucosa, notably eosinophils (Eo), which are a main source of CysLT. Montelukast (MTK), a CysLT receptor antagonist used in asthma treatment, diminishes the inflammation caused by CysLT. In this study, we evaluated the effect of MTK on calcium ionophore (A23187)induced LTC4 release from isolated Eo. Asthmatics' blood Eo (n = 4) were incubated with MTK or the Pertussis toxin (PTX) which inactivates Gi-proteins putatively coupled to CysLT receptor. Eo were then stimulated with an increasing concentration of A23187 (0.1-2 uM). LTC4 levels were determined in cell-free supernatants by enzyme-linked immunoassay. MTK inhibits, in a concentration-dependant manner, the A23187-induced (0.1 uM) LTC4 biosynthesis with an IC50 of 0.14 uM and abrogated it at a concentration of 1uM. Interestingly, increasing concentration of A23187 (up to 2 uM) did not prevent the inhibitory effect of MTK on LTC4 biosynthesis. PTX (25 ng/ml) decreased the A23187-induced LTC4 release by only 30% (p=0.03); such an effect was not sufficient to support a possible inhibition of LTC4 autocrine stimulation by MTK. These data suggest that MTK decreases LTC4 release by a mechanism unrelated to CysLT receptor blockade. To confirm that this inhibition is relevant in vivo, LT release will have to be evaluated ex vivo in blood of asthmatics taking MTK.

PYRIDINE NUCLEOTIDES AND OXIDATIVE STRESS REGULATE 5-OXO-ETE SYNTHESIS IN AGING NEUTROPHILS
F Graham 1 , S Gravel 1 , KR Erlemann 1 , J Rokach 2 , WS Powell 1 1 Meakins-Christie Laboratories, McGill University, Montreal, Quebec; 2 Department of Chemistry, Florida Institute of Technology, Melbourne, Florida, USA 5-oxo-ETE is a recently discovered 5-LO product that may have an important role in asthma due to its chemoattractant properties on eosinophils and neutrophils. It is synthesized by the action of the microsomal enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH) on the 5-LO product 5-HETE in the presence of NADP+. Under normal conditions, 5-oxo-ETE synthesis is inhibited by a high intracellular ratio of NADPH to NADP+ but we speculate that as neutrophils undergo aging, the levels of NADP+ increase, which favors the formation of 5-oxo-ETE. To investigate this hypothesis, we incubated neutrophils for 24 h in the presence of 10% FBS. At this time, 5-oxo-ETE synthesis was 15 ± 4 times higher compared to freshly isolated neutrophils (p <0.001). The increase in 5-oxo-ETE synthesis was paralleled with a dramatic increase in intracellular pyridine nucleotides, measured by HPLC. NADP+ (pmol/10 6 cells) rose from 1.2 ± 0.5 in freshly isolated neutrophils to 18 ± 6 after 24 h, whereas NADPH levels remained nearly constant. NAD+ (pmol/10 6 cells) rose from 17 ± 2 to 136 ± 30 over 24 h, suggesting that the increase in NADP+ could be coming from the activation of neutrophil NAD kinase. The increase in 5-oxo-ETE synthesis was partially inhibited by the survival factor GM-CSF (65 ± 6%), LPS (54 ± 7%), and catalase (53 ± 13%), all of which also reduced the ratio of NADP+/NADPH (0.4 ± 0.1, 0.7 ± 0.4, and 0.5 ± 0.3, respectively) compared to vehicle-treated controls (2.0 ± 0.5). We conclude that in aging neutrophils apoptosis-associated oxidative stress increases NADP levels and 5-oxo-ETE synthesis, which could prolong inflammation due to its chemoattractant effects. Supported by grants from the CIHR, the HSFQ, the QRHTP and the NIH IL-33, a recently described IL-1 cytokine family member, promotes Th2 inflammation but evidence on implications of this cytokine in asthma is lacking. IL-33 is mainly expressed by structural cells including ASMC, but whether pro-inflammatory cytokines and anti-inflammatory drugs modulate its expression in ASMC is unknown. METHODS: Endobronchial biopsies were collected from adults with mild (n = 8), moderate (8), severe (9) asthma and from control subjects (5). Primary ASMC were cultured with or without inflammatory cytokines, Dexamethasone (DEX) or Mithramycin A (MMA). IL-33 expression was investigated by real-time quantitative RT-PCR, immunocytochemistry (ICC) and western blotting. RESULTS: Higher levels of IL-33 transcripts were detected in biopsies from asthmatic patients (mild, moderate and severe) compared to control subjects (p = 0.932, 0.0186 and 0.002, respectively). ICC staining seemed to correlate with transcript levels. In ASMC, TNFα upregulated IL-33 expression in a time-and dose-dependent manner. MMA reduced the TNFα-induced IL-33 upregulation, whereas DEX did not display significant effect. CONCLUSIONS: IL-33 expression increases with asthma severity in bronchial biopsies. In cultured ASMC, TNFα is a potent inducer of IL-33 expression. IL-33 might consist of an alternative therapeutic target for drugs acting on respiratory disease states which are refractory to corticosteroid therapy, such as severe asthma. IL-33 consists of novel Th2 mediator but its precise role in lung inflammatory diseases remains unclear. La Dystrophie Musculaire de Duchenne (DMD) est une maladie neuromusculaire dégénérative causée par la perte de la dystrophine, qui entraîne le décès précoce des patients suite à des dysfonctionnements respiratoires. Une réponse inflammatoire exacerbée et une dérégulation de l'homéostasie Ca2+ sont les caractéristiques pathologiques des muscles DMD. Nous proposons que ces deux phénomènes soient liés à la voie de signalisation des récepteurs Toll-like (TLRs), récepteurs impliqués dans l'immunité innée. Dans cette étude, nous nous sommes intéressés aux TLR-2 et -4, qui reconnaissent respectivement les PGN et les LPS, particules bactériennes. Nos résultats montrent que les muscles de souris saines et mdx (modèle animal de la DMD) expriment les TLR-2 et -4. Afin de déterminer le rôle de ces récepteurs dans l'inflammation, nous avons étudié l'expression des cytokines induite par leur stimulation. Comparés aux myotubes sains, les myotubes mdx montrent une expression plus importante de cytokines suite à la stimulation avec les PGN. Cependant aucune différence n'est observée lorsque les myotubes sont stimulés avec les LPS. Après une incubation des myotubes sains et mdx avec les PGN et LPS, le taux de Ca2+ intracellulaire a été mesuré. Nos résultats montrent un taux de Ca2+ plus élevé dans les myotubes mdx. L'utilisation d'un inhibiteur (FK506) de la calcineurine activée par l'augmentation de Ca2+, abolit l'expression de cytokines induite par les PGN mais seulement dans les myotubes sains. En conclusion, la voie de signalisation médiée par les TLR-2 est exacerbée et régulée différemment dans les myotubes mdx. Financé par : CIHR et RSRQ

COMPARAISON DE L'EXPRESSION GÉNÉTIQUE ET PROTÉIQUE DU HSP60 PAR LES MACROPHAGES ALVÉOLAIRES DE SUJETS TÉMOINS ET ASTHMATIQUES ALLERGIQUES
