Mesenchymal Stem Cells In OVA-Sensitized And -Challenged Mice Produce Immunomodulatory Cytokines
C19. NEW DISCOVERIES IN MECHANISMS OF LUNG DISEASE: LATE BREAKING ABSTRACTS
Rationale: Recent studies have indicated the presence of mesenchymal stem cells (MSCs) in human lung diseases. Excess airway smooth muscle, myofibroblasts and activated fibroblasts have been noted in asthma, suggesting that mesenchymal progenitor cells are involved in asthma pathogenesis. We therefore sought to determine whether MSCs are present in ovalbumin (OVA)-sensitized and challenged mice. Methods: Balb/c mice were sensitized and challenged with PBS or OVA over a 25 day period. Flow
... try, clonogenicity, and differentiation potential, were used to analyze the emergence of MSCs. Results: A CD45-negative subset of cells expressed MSC antigens including Stro-1, Sca-1, CD73 and CD105. Selection for these and against CD45 yielded a pluripotent population of cells. Lungs from OVA-treated mice demonstrated a significantly greater average colony forming unit-fibroblast (CFU-F) than control mice. Sorted cells differed significantly from total lung adherent cell populations, exhibiting a pattern of gene expression nearly identical to bone marrow-derived MSCs. The sorted lung cells expressed significantly more osteopontin and proliferin than total lung adherent cell fibroblasts. Cultured MSCs also produced significant levels of IL-6, MCP-1/CCL2, KC/CXCL1, RANTES/CCL5 and eotaxin/CCL11, and co-culture experiments showed that MSCs increased the production of these cytokines by splenocytes from OVA-treated mice. Finally, we isolated cells from the BAL of a human asthma patient with identical patterns of cell surface markers and pluripotentiality. Conclusions: Allergen sensitization and challenge is accompanied by an influx of MSCs into the lungs that may regulate inflammatory and fibrotic responses. Rationale: There are currently no effective treatments for patients with severe asthma associated with neutrophilic bronchial inflammation. CXCR2 is highly expressed on neutrophils and mediates chemotaxis to inflammatory sites. We investigated the use of SCH527123, a selective CXCR2 antagonist, inhibiting neutrophil migration in patients with severe neutrophilic asthma (SNA). Design: In a randomized, double blind, parallel study, 34 patients with severe asthma (GINA 2008) and sputum neutrophils > 40% were randomized to SCH527123, 30 mg daily PO or placebo for 4 weeks. Primary endpoints were safety and changes in blood neutrophil counts, secondary endpoints were changes in sputum neutrophils, ACQ score, minor and major exacerbations and lung function. Background: Evidence from systematic reviews demonstrates the effectiveness of oral corticosteroids in the treatment of acute asthma in school-aged children when administered following physician review. As a result, the use of parent initiated corticosteroids is becoming a more widely accepted practice. There is a paucity of published evidence to support this practice. Objective: To evaluate the efficacy of a short course of parent-initiated oral prednisolone for acute asthma in school-aged children. Methods: A population-based sampling strategy was used to recruit children aged 5-12 years with a history of four or more episodes of acute asthma in the preceding year. Episodes of acute asthma, rather than participants, were randomised to receive a short course of parent-initiated prednisolone (1mg/kg/day) or placebo. Parents initiated a course of treatment if they felt, from previous experience, the episode to be a more severe attack, or if the symptoms were not improving after 6 to 8 hours with regular use of reliever medication. The primary end point: a validated 7-day daytime symptom score (DTSS). Other end points: a validated 7-night night time symptom score (NTSS), health resource utilisation (HRU), and school absenteeism. Results 230 children were enrolled in the study. Over a 3 year period 131 participants contributed 155 episodes of acute asthma randomised to parent-initiated treatment with prednisolone and 153 episodes randomised to treatment with placebo. Treatment with prednisolone reduced the mean DTSS by 15% (95% CI 2% to 26%, p=0.022) and the mean NTSS by 16% (95% CI 0% to 30%, p=0.050). The number of episodes requiring a HRU was 48 (31%) in those treated with prednisolone and 69 (45%) in those treated with placebo (OR 0.54. 