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1.  Deletion of the Gene Encoding Calcitonin and Calcitonin Gene–Related Peptide α Does Not Affect the Outcome of Severe Infection in Mice 
Procalcitonin (PCT) is expressed in nonthryoidal tissues of humans during severe infections. Serum PCT levels are measured to diagnose and guide therapy, and there is some evidence that PCT may also contribute to the pathogenesis of sepsis. We tested whether disruption of the gene encoding PCT in mice affected the course of sepsis. Mice with exons 2–5 of the gene encoding calcitonin/calcitonin gene–related polypeptide α (Calca) knocked out and congenic C57BL/6J control mice were challenged with aerosolized Streptococcus pneumoniae or Pseudomonas aeruginosa, or injected intraperitoneally with S. pneumoniae. There were no significant differences in the survival of knockout and control mice in the two pneumonia models, and no significant differences in weight loss, splenic bacterial counts, or blood leukocyte levels in the peritoneal sepsis model. To verify disruption of the Calca gene in knockout mice, the absence of calcitonin in the serum of knockout mice and its presence and inducibility in control mice were confirmed. To evaluate PCT expression in nonthyroidal tissues of control mice, transcripts were measured in multiple organs. PCT transcripts were not significantly expressed in liver or spleen of control mice challenged with aerosolized P. aeruginosa or intraperitoneal endotoxin, and were expressed in lung only at low levels, even though serum IL-6 rose 3,548-fold. We conclude that mice are not an ideal loss-of-function model to test the role of PCT in the pathogenesis of sepsis because of low nonendocrine PCT expression during infection and inflammation. Nonetheless, our studies demonstrate that nonendocrine PCT expression is not necessary for adverse outcomes from sepsis.
doi:10.1165/rcmb.2012-0489OC
PMCID: PMC3727888  PMID: 23526213
procalcitonin; calcitonin; sepsis; pneumonia
2.  Safety, tolerability, and biomarkers of the treatment of mice with aerosolized Toll-like receptor ligands 
We have previously discovered a synergistically therapeutic combination of two Toll-like receptor ligands, an oligodeoxynucleotide (ODN) and Pam2CSK4. Aerosolization of these ligands stimulates innate immunity within the lungs to prevent pneumonia from bacterial and viral pathogens. Here we examined the safety and tolerability of this treatment in mice, and characterized the expression of biomarkers of innate immune activation. We found that neutrophils appeared in lung lavage fluid 4 h after treatment, reached a peak at 48 h, and resolved by 7 days. The peak of neutrophil influx was accompanied by a small increase in lung permeability. Despite the abundance of neutrophils in lung lavage fluid, only rare neutrophils were visible histopathologically in the interstitium surrounding bronchi and veins and none were visible in alveolar airspaces. The cytokines interleukin 6 (IL-6), tumour necrosis factor, and Chemokine (C-X-C motif) ligand 2 rose several hundred-fold in lung lavage fluid 4 h after treatment in a dose-dependent and synergistic manner, providing useful biomarkers of lung activation. IL-6 rose fivefold in serum with delayed kinetics compared to its rise in lavage fluid, and might serve as a systemic biomarker of immune activation of the lungs. The dose–response relationship of lavage fluid cytokines was preserved in mice that underwent myeloablative treatment with cytosine arabinoside to model the treatment of hematologic malignancy. There were no overt signs of distress in mice treated with ODN/Pam2CSK4 in doses up to eightfold the therapeutic dose, and no changes in temperature, respiratory rate, or behavioral signs of sickness including sugar water preference, food disappearance, cage exploration or social interaction, though there was a small degree of transient weight loss. We conclude that treatment with aerosolized ODN/Pam2CSK4 is well tolerated in mice, and that innate immune activation of the lungs can be monitored by the measurement of inflammatory cytokines in lung lavage fluid and serum.
doi:10.3389/fphar.2014.00008
PMCID: PMC3915096  PMID: 24567720
pneumonia; innate immunity; Toll-like receptor; oligodeoxynucleotide; lipopeptide; aerosol; myeloablation
3.  Regulated Mucin Secretion from Airway Epithelial Cells 
Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3 × 106 Da per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ∼1 μm in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the generation of DAG and of IP3 that releases calcium from apical ER. Stimulated secretion requires activation of the low affinity calcium sensor Synaptotagmin-2, while a corresponding high affinity calcium sensor in basal secretion is not known. The core exocytic machinery is comprised of the SNARE proteins VAMP8, SNAP23, and an unknown Syntaxin protein, together with the scaffolding protein Munc18b. Common and distinct features of this exocytic system in comparison to neuroendocrine cells and neurons are highlighted.
doi:10.3389/fendo.2013.00129
PMCID: PMC3776272  PMID: 24065956
secretion; exocytosis; mucin; mucus; MARCKS; Munc18; Munc13; synaptotagmin
4.  Pneumonia during Remission Induction Chemotherapy in Patients with Acute Leukemia 
Background: Pneumonia is a major cause of death during induction chemotherapy for acute leukemia. The purpose of this study was to quantify the incidence, risk factors, and outcomes of pneumonia in patients with acute leukemia.