A-M Madore 1 , V Turmel 2 , M Laviolette 1,2 , E Bissonnette 1,2 , C Laprise 3 1 Université Laval Québec, Québec; 2 Unité de recherche en pneumologie, Hôpital Laval, Québec, Québec; 3 Université du Québec à Chicoutimi, Chicoutimi, Québec CONTEXTE: Jusqu'à maintenant les études n'ont identifié qu'une mince proportion des déterminants génétiques et voies biologiques définissant l'implication des macrophages alvéolaires (MA) dans l'asthme. L'étude du transcriptome des MA de sujets témoins (T) et de sujets asthmatiques allergiques (AA) ont permis de cibler plusieurs gènes d'intérêt dont 3 ont été validés en PCR en temps réel: HSPD1, SERPINH1 et PRNP. Parmi ceux-ci, HSPD1 code pour la protéine HSP60, qui est impliquée dans la régulation de la réponse immunitaire. OBJECTIFS: 1) démontrer la production d'HSP60 par les MA de sujets T et AA par immunocytochimie; 2) comparer l'expression protéique d'HSP60 par western blot et ELISA à l'expression génétique préalablement observée. MÉTHODE: Les MA de lavages bronchoalvéolaires de sujets T (sans asthme ni allergie) et de sujets AA ont été isolés par adhérence. Une partie d'entre eux a été fixée sur lames pour l'immunocytochimie alors qu'une autre a été utilisée pour l'obtention d'extraits protéiques pour le western blot et l'ELISA. RÉSULTATS: L'immunocytochimie a confirmé la production d'HSP60 par les MA des sujets T et AA. Les extraits protéiques pour le western blot se sont avérés inadéquats mais les ELISA indiquent une différence To measure the expression of TNF in uvular tissue of nonapneic snorers and apneic patients. METHODS: Forty patients who underwent an UPPP were enrolled and divided into three groups. The first group was composed of non-apneic snorers (n = 14) and the second group (OSA 1; n = 14) consisted of patients whose body mass index was similar. The third group (OSA 2; n = 12) consisted of OSA patients whose BMI was higher than the one of the other groups. Following UPPP, the resected tissues were divided into two sections (proximal and distal). RESULTS: TNF is highly expressed in the epithelium of the uvula and in the musculus uvulae (MU). Western blotting of whole proximal uvula (n=3) revealed that TNF expression was significantly higher in apneic patients compared to snorers (Snorers: 100.5 ± 2.99% ; OSA 1: 127.07 ± 6.91%; OSA 2: 140.75 ± 10.97%; P=0.01). TNF expression in muscular area was higher in OSA 2 group compared to both OSA 1 and snorer's groups. This TNF expression was correlated to body mass index (r = 0.8702; p = 0.02) CONCLUSION: Heaviest OSA patients have a higher TNF expression in Musculus Uvulae than less obese OSA and non-apneic snorers. This suggests that in heaviest OSA patients Musculus Uvulae is the main TNF source. There is growing evidence that inflammatory mechanisms are important in the pathophysiology of OSA. We recently reported increased inflammatory cell infiltration in the upper airway (UA) of OSA patients. Alterations in upper airway tissue mechanical properties in OSA have also been reported. Our aim was to test the hypothesis that both pro-fibrotic cytokine expression and connective tissue deposition are increased in the mucosal compartment of upper airway tissue in severe vs. mild OSA. METHODS: We evaluated UA surgical tissue from two OSA groups of similar age and BMI: mild (n = 17, mean AHI = 16.1 ± 4.1 (SD) events/h), and severe (n = 25, AHI = 63.0 ± 27.6). Ribonuclease protection assays were performed on total RNA extracted from mucosa-predominant tissue specimens to determine levels of message for a panel of pro-fibrotic cytokines including: Interleukin (IL)-1alpha, IL-6 and Transforming Growth Factor (TGF)-beta. We also performed van Gieson staining for connective tissue on frozen sections which were then subjected to morphometric analysis (ImagePro).

SEX DIFFERENCES IN THE RELATIONSHIP BETWEEN BODY FAT DISTRIBUTION AND ASTHMA CONTROL
M Piccioni 1,2,3 , KL Lavoie 1,2 , A Rizk 1,2,3 , A Wright 1,2,3 , SL Bacon 1,2,3 1 Montreal Behavioral Medicine Centre; 2 Hôpital du Sacré-Coeur de Montreal, Montreal, Quebec; 3 Department of Exercise Science, Concordia University, Montreal, Quebec BACKGROUND: Previous research, has suggested that sex plays an important role in asthma control, with women having worse control than men. As well, obesity has been shown to influence asthma control, particularly in women. However, little is known about the impact of sex and body fat distribution on asthma control. PURPOSE: The current study assessed sex differences in the effects of central adiposity and total body fat on asthma control. METHODS: 51 patients with physician diagnosed asthma, M±SD age=46±13 yrs, 42% male, completed the asthma control questionnaire (ACQ) and provided demographic information, including height, weight, and waist circumference (WC). Two general linear models were conducted to assess the relationship between adiposity and asthma control. Separate models were run for the main and interaction effects of body mass index (BMI) and sex on ACQ total and its subscales, and WC and sex on ACQ. RESULTS: In general, there was a main effect of sex and an interaction effect of sex and BMI on ACQ total and subscale scores. In addition, there Abstracts were main effects of sex and interactions of sex and WC on ACQ scores. All analyses used age as a covariate. The main effect of sex revealed that men exhibited worse asthma control than women. The sex X WC interaction suggested that increasing WC was associated with worse control in men, with no effect in women. In contrast, the sex X BMI interaction suggested that increasing BMI was associated with better control in women, with no effect in men. CONCLUSION: Further research is needed in larger samples to establish mechanisms by which central adiposity and total body fat influences asthma control and the variation that exists between sexes. Abnormalities of the lung predate onset of symptoms. Our objective is to determine how genes that regulate lung development contribute to asthma susceptibility. METHODS: We measured mRNA (real-time PCR) and protein (immunohistochemistry) expression of genes involved in mesenchymalepithelial signaling and airway muscle maturation. Goblet cells were assessed with PAS stain. Lung function was measured using the flexiVent small animal ventilator. RESULTS: Asthma susceptible (Brown Norway, BN), hyperresponsive (Fisher) and normoresponsive (Lewis) rats showed differences in lung function at postnatal day 14. These rat models also exhibited differential developmental expression in the lung of a ) epidermal and platelet-derived growth factor receptors (associated with lung remodeling in asthma); b) glucocorticoid receptor (associated with steroid responsiveness) c) total and fast myosin (SM-B, associated with airway narrowing). At postnatal day 14 Fisher and BN pups had increased total tracheal myosin. SM-B was upregulated in Fisher and down-regulated in BN pups. Newborn BN rats displayed tracheal goblet cell hyperplasia. CONCLUSIONS: Asthma susceptibility is associated with significant differences in lung function and respiratory gene expression in the newborn period. Increased SM-B/total myosin leads to faster muscle shortening. Increased SM-B/total myosin in developing lung leading to increased smooth muscle contraction may contribute to innate hyperresponsiveness. Neonatal goblet cell hyperplasia, characteristic of asthmatic airways, associates with the asthma susceptible phenotype of BN rat. Clarification of genetic mechanisms that contribute to asthma-susceptibility will be essential to the design of therapeutic targets for early intervention and primary prevention.