95%CI 0.34,0.86) The number of episodes needed to treat to prevent an HRU was 7.1, (95% CI 4.0 to 30.3). School absenteeism was reduced by 0.4 days (95% CI -0.8 to -0.0 days, p=0.045). Conclusion A short course of oral prednisolone initiated by parents when their child suffers an episode of acute asthma may reduce asthma symptoms, HRU, and school absenteeism. Parent-initiated prednisolone is an appropriate strategy for the management of more severe episodes of acute asthma in school-aged children. The modest benefits of this strategy must be balanced against potential side effects of repeated short courses of oral corticosteroid. Rationale: Previous studies of immigrants in USA, Australia, New Zealand and Sweden have suggested that migration to a country with a high prevalence of asthma is a risk factor for developing asthma. ISAAC Phase Three (2001)(2002)(2003)(2004)(2005) provided an opportunity to examine the effect of immigration on the prevalence of asthma, eczema and rhinoconjunctivitis worldwide. Methods: ISAAC Phase Three was a questionnaire based survey of 6-7 year old children and 13-14 year old adolescents worldwide to determine the prevalence of asthma, eczema and rhinoconjunctivitis. A questionnaire was included which contained questions about immigration and duration of residence in the adopted country. Analysis was limited to those centers with at least 5% immigration and those subjects with complete covariate data. Odds ratios and 95% CI were calculated, adjusted for region of the world, gender, GNI and language. Results: Complete data were available for 43,143 children from 15 centers in 8 countries and 89,482 adolescents from 33 centers in 22 countries. Average immigration rate was 8.9% for children and 15.9% for adolescents. Overall, immigrants had fewer symptoms of asthma and eczema in children and for asthma and rhinoconjunctivitis in adolescents (Table 1) . Odds Ratios (95%CI) for risk of asthma, rhinitis and eczema if not born in country of residence 6-7 year old n = 43,143 13-14 year old n = 89,482 Current wheeze 0.73 (0.65,0.83) 0.81 (0.73,0.76) Asthma ever 0.66 (0.58,0.74) 0.69 (0.64,0.75) Current rhinoconjunctivitis 0.88 (0.76,1.01) 0.85 (0.79,0.93) Hayfever ever 1.06 (0.93,1.22) 0.93 (0.85,1.00) Current eczema 0.71 (0.62,0.81) 0.94 (0.85,1.04) Eczema ever 0.57 (0.51,0.64) 0.79 (0.71,0.87) Introduction: Human rhinovirus (HRV), a single-stranded (ss) RNA virus, causes exacerbation of chronic lower airway diseases including asthma. HRV infection triggers innate immune responses, including the production of interferons (IFNs) and proinflammatory chemokines. Methods: We investigated the requirement of Toll-like receptor (TLR)-3, which recognizes viral double-stranded RNA formed upon viral replication, for HRV-induced airway responses. Wild type (wt) B6129SF2/J and TLR3-deficient (TLR3-/-) mice were inoculated intranasally with HRV1B, a minor group virus which replicates in mouse lungs. Results: One day after infection with HRV1B, TLR3 -/-mice showed significantly decreased expression of the neutrophil chemoattractants KC/CXCL1 and MIP-2/CXCL2, and decreased whole lung neutrophil counts compared to HRV-infected wt mice. TLR3-/mice also displayed significantly decreased airway cholinergic responsiveness after HRV infection. However, compared to wt mice, TLR3 -/-mice showed no significant differences in viral titer, viral RNA or type I IFN expression. TLR3 -/-mice showed partial reductions in type II and III IFN expression. Conclusions: We conclude that TLR3 is required for HRV-induced airways neutrophilic inflammation and hyperresponsiveness but not viral clearance in B6129SF2/J mice. In other words, in this model. TLR3-driven innate immune responses to HRV are paradoxically maladaptive following experimental infection. Context: Because of the high mortality and morbidity in the ICU, palliative care is an important component of intensive care. There is currently debate over whether the best approach to improving ICU palliative care is to educate ICU clinicians, incorporate palliative care consultants, or both. Objective: Evaluate the effectiveness of a multi-faceted quality-improvement intervention to improve the quality of palliative care in the ICU by educating and supporting ICU clinicians. Design: A cluster-randomized trial of 12 hospitals assigned to intervention or usual care. Intervention: The intervention targeted ICU clinicians with 5 components: clinician education, local champions, academic detailing, clinician feedback, and system support. Outcomes and Analysis: Outcomes were assessed surveying family members and nurses of patients who died in the ICU or within 30 hours of transfer from the ICU. Families completed Quality of Dying and Death (QODD) and family satisfaction surveys. Nurses completed the QODD. Data were collected before and after the intervention/control period at each hospital. We used robust linear and Cox regression to test for intervention effects, controlling for site, patient, family, and nurse variables. Results: There were 2318 patient deaths. The patient-based survey completion rate for families was 43% (822/1924) and for nurses was 50% (636/1269). The primary outcome, family-QODD, showed no change with the intervention (p=0.23). There was also no change in family satisfaction (p=0.64) or the nurse-QODD (p=0.81). There was