HETEROZYGOUS LGL1 KNOCKOUT MICE DISPLAY IMPAIRED ALVEOLARIZATION AND FEATURES OF LUNG INJURY
L Ribeiro 1,2 , J Lie 1,2 , I Mandeville 2 , K Nadeau 1,2 , S Cornejo 1,2 , NB Sweezey 3 , F Kaplan 1,2 1 Department of Human Genetics and Pediatrics, McGill University; 2 Montreal Children's Hospital Research Institute; 3 The Hospital for Sick Children Research Institute, Toronto, Ontario Bronchopulmonary dysplasia (BPD), a leading cause of morbidity in prematurely born infants, is associated with arrested alveolar development characterized by distal airspace enlargement often accompanied by goblet cell hyperplasia. Glucocorticoids (GC), formerly the treatment/ preventive agent of choice for BPD, is now known to impair lung and brain development. In a search for downstream targets of GCs that stimulate alveolarization without attendant GC toxicity, we cloned Lgl1, encoding a mesenchyme-enriched secreted glycoprotein (lgl1). Reduced levels of lgl1 in O2 toxicity models of BPD were normalized during recovery.

OBJECTIVE:
To determine whether deficiency of lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury. METHODS: An Lgl1 knockout mouse was generated by replacement of exon 2 of the Lgl1 gene with a neomycin cassette. Absence of lgl1 was lethal prior to lung formation. We compared lung morphology, Lgl1 RNA and lgl1 protein, tracheal goblet cells, elastin fibers and lung function in heterozygous Lgl1+/-and wild type mice. RESULTS: At embryonic day 18.5, Lgl1+/-lungs had reduced levels of Lgl1 RNA and lgl1 protein. Postnatal Lgl1+/-lungs (days 1-14) displayed delayed septation, distal airspace enlargement, increased interstitium, goblet cell hyperplasia, and fragmented elastin fibers. At one month of age, lungs of Lgl1+/+ and Lgl1+/-mice were indistinguishable. CONCLUSIONS: Lgl1 is essential for viability, and is required for developmental processes preceding and during lung formation. Newborn Lgl1+/-mice display features of delayed lung maturation and inflammatory injury. Lgl1 deficiency states may contribute to BPD.

ROLE OF THE TACHYKINERGIC SYSTEM IN FC(EPSILON)RI AGGREGATION INDUCED LEUKOTRIENE C4 (LTC4) PRODUCTION: INTERPLAY WITH THE INHIBITORY ACTION OF GLUCOCORTICOIDS S Favret, L Castellanos, K Maghni
Research Center, Sacré-Coeur Hospital of Montreal, Laboratory of Asthma Neuroimmunology, Université de Montréal, Québec BACKGROUND: Although basophils are a major source of LTC4, the underlying molecular mechanisms of FceRI clustering-induced LTC4 synthesis and release is mostly unknown. Recently, we found that (a) the rat basophilic cell line (RBL-2H3) express both substance P (SP) and its receptor, the neurokinin-1 receptor (NK-1R), (b) the NK-1R is activated in an autocrine manner in response to FceRI clustering, and (c) blocking NK-1R activation with the selective antagonist L-703,606 decreased basophil degranulation. AIM: To determine the role of the tachykinergic system SP/NK-1R in the production of LTC4 in IgE-dependent activation of basophils. METHODS: IgE-sensitized RBL-2H3 cells were treated or not with L-703,606 or glucocorticoids prior to FceRI clustering. The production of LTC4 was measured using the Luminex technology. RESULTS: The kinetic study indicates that LTC4 production occurred as early as 2 min following FceRI clustering, and that most of the LTC4 synthesized is released by basophils. Interestingly, blocking NK-1R activation reduced the synthesis of the LTC4. The glucocorticoids, dexamethasone and fluticasone, also reduced FceRI clustering-induced LTC4 synthesis. Very interestingly, the combination of glucocorticoids and the NK-1R antagonist has further inhibited the production of LTC4 which suggests a synergic interplay between these two inhibitory pathways. CONCLUSION: Our study provides the first evidence that the tachykinergic system plays a role in the production of LTC4 in response to FceRI clustering. The combination of a NK-1R antagonist with a glucocorticoid may be an interesting medication for the treatment of allergic diseases. Supported by the CIHR 37 ALOX15 : UN NOUVEAU DÉTERMINANT GÉNÉTIQUE DE L'ASTHME IDENTIFIÉ AVEC LES PUCES À ADN K Tremblay 1,2 , D Daley 3 , A Chamberland 2 , M Lemire 4 , A Montpetit 5 , M Laviolette 1 , A James 6 , M Chan-Yeung 7 , A Becker 3 , AJ Sandford 3 , PD Paré 3 , TJ Hudson 4 , C Laprise 2 1 Université Laval; 2 UQAC; 3 Université de Colombie-Britannique; 4 Institut de recherche sur le cancer de l'Ontario; 5 Centre d'Innovation Génome Québec et Université McGill; 6 Université de l'Australie de l'Ouest; 7 Université du Manitoba CONTEXTE: L'asthme implique l'interaction des gènes et de l'environnement dans son développement. Une étude du profil d'expression effectuée avec les puces à ADN a identifié des gènes différemment exprimés dans le tissu bronchique de sujets asthmatiques comparativement à des sujets témoins. Parmi eux, nous avons choisi huit gènes de molécules immunes (ALOX15, CD14, CD27, CXCL12, IL2RB, IL7R, NOS2A, SFRP1) comme gènes candidats pour l'asthme. BUT: Effectuer une étude d'association entre des polymorphismes (SNP) de ces gènes et quatre phénotypes de l'asthme: asthme, atopie, hyperréactivité bronchique (HRB) et asthme allergique. MÉTHODE: 77 SNP répartis dans les huit gènes ont été génotypés sur une plate-forme Illumina dans trois échantillons familiaux d'asthme indépendants provenant du Canada (n = 866, 1523 et 2052). Un test de déséquilibre de transmission a été effectué avec le logiciel FBAT et une correction Bonferroni a été appliquée sur les valeurs p. RÉSULTATS: Après corrections statistiques, deux associations demeurent significatives : entre ALOX15 et l'HRB dans l'échantillon de Vancouver (p corrigée = 0,02) et entre ALOX15 et l'asthme allergique dans l'échantillon du Manitoba (p corrigée = 0,049). Dans les deux cas, un effet de protection pour les allèles mineurs a été suggéré. CONCLUSION: Ces résultats démontrent l'efficacité des puces à ADN à cibler des gènes candidats pour l'asthme en ayant permis de trouver une nouvelle association pour ALOX15 dans deux échantillons indépendants. Le potentiel anti-inflammatoire de ce gène dans la pathophysiologie relative à l'asthme n'est pas encore connu et reste à être déterminé par des études fonctionnelles subséquentes. Recurrent apnea increases sensitivity of chemoreceptors and enhances hypoxic ventilatory response (HVR), but it remains unknown whether these alterations are sex-specific in newborn. Accordingly, we tested the hypothesis that sexual dimorphism in HVR occurs also in newborn rats. METHODS: We established a model of neonatal apnea by exposing rat pups with their dams to chronic intermittent hypoxia (IH: hypoxia 100 s / nadir 5% O2; reoxygenation at 21% O2 in 140 s; every 5 min during 1 h followed by 1 h under normoxia, repeated 24h) during the first 10-postnatal days. After exposure to neonatal IH or normoxia as control group, minute ventilation (VE) was measured in male and female rat pups using whole-body plethysmography. To assess HVR, changes in VE were monitored during a decrease in inspired oxygen level from 21% to 5% in 60 seconds and expressed in % of baseline (mean+/-sem). Inspired oxygen threshold and magnitude of HVR were evaluated. RESULTS: In male, HVR was higher in pups exposed to neonatal IH than in normoxic pups (+57+/-13% vs. +32+/-8% of baseline value, respectively). In female, HVR was similar between neonatal IH and normoxic pups (+35+/-13% vs. +42+/-7% of baseline value, respectively). CONCLUSION: This study shows that neonatal IH induces a malespecific enhancement of HVR in rat pups. This study suggests that neonatal apnea contribute to respiratory disorder breathing in sex specific manner in infant. Abstracts which eliminates confounding inflammation due to surgical procedure. Understanding the time course of the infection with P.aeruginosa using the non-surgical method is important in setting up any further study, in particular, if therapeutic intervention is planned to apply. METHODS: Female C57BL/6 mice were infected with 10 6 or 5 x 10 5 CFUs/mice of P. aeruginosa in agar beads. Mortality, daily body weight, total and differential leukocyte count in broncheoalveolar lavage fluid (BALF) were assessed at day 3 and 7 post-infection. Bacterial burden in lungs was tested at 6 hrs, 24 hrs, 3 and 7 days. RESULTS: There was a considerable mortality by day 3 post-infection (32.3 %, 12 out of 33) when mice were infected with 10 6 CFUs meanwhile all the mice survived the infection when the dose was decreased to 5 x 105 CFUs. Body weight loss peaked (-12%) at day 2 or 3 (in both doses) and baseline body weight was regained by day 7 post-infection. P. Aeruginosa at 10 6 CFUs induced 11.6-fold increase in total leukocytes in BALF at day 3 with neutrophils constituting 85.2 ± 1.7 % and macrophages 13.9 ± % vs. 1 ± 0.5 % neutrophils and 97.2 ± 0.5% macrophages in BALF of sterilized beads instilled controls. There was evidence of sustained inflammation at 7 days. 5x105 CFUs induced 4.9-fold increase in total leukocyte count in BALF. Bacterial burden was detected only in lungs of 3 out of 18 infected mice independently of the dose or time point (3 or 7 days). At 24 hrs post-infection bacterial burden was detected in 2 out of 3 infected mice (both doses). At 6 hrs post-infection all mice infected with10 (6) CFUs showed high bacterial burden (6.8 ± 0.2 log CFUs/lung, N= 5). CONCLUSION: 1) We confirm that this model induces a reliable airway inflammation. 2) Bacterial burden can be better studied at early time point (6-12 hours post-infection). 3) We plan to study whether an intermediate dose (7-8 x 105 CFUs) will minimize mortality while prominent inflammation will be still induced. Des mesures pharmaco-mécaniques couplées à des analyses par Western Blot ont été réalisées sur des tissus contrôles ou prétraités avec 10 ng/ml de TNFa ou au TNFa + 100 nM de 14,15-EET provenant de 16 résections pulmonaires. Les mesures de tension mécaniques révèlent que le 14,15-EET réduit la réactivité des bronchioles traitées au TNFa à différents agonistes contracturants et restore leurs réponses relaxantes à l'isoprotérénol. L'effet anti-inflammatoire de l'EET pourrait s'expiquer par une réduction de l'activation de NFkB, tel que démontré indirectement par immuno-marquage via la stabilisation de la protéine IkBa. Des expériences réalisées sur des tissus perméabilisés à la b-escin ont permis de mesurer la sensibilité des myofilaments au Ca2+ et de démontrer que le 14,15-EET prévient le développement de l'hypersensibilité au Ca2+ induite par le TNFa. De plus, à pCa 6, l'EET réduit de façon significative le tonus induit par le PDBu, un activateur de PKC. Ce résultat est corrélé avec une diminution du niveau de phosphorylation et de l'expression de la CPI-17, une protéine impliquée dans la régulation fine du tonus bronchique, par le 14,15-EET sur les bronchioles traitées au TNFa. Ces résultats démontrent que le 14,15-EET est un modulateur de la réactivité bronchique via la régulation de facteurs nucléaires et d'une protéine spécifique modulant la sensibilité au Ca2+ des muscles lisses. A growing body of evidence suggests that exogenous nitric oxide (NO) may have beneficial roles in airway diseases such as asthma and COPD. TPI 1020 is a novel respiratory anti-inflammatory drug which incorporates an NO-donating moiety and displays broncho-dilating activities in the lungs. PURPOSE: The aim of this study was to compare TPI 1020 to budesonide in animal models of neutrophil-mediated lung inflammation and bronchoconstriction. METHODS: Mice were treated intra-tracheally with dry powder formulations of TPI 1020 or budesonide at equimolar concentrations, prior to challenge with lipopolysaccharide (LPS). Neutrophil recruitment and inflammatory mediators were measured in lung lavages. The bronchodilating activity of TPI 1020 was assessed in guinea pigs challenged with histamine. RESULTS: In mice, TPI 1020 (8.0 mg/kg) was found to inhibit the LPSinduced neutrophil recruitment by 33%. In addition, TPI 1020 significantly decreased the secretion of TNFa (83%, p<0.001), MCP-1 (49%, p<0.05) and pro-MMP-9 (84%, p<0.05) in the lung while it had no effect on KC levels. Budesonide (5.6 mg/kg) had a weaker effect on neutrophil recruitment (19%), secretion of TNFa (71%, p<0.001), and pro-MMP-9 (54%), and had no effect on MCP-1 and KC secretion. In guinea pigs, TPI 1020 had a greater protective effect (90%) than budesonide (30%) against broncho-constriction induced by histamine. CONCLUSION: Together, these data indicate that TPI 1020 exerts broader anti-inflammatory and broncho-dilating effects as compared to budesonide, and demonstrates the potential for unique anti-inflammatory effects on prognostic indicators that are believed to be relevant in COPD.