a reduction in ICU days prior to death associated with the intervention, but this was not statistically significant (HR=0.86; p=0.07). Among those patients who underwent withdrawal of life support, the intervention was not associated with a significant reduction in time from ICU admission to withdrawal (HR=0.93, p=0.49). Palliative care consultation was not available in 5 hospitals and in early development in 6 hospitals; only 6% of patients dying in the ICU received palliative care consultation and there was no effect of the intervention on palliative care consultation. CONCLUSIONS: Since no effective treatment for IPF exists, selective antagonism of ETA suggests a potential novel therapeutic approach for pulmonary fibrosis. Research Funding Source: Gilead Sciences, Inc. Conclusions: Hypoxia significantly alters translation and transcription of the adult rat AEC genes in complex patterns. Further studies are needed to test which of the putative miRNA actually mediate regulation of mRNA translation with hypoxic stress. Rationale: Caveolin-1 (cav-1) is the marker protein of caveolae, which are a specialized subset of lipid rafts. Cav-1 is highly expressed within the lung in epithelial cells, endothelial cells, various immune cells, and airway smooth muscle (ASM). Caveolae are thought of as membrane platforms that can concentrate and organize signaling proteins, leading to more rapid and effective activation of the signaling cascade. Additionally, it has been shown that cav-1 directly participates in various signaling pathways. We have previously reported (Brown et al., Am. J. Respir. Crit. Care Med., Apr 2009; 179: A5093) that the caveolin-1 deficient (cav-1 -/-) mice exposed to aerosolized lipopolysaccharide (LPS) have decreased airway hyperresponsiveness (AHR) to methacholine challenge as compared to wild-type (WT) mice. We also showed that this decrease does not directly correlate with the inflammatory response, as inflammatory cytokine and chemokine levels were increased in the bronchoalveolar lavage fluid of cav-1 -/mice compared to WT mice. The reduced AHR was instead attributed to decreased contraction of ASM observed in an ex vivo challenge of isolated bronchial rings. In order to investigate the mechanism behind this decreased contraction, we measured cholinergic agonist-induced calcium responses in freshly isolated ASM cells and muscarinic receptor expression in lungs from cav-1 -/mice and their littermate WT controls. Rationale: Vitamin E and selenium play a role in antioxidant defenses and may decrease oxidant damage to tissues and thereby reduce the risk of COPD. Prior observational epidemiologic studies support this hypothesis, but no large randomized trials have assessed supplementation with selenium, and one trial (Heart Protection Study) found no effect of combined vitamins E, C and beta-carotene on COPD hospitalizations. The effect of selenium, vitamin E or both on COPD risk was assessed in the Selenium and Vitamin E Cancer Prevention Trial (SELECT). Methods: SELECT was a double-blind, randomized, placebo-controlled trial of 35,533 men (median age 62.4 yrs) at 427 sites in the US, Canada, and Puerto Rico testing the single or joint effects of vitamin E (400 IU/day all rac-α-tocopherol acetate) and selenium (200 µg/d selenomethionine) in a 2x2 factorial design. As of 10/23/08, the end of supplementation in SELECT, the median length of follow-up was 5.46 years (range 4.17-7.33 years). The endpoint for the current study was assessed by participant report on bi-annual medical history questionnaires of incident physician-diagnosed emphysema, COPD and/or chronic bronchitis in 32,371 eligible men [excluding men with inadequate data (2,094) or with prevalent lung disease at baseline (1,068)]. Results: Hazard ratios [95% confidence intervals (CIs)] for COPD were 1.08 (95% CI 0.91, 1.28) for vitamin E, 1.01 (95% CI 0.85, 1.20) for selenium, and 0.94 (95% CI 0.79, 1.12) for combined vitamin E and selenium, all versus placebo/placebo. Findings were unchanged in models that included participants with a baseline report of prevalent lung disease, and Kaplan-Meier curves show no separation over time in probability of event by treatment group. The cumulative incidence of COPD was 3.27% over the follow-up period, and current smokers were at increased risk compared to never smokers (HR 7.22; 95% CI 5.99, 8.72). Conclusion: Selenium or vitamin E, alone or in combination, did not prevent COPD in a population of relatively healthy men. Although it is possible that effects may have been missed due to the short duration of treatment and suboptimal endpoint ascertainment, this study is a strong test of the hypothesis that antioxidants decrease the risk of physiciandiagnosed COPD. Statistical power to test for subgroup effects in smokers is limited, although such a test follows directly from the study hypothesis. A