STUDIES OF POTENTIAL CFTR THERAPEUTICS IDENTIFIED BY HIGHTHROUGHPUT SCREENING
R Robert, GW Carlile, C Pavel, N Liu, SM Anjos, J Liao, Y Luo, D Zhang, K Teske, DY Thomas, JW Hanrahan McGill University, Montreal, Quebec BACKGROUND: Cystic Fibrosis (CF) is the result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation is the deletion of phenylalanine 508 (F508del) that causes mild misfolding of CFTR and its retention in the endoplasmic reticulum (ER) leading to a drastic decrease in cAMP-stimulated Cl-permeability at the cell surface. Most evidence suggests that even partial correction (6-15%) of the ER-retained F508del-CFTR could provide therapeutic benefit for CF patient. PURPOSE: To identify small molecule correctors of abnormal F508del-CFTR processing, we developed and used a robust cell-based assay using F508del-CFTR with three hemagglutinin (3HA) tags inserted into the fourth external loop. METHODS: Baby Hamster Kidney (BHK) cells expressing 3HA tagged F508del-CFTR were incubated with test compounds at 10μM for 24 hours at 37°C. One of the hits we identified was the phosphodiesterase inhibitor sildenafil which has previously been reported to increase CFTR activity in nasal epithelia. A computional search for similar structures yielded analogues of sildenafil carrying the distinctive sulfonyl-piperazine group. We evaluated these analogues and found that one, called KM60, was surprisingly potent. It partially restored F508del trafficking and increased maturation significantly when BHK cells were treated with 10nM for 24 h or 10μM for 2 h. Partial correction was confirmed by the appearance of mature CFTR in Western blots and by halide flux, patch-clamp and shortcircuit current measurements in unpolarized BHK cells, monolayers of human airway epithelial cells, and intestines isolated from F508del mice treated ex-vivo and in-vivo. The matrix metalloprotease (MMPs) are an enzyme family, which have an important role in the degradation of extracellular matrix. Amongst MMPs, MMP-9 has been implicated in airway remodeling in asthma. Our previous findings indicated that MMP-9 is not expressed by HBSMC but it appears to bind to these cells as a function of cell confluency. Recent studies have reported that MMP-9 is a ligand for the CD44 receptor. However, little is known about the expression and the role of CD44 in HBSMC, and there is no data on its possible interaction with MMP-9. AIMS: To determine (a) the expression of CD44 in HBSMC as a function of cell confluency, (b) its functionality and (c) its potential interaction with MMP-9. METHODOLOGY: HBSMC were purchased from Cambrex. CD44 expression was determined by RT-PCR, Western blot (WB), and flow cytometry. RESULTS: RT-PCR analysis showed that CD44 is expressed in HBSMC, at least, as the v6 and v8 isoforms. WB analysis confirmed the expression of CD44. Flow cytometric analysis indicated that, similarly to cell-bound MMP-9, CD44 expression decreased as a function of cell confluency. These results were confirmed by semi-quantitative WB analysis. The functionality of CD44 was demonstrated through its internalization in the presence of hyaluronan, one of its orthosteric ligands. CONCLUSION: Our study indicates that HBSMC express a functional CD44 receptor. Furthermore, changes in its level of expression suggest a role for CD44 in cell growth. However, whether MMP-9 is an orthosteric ligand for CD44 and regulates HBSMC function remain to be determined. La fumée de cigarette induit des changements inflammatoires au niveau de la muqueuse bronchique. Les cellules épithéliales bronchiques et les macrophages alvéolaires sont les premières cellules à entrer en contact avec la fumée de cigarettes. Les macrophages alvéolaires sont impliqués dans la défense immune. L'interaction entre ces deux types cellulaires suite à une exposition à la fumée de cigarette est à mieux documenter. BUT: Déterminer les interactions possibles entre les cellules épithéliales bronchiques (NRBE) et les macrophages alvéolaires (NR8383), sur la libération de médiateurs inflammatoires, à la suite d'une exposition de 5 jours à un extrait de fumée de cigarette (EFC). MÉTHODES: Des NRBE et des NR8383 ont été utilisés dans un modèle de co-culture. L'EFC a été obtenu avec l'aide d'un échantillonneur d'air (AGI-30). Tous les jours, durant 5 jours, 1 ml de milieu a été récupéré pour le dosage ultérieur de la Macrophage Chemotactic Protein-1 (MCP-1) et de l'interleukine-10 (IL-10) et 1 ml de milieu avec une concentration de 3 % d'EFC ou de contrôle air a été ajouté. RÉSULTATS: Les NR8383 exposées à l'EFC démontrent une diminution de la libération de l'IL-10 comparé aux cellules contrôles. Dans le modèle de co-culture, une diminution de la libération de l'IL-10 moins importante a été observée chez les cellules exposées à l'EFC comparées aux cellules contrôles après 72 heures d'exposition.

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LGL1 MRNA LEVELS ARE REGULATED BY GLUCOCORTICOID, RETINOIC ACID, VITAMIN D AND TGF-B1 IN POSTNATAL LUNG CELL CULTURES K Nadeau 1,2 , L Oyewumi 3 , NB Sweezey 3 , F Kaplan 1,2 1 Department of Human Genetics, McGill University, Montreal, Quebec; 2 Montreal Children's Hospital Research Institute, Montreal, Quebec; 3 Hospital for Sick Children, Toronto, Ontario RATIONALE: Bronchopulmonary dysplasia (BPD), characterized by arrested lung growth and alveolarization, is a leading cause of morbidity in premature infants. Glucocorticoid (GC), retinoic acid (RA) and transforming growth factor-1 (TGF-B1) regulate lung development and are implicated in the pathology of BPD. Late-Gestation-Lung 1 (LGL1) is reduced in O2 toxicity models of BPD and normalizes during recovery.
LGL1 transgenic mice show advanced lung maturation. Analysis of the LGL1 promoter identified binding sites for GC receptor, RA, vit D and SMAD 3/4 (mediates TGF-B1 signaling). We hypothesized that GC, RA, vit D and TGF-B1 regulate LGL1 expression during late lung development. METHODS: Fibroblasts isolated from fetal and neonatal rat lung were treated with 10-7 M GC, 10-5 M RA, 10-8M vit D, 25 ng/ml TGF-B1or combinations of these for up to 72 hr (n>4). LGL1 mRNA was quantified by real-time RT-PCR. Embryonic day (E) 20 and postnatal day (PN) 7 rat lung fibroblasts transfected with an LGL1 promoter-driven luciferase expression construct were similarly treated and assayed for luciferase activity (n=4). RESULTS: GC treatment for 24 hr stimulated LGL1 mRNA from E18 until PN14 (range 7-115 fold), with maximal increase observed from PN1 to PN4. RA treatment for 48-72 hr, but not 24 hr, led to a reduction in LGL1 mRNA (5 fold). TGF-B1 and vit D caused a decrease in LGL1 mRNA. Luciferase assays confirmed these results. CONCLUSION: GC and RA have opposing effects on lung maturation and repair in the face of O2 injury. TGF-B1 influences lung growth and inflammation. Our findings suggest that these agents modulate the expression of LGL1 during lung maturation and in neonatal lung.

INVOLVEMENT OF K + CHANNELS AND EGF IN REPAIR PROCESSES OF NON-CF AND CF BRONCHIAL EPITHELIA
NTN Trinh 1,3 , A Privé 3 , É Maillé 3 , J Noël 2 , E Brochiero 1,2,3 1 Département de Médecine; 2 Département de Physiologie (GÉPROM); 3 Université de Montréal and Centre de recherche du CHUM-Hôtel-Dieu, Montréal, Québec Severe lesions of airway epithelia are observed in cystic fibrosis (CF) patients. Epithelial repair then involve cell migration and proliferation processes. Their regulatory mechanisms, controlled partially by growth factors (EGF) secreted by epithelial cells or fibroblasts, are not well defined. A model of mechanical wound of non-CF (NuLi) and CF (CuFi) bronchial cell monolayers was employed to study their repair mechanisms. We first verified that wound-healing was stimulated by EGF (3-fold increase) and reduced (by half) upon EGF titration with EGF antibody. Normal NuLi monolayers repair faster than CF CuFi, which agrees with a higher activation of EGFR observed in NuLi cells. Furthermore, we recently showed that EGF-stimulated alveolar repair was largely dependent of K + channel activity. Similarly, EGF-stimulated bronchial woundhealing was highly decreased with K ATP and KvLQT1 channel inhibitors. We tested the hypothesis that an alteration in K + channel expression and/or function could explain the delayed CF bronchial epithelia repair. We observed that K ATP and KvLQT1 protein expression was 40 to 70% lower in CuFi than in NuLi cells in agreement with the smaller K + currents measured in CuFi cells. In CONCLUSION, we demonstrated that EGFR activation and K + channel activity could play a crucial role in bronchial epithelia repair. Furthermore, our results suggested that a lower EGFR signaling coupled with a decrease in K + channels expression/function could be responsible for the delay in CF epithelia repair. Deletion of a phenylalanine at position 508 causes a misfolding of the CFTR chloride channel (dF508-CFTR) and is the most common cause of cystic fibrosis (CF). The mutant is degraded in the endoplasmic reticulum (ER) rather than trafficked to the apical membrane of epithelial cells, leading to impaired Cl-conductance and mucociliary clearance, and other epithelial problems that are not well understood. AIM: To better understand the epithelial abnormalities observed in CF. METHODS: Using a new cell culture method and live cell confocal imaging, we found that adenovirally transduced wild-type (WT) CFTR tagged with green fluorescent protein (GFP) can traffic to the apical membrane of polarized CF airway epithelial cell (CFBE41o-) monolayers whereas the GFP-tagged mutant protein has a distribution consistent with the ER. Apical expression of WT-CFTR is correlated with an increase in the trans-epithelial resistance (TER) (518 +/-69 ohm•cm2 vs control: 381 +/-44 ohm•cm2, n=6, p<0.05). Expression of GFP-dF508-CFTR does not affect TER (353 +/-69 ohm•cm2, n=6, p=1). In non-CF polarized CALU-3 cells, GFP-CFTR, but not GFP-dF508-CFTR, is trafficked to the apical membrane, but TER is not altered (1800 +/-378.9 ohms•cm2 vs control: 1243 +/-243 ohms•cm2, n=4, p=0.25). This shows that the barrier function of CF epithelial cells is enhanced by expression of WT CFTR, and that CFTR apical targeting is important for cell polarization. Defective trafficking of dF508-CFTR may impair the barrier function of airway epithelium and its ability regenerate in CF.