spirometry endpoint, currently being evaluated, may give further insight into these questions in a subgroup enriched with smokers. Rationale: Stat3 is constitutively activated in various cancers, including lung adenocarcinoma. Because the activation of Stat3 in cancer cell has been reported to promote survival and proliferation of the cell in a cell-autonomous manner and suppress the anti-cancer inflammation in the tumor microenvironment, Stat3 signal pathway is the attractive therapeutic target for cancer. To understand the role of Stat3 in the mouse model of lung adenocarcinoma is of considerable relevance for developing the therapy targeting Stat3 signal pathway for human lung adenocarcinoma. Methods: Stat3 was conditionally deleted from pulmonary epithelial cells of transgenic mice in vivo, producing Stat3 ∠†/∠† mice. Stat3 ∠†/∠† and control (Stat3 flox/flox ) mice were injected once intraperitoneally with 1mg/g body weight urethane. The total volume of lung tumor was measured by microCT after 4 months. Histological analysis and bronchoalveolar lavage (BAL) were performed after 6 months. Tumors were microdissected from the lung for RNA or protein extraction. Microarray analysis, RT-PCR and Western blot were performed. Results: The total tumor volume (TV) in Stat3 ∠†/∠† (2.9mm 3 /mouse) was significantly smaller compared to that of Stat3 flox/flox (6.5mm 3 /mouse). Immunohistochemistry (IHC) for phospho-Stat3 showed that Stat3 is activated in the inflammatory cells which surrounded the tumor in Stat3 ∠†/∠† . IHC for both surfactant protein-C and clara cell secretory protein indicated that the origin of the tumor cell was type II alveolar epithelial cell. Total cell count in BAL (TC) increased in proportion to TV in both mice. Howerver, the ratio of TC to TV (TC/TV) was significantly higher in Stat3 ∠†/∠† compared to that of Stat3 flox/flox . Microarray analysis showed that the genes regulating inflammatory response were significantly up-regulated in the tumor of Stat3 ∠†/∠† . Conclusions: Stat3 signal is required during the tumorigenesis of the lung. The abscence of Stat3 signal in tumor cell induced the increased inflammation in the lung. The target genes of Stat3 which affect survival, proliferation and immune-regulation of the lung tumor cell should be searched. RATIONALE: Staphylococcus aureus are an important cause of severe pneumonia especially in hospital settings and as a potentially fatal complication of influenza. Nasal colonization with S. aureus increases the likelihood of infection, yet is unclear how mucosal organisms once aspirated, cross an intact epithelial barrier. The staphylococcal surface protein, protein A (SpA), binds EGFR and activates several signaling cascades. EGFR has a central role in cell development and motility and is linked to both actin and E-cadherin. EGFR initiated MAPK activity is linked to activation of Rho/ROCK/ MLC induced actinomyosin contraction and to epithelial proteases. METHODS: We postulated that staphylococci could stimulate contraction of the epithelial cytoskeleton as well as cleavage of junctional proteins, such as E-cadherin and occludin, thus creating a gap in the paracellular junctions sufficient to enable staphylococcal translocation. RESULTS: Using strain Newman and the epidemic USA300 MRSA strain of S. aureus and isogenic spa null mutants, we demonstrate disruption of the actin cytoskeleton in polarized human airway monolayers in response to S. aureus strains expressing spa, but not the mutants unless complemented with the SpA peptide. Bacterial translocation and permeability to 10k MW fluorescent dextran similarly was limited to SpA+ strains and correlated with SpA+ induced calpain activity. SpA-exposed monolayers had activated RhoA and phosphorylated MLC. The SpA+ strains also induced cleavage of the extracellular portions of E-cadherin and occludin that span the paracellular junctions. Monolayers treated with EGFR inhibitor BPDQ, the ERK inhibitor U0126, the ROCK inhibitor Y27632 or the calpain inhibitor calpeptin had significantly (p<0.05) decreased staphylococcal transmigration; by confocal imaging these monolayers were found to be protected from staphylococcal induced disruption of the cytoskeleton. In a mouse model of staphylococcal pneumonia, mice pretreated with the EGFR inhibitor AG14578 had significantly less bacteremia than did the untreated controls (p=0.017). CONCLUSION: These studies suggest that activation of the EGFR-ERK/RhoA/MLC and EGFR-ERK calpain pathways by S. aureus protein A enable these non-motile organisms to pass through the paracellular junctions and gain access to the subepithelia space and disseminate into the bloodstream. Thus in addition to the other potent virulence factors of S. aureus, the protein A-EGFR interaction facilitates staphylococcal invasion through epithelial and likely through endothelial barriers as well.