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TPI 1100: AN INNOVATIVE RNA-TARGETING DRUG FOR CHRONIC RESPIRATORY DISEASES H D'Anjou, M Fortin, A Balassy, J Gougeon, R Séguin, L Paquet, P Renzi, N Ferrari Topigen Parmaceuticals Inc, Montreal, Quebec BACKGROUND: While the importance of phosphodiesterases (PDE) in the regulation of cell function is now widely accepted as a pathway to potential respiratory drug candidates, data from large, long-term clinical trials with the first selective PDE4 oral drugs have significantly tempered expectations due to serious dose limiting side effects. Topigen has developed TPI 1100, a combination of two antisense oligonucleotides (AON) targeting PDE subtypes 4B, 4D and 7A, which are major regulators of cAMP metabolism in lung tissue and immune cells. PURPOSE: The aim of this study was to investigate the potential of an antisense therapy directed against specific PDE isoforms for reducing lung inflammation. METHODS: TPI 1100 was transfected in lung epithelial cells (A549 or NHBE) and PDE gene expression was measured by RT-PCR. Cells were stimulated with IL-1β following treatment with TPI 1100 and the quantification of IL-8, MCP-1 and GM-CSF protein levels were performed by ELISA. The effect of PDE inhibition on lung inflammation was assessed in a sub-acute cigarette smoke model in mice. RESULTS: Transfected in lung epithelial cells, TPI 1100 nearly abolished PDE mRNA expression and reduced by at least 50% the secretion level of inflammatory markers (IL-8, MCP-1 and GM-CSF) following stimulation with IL-1β. In mice, TPI 1100 reduced target gene expression in lung lavage cells and blocked neutrophil recruitment (68%; p<0.05) induced by sub-acute exposure to cigarette smoke. CONCLUSION: Delivered directly to the lungs, TPI 1100 represents a novel RNA-antagonist therapy for COPD without the systemic side effects widely associated with known small molecule inhibitors of PDE. Increasing dialysis beyond conventional thrice-weekly schedules can improve SA, but is impractical. Hemodiafiltration (HDF) is an adjunct to Conventional Hemodialysis (CHD) which can increase dialysis efficiency during conventional schedules. The aim of the present study was to compare SA severity during 3 months of CHD vs. HDF while maintaining a conventional 3 day per week schedule. METHODS: 15 clinically stable CHD patients identified with SA on an overnight polysomnograpm (apnea-hypopnea hypopnea index (AHI) > 15 events/h) have been randomized to two 3-month periods of CHD and HDF, in random order. Follow-up complete in-home PSG has been performed at the end of each 3-month intervention period. Full dialysis details and body weight are recorded at each dialysis session, and Creatinine clearance and B2 microglobulin are periodically assessed. 24h blood pressure monitoring, Epworth score, generic and sleep-related quality of life indices are determined at baseline and the end of each intervention period. RESULTS: 5 men and 1 woman (mean age 59 SD 11 ) of the 15 enrolled have completed the protocol. The mean AHI with CHD is 46.3 SD 14.9 compared to 39.9 SD 15 events/h on HDF (p = .56). CONCLUSIONS: Our pilot data thus far indicates that the study is design is feasible; however data collection on the remaining recruits will have to be completed before further conclusions can be drawn. Upper airway stabilizing muscles play a crucial role in the maintenance of UA patency. Transcranial magnetic stimulation allows the investigation of respiratory muscles' corticomotor activation process. This technique has also been used to evaluate the genioglossus corticomotor response. The aims of this study were to characterize the response of different upper airway stabilizing muscles to focal cortical stimulation of the genioglossus. METHODS: Alae nasi, genioglossus, levator palatini, palatoglossus and diaphragm motor evoked potential responses to transcranial magnetic stimulation were recorded during expiration, tidal inspiration and deep inspiration in 9 normal awake subjects. RESULTS: A concomitant response of the four studied upper airway muscles was observed in the majority of cortical stimuli. The response of these muscles was independent of the diaphragmatic one that was only occasionally observed. Significant positive relationships were found between alae nasi, levator palatini and palatoglossus motor evoked potential latencies and amplitudes and the corresponding values of the genioglossus. Il est généralement reconnu que la maîtrise de l'asthme est plus difficile à obtenir chez la personne obèse, mais l'influence de l'obésité sur la sévérité de la maladie demeure controversée. Nous avons émis l'hypothèse que l'inflammation systémique liée à l'obésité, via un effet sur l'inflammation bronchique, a un impact sur la sévérité et la maîtrise de l'asthme. MÉTHODES: 44 sujets obèses (IMC>30) et 44 non-obèses avec un diagnostic d'asthme ont eu un questionnaire sur le contrôle de l'asthme, un prélèvement sanguin, une analyse de l'expectoration induite (EI) et une mesure de l'IMC et du tour de taille. Les niveaux de leptine et d'adiponectine sanguins et de leptine dans l'EI ont été mesurés par ELISA. RÉSULTATS: Les taux sanguins de leptine étaient significativement plus élevés (p<0,0001) et ceux d'adiponectine plus bas (p=0,0002) chez les asthmatiques obèses comparés aux non-obèses. Le taux de leptine corrélait significativement avec la maîtrise de l'asthme (r=0,25, p=0,01). Les obèses utilisant des corticostéroïdes inhalés pour traiter leur asthme avaient un plus haut taux de leptine (p=0,01) dans le sang que ceux avec bronchodilatateur seul. Le décompte cellulaire de l'EI était comparable dans les 2 groupes. Les niveaux d'éosinophiles et de leptine dans l'EI corrélaient respectivement avec la mesure du tour de taille 36,p=0,01) et l'IMC (r=0,41,p=0,008). CONCLUSION: L'augmentation de la leptine liée à l'obésité pourrait avoir un rôle à jouer dans la sévérité, le contrôle et le type d'inflammation bronchique retrouvés chez la personne asthmatique obèse.

ARE RESPIRATORY SYMPTOMS SUGGESTIVE OF WORK-RELATED ASTHMA RELIABLE?
S Chiry 1 , J Lepage 2 , A Forget 1 , D Bégin 3 , S Chaboillez 1 , L-P Boulet 2 , M Gérin 3 , C Lemière 1 ¹Pneumologie-recherche, Hôpital du Sacré-Coeur de Montréal; ²Institut de cardiologie et de pneumologie de l'Université Laval, Hôpital Laval; ³Département de santé environnementale et santé au travail, Université de Montréal, Montréal Québec BACKGROUND: A proportion of subjects with work-related respiratory symptoms (WRS) may be misclassified as asthmatic subjects. We sought to assess the proportion of confirmed diagnoses of WRA in a cohort of workers with respiratory symptoms suggestive of WRA and to identify the types of occupations associated with WRA and WRS. METHODS: Prospective observational study of workers referred for symptoms suggestive of WRA over a one-year period. Detailed medical and occupational questionnaires were asked. Pulmonary function tests were performed in order to confirm the diagnosis of asthma. Specific inhalation challenges to occupational agents were performed in order to distinguish occupational asthma (OA) from work-exacerbated asthma (WEA). RESULTS: One hundred and twenty workers were investigated: 51 had WRA (OA: 33; WEA: 18) and 69 had WRS. Although 70 subjects had a previous diagnosis of asthma made by a physician, this diagnosis was confirmed in only 51 subjects. Seventy four percent of subjects with WRS but without asthma were taking anti-asthma medications. Workers were mostly employed in the manufacturing sector (64.7% with WRA and 71% with WRS) which differs greatly from the working Canadian and Quebec population where only 12.8% and 15.4% of workers respectively work in the manufacturing sector. CONCLUSIONS: Although a large proportion of workers complain of work-related respiratory symptoms, a minority of them have WRA. To study the prevalence of airway hyperresponsiveness in elite swimmers and prove the necessity to quickly create a health follow-up of Canadian's athletes.. METHODS: National to international swimmers (n=20; 18.9 2.5 years; 8 men, 12 women) answered to a questionnaire on their exercise symptoms and performed eucapnic voluntary hyperventilation (HVE) and methacholine (MT) challenges. Forced expiratory volume in one second (FEV 1 ) was regularly measured for 15 min after the HVE. A swimmer was considered as having an AHR if the FEV 1 fall (HVEf) reached 10% or more or if the MT PC 20 was 4 mg.ml -1 or less, according to the IOC-MC criteria. RESULTS: Only one athlete had a physician-diagnosed asthma before the study. Six swimmers had a PC 20 <4 mg.ml -1 (40%) and 14 swimmers (70%) reached a HVEf of 10% at least. In total, 75% of the swimmers had a positive diagnostic regarding to International Olympic. Sixteen swimmers (80%) reported at least 2 asthma symptoms during effort. The PC 20 was correlated neither to asthma symptoms during effort, nor with the number of training hours. The HVEf was positively correlated to the PC 20 (p<0.05) and the number of hours training in the swimming pool (p p<0.05), but not with respiratory symptoms during effort. CONCLUSION: According to IOC-MC criteria, particularly from HVE test, 75% of elite swimmers would respond to the criteria for asthma and be allowed to use β2-agonists, suggesting that assessment of AHR should be Abstracts more frequently done in swimmers. Further studies are needed to understand why respiratory symptoms do not correlate with these challenges.

EFFECTS OF INHALED CORTICOSTEROIDS ON WEIGHT GAIN AMONG PATIENTS WITH ASTHMA
A Rizk 1,2 , KL Lavoie 1 , M Joseph 1 , A Wright 1,2 , H Favreau 1 , SL Bacon 1,2 1 Montreal Behavioural Medicine Centre, Hôpital du Sacré-Coeur de Montréal, Montréal, Québec; 2 Department of Exercise Science, Concordia University, Montreal, Quebec Inhaled corticosteroids (ICS) play an important role in the management of asthma symptoms by reducing airway inflammation, one of the hallmark physiological characteristics of this respiratory illness. Several studies have shown that asthma is related to body weight and weight gain and asthma exacerbation, and that this relationship might be sex specific. Corticosteroids have been associated with weight gain. However, little is known about the effect of ICS use on weight gain among patients with asthma. We hypothesized that the use of ICS will trigger weight gain among patients with asthma, and specifically, women with asthma will have greater weight gain compared to men. In total 169 adult patients with asthma provided details of their medical history (including ICS use) and demographic information (including weight and height). All participants provided the same information at one year. General linear models revealed a main effect of ICS dose (F=5.23, P=.024), sex (F=3.87, P=.051), as well as the interaction between ICS dose and sex (F=4.69, P=.032) on BMI change, controlling for age, smoking status, level of education and baseline BMI. Results indicate that a greater ICS dose was associated with increased weight gain at 1 year and women had greater weight gain than men. Further analysis of the interaction indicated that women had greater weight gain with increased ICS dose; whist men had decreased weight gain Abstracts WHO standard treatment regimens, failure rates with initial therapy and retreatment were significantly higher in countries with the highest MDR prevalence (p<0.0001). CONCLUSION: Prevalence of MDR in new cases, is highly associated with rates of failure of initial therapy and retreatment. Current recommended standardized treatment regimens should be re-evaluated in countries with high prevalence of MDR-TB. This study tried to validate the relationship of the GOLD staging system with respiratory symptoms presentation in a clientele at risk of COPD. METHODS: A program for early detection of COPD was introduced in 11 family medicine practice clinics. Patients selected for screening did not have a previous diagnosis of COPD or asthma and were not under regular respiratory medication. Post-BD spirometry was performed on patients over the age of 40, having a minimum of 10 pack-years smoking history and at least one respiratory symptom (cough, cough with sputum, shortness of breath of at least 2 in the MRC scale, wheezing or frequent respiratory infections). COPD was classified based on the GOLD Standards (2001). RESULTS: We screened 240 eligible subjects (mean age 55.7 years, smoking history 33.7 pack-years). After performing spirometry, we discovered 31 new cases of COPD (GOLD 0=191, GOLD I=18, GOLD II=31). We found a trend indicating higher prevalence of COPD among subjects presenting 2 or more respiratory symptoms as compared to those with only 1 symptom (15% vs. 8%, p=0.18). No statistically significant differences were seen among COPD stages in terms of number of respiratory symptoms present, type of symptoms or severity of shortness of breath. CONCLUSIONS: The recent GOLD update (2006) has eliminated an important COPD stage, "0: at risk". The incidence of chronic respiratory symptoms in subjects negative to COPD represents an unmet need for treatment. As well, spirometry remains absolutely necessary to establish a diagnosis of COPD; symptom presentation is not a reliable indicator of the presence of this condition. C Fournier 1 , F Chagnon 1 , B Hogue 1 , DM Payet 1 , P Charrette 1 , S Thomas 2 , P Delaney 2 , O Lesur 1 1 Université de Sherbrooke, Sherbrooke, Québec; 2 Optiscan Pty Ltd Victoria, Australia PRÉMISSES: Les instruments d'évaluation d'une amélioration clinique du SDRA qui aident le clinicien au chevet à se convaincre que le poumon guérit, sont peu sensibles et peu spécifiques. La biopsie pulmonaire répétée serait idéale mais est grevée de risques et de complications inacceptables pour des patients en condition aussi critique. Le monitorage direct de la réparation pulmonaire pourrait être accessible par technique de micro imagerie, et ainsi permettre de changer la conduite du clinicien sur des critères objectifs sensibles et spécifiques. OBJECTIFS / MÉTHODES GÉNÉRAUX: Développer la micro imagerie laser de fluorescence confocale in vivo (Optiscan V) du poumon à l'aide d'un contraste systémique ou local par un agent fluorophore émettant dans la gamme d'excitation/émission de le fluorescéine (FITC), et permettant en temps réel de visionner par application directe sur la plèvre viscérale pulmonaire (profondeur de champs 250μm, résolution optique de 7μm par ajustements de 4μm/sec, résolution d'images est de 1024x1024 pixels). Un marquage nucléaire de l'endothelium vasculaire est assuré par Acridine Orange, associé à un contraste plasmatique par infusion de Dextran-FITC, et à une instillation intra-trachéale de lectine épithéliale (R Communis-FITC), chez des rats. Le laser est introduit et sécurisé sur la plèvre viscérale par une approche laparotomique et trans-diaphragmatique. RÉSULTATS: Des essais préliminaires sur poumons normaux et plusieurs modèles : aigu (acide chlorydrique), subaigü (hyperoxie), chronique (Bléomycine), sont en cours, afin de déterminer des paramètres distinctifs de réparation ou de non réparation pulmonaire. Des marqueurs fluorescents ciblant la circulation pulmonaire, l'épithélium, l'interstitium, la perméabilité de la barrière alvéolo-capillaire sont en développement. CONCLUSION: Une technique d'imagerie intra-vitale pulmonaire est en développement afin de déterminer ultimement des paramètres de monitorage et de pronostic des agressions pulmonaires aigues de type SDRSA et de les appliquer au chevet des patients atteints aux Soins Intensifs. Support: RSR-FRSQ et CRC UDS Abstracts