Methods: We conducted a retrospective cohort study of 801 patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or acute lymphocytic leukemia (ALL) who underwent induction chemotherapy.
Measurements and Main Results: Pneumonia was present at induction start in 85 patients (11%). Of the 716 remaining patients, 148 (21%) developed pneumonia. The incidence rate of pneumonia was higher in MDS and AML than in ALL (0.013 vs. 0.008 vs. 0.003 pneumonias per day, respectively; P < 0.001). In multivariate analysis, age greater than or equal to 60 years, AML, low platelet count, low albumin level, neutropenia, and neutrophil count greater than 7,300 were risk factors. The case fatality rate of pneumonia was 17% (40 of 233). Competing risk analysis demonstrated that in the absence of pneumonia, death was rare: 28-day mortality was 6.2% for all patients but only 1.26% in those without pneumonia. Compared with patients without pneumonia, patients with pneumonia had more intensive care unit days, longer hospital stays, and 49% higher costs (P < 0.001).
Conclusions: Pneumonia after induction chemotherapy for acute leukemia continues to be common, and it is the most important determinant of early mortality after induction chemotherapy. Given the high incidence, morbidity, mortality, and cost of pneumonia, interventions aimed at prevention are warranted in patients with acute leukemia.
doi:10.1513/AnnalsATS.201304-097OC
PMCID: PMC3960911  PMID: 23987587
pneumonia; opportunistic infections; leukemia; fungal pneumonia; epidemiology
5.  Muc5b Is Required for Airway Defense 
Nature  2013;505(7483):412-416.
Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them via mucociliary clearance (MCC)1,2. However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases1. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus1,3. Genetic variants are linked to diverse lung diseases4-6, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in the lungs. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally7. Apoptotic macrophages accumulated, phagocytosis was impaired, and IL-23 production was reduced inMuc5b−/− mice. By contrast, in Muc5b transgenic (Tg) mice, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum1,8. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%9-11. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.
doi:10.1038/nature12807
PMCID: PMC4001806  PMID: 24317696
6.  Airway Mucus Function and Dysfunction 
The New England journal of medicine  2010;363(23):2233-2247.
doi:10.1056/NEJMra0910061
PMCID: PMC4048736  PMID: 21121836
7.  Toll-Like Receptor–2/6 and Toll-Like Receptor–9 Agonists Suppress Viral Replication but Not Airway Hyperreactivity in Guinea Pigs 
Respiratory virus infections cause airway hyperreactivity (AHR). Preventative strategies for virus-induced AHR remain limited. Toll-like receptors (TLRs) have been suggested as a therapeutic target because of their central role in triggering antiviral immune responses. Previous studies showed that concurrent treatment with TLR2/6 and TLR9 agonists reduced lethality and the microbial burden in murine models of bacterial and viral pneumonia. This study investigated the effects of TLR2/6 and TLR9 agonist pretreatment on parainfluenza virus pneumonia and virus-induced AHR in guinea pigs in vivo. Synthetic TLR2/6 lipopeptide agonist Pam2CSK4 and Class C oligodeoxynucleotide TLR9 agonist ODN2395, administered in combination 24 hours before virus infection, significantly reduced viral replication in the lung. Despite a fivefold reduction in viral titers, concurrent TLR2/6 and TLR9 agonist pretreatment did not prevent virus-induced AHR or virus-induced inhibitory M2 muscarinic receptor dysfunction. Interestingly, the TLR agonists independently caused non–M2-dependent AHR. These data confirm the therapeutic antiviral potential of TLR agonists, while suggesting that virus inhibition may be insufficient to prevent virus-induced airway pathophysiology. Furthermore, TLR agonists independently cause AHR, albeit through a distinctly different mechanism from that of parainfluenza virus.
doi:10.1165/rcmb.2012-0498OC
PMCID: PMC3727870  PMID: 23449736
Toll-like receptor; airway hyperreactivity; muscarinic receptor; parainfluenza virus
8.  Mucus hypersecretion in asthma: causes and effects 
Purpose of review
Airway mucus plugging has long been recognized as a principal cause of death in asthma. However, molecular mechanisms of mucin overproduction and secretion have not been understood until recently. These mechanisms are reviewed together with ongoing investigations relating them to lung pathophysiology.
Recent findings
Of the five secreted gel-forming mucins in mammals, only MUC5AC and MUC5B are produced in significant quantities in intrapulmonary airways. MUC5B is the principal gel-forming mucin at baseline in small airways of humans and mice, and therefore likely performs most homeostatic clearance functions. MUC5AC is the principal gel-forming mucin upregulated in airway inflammation and is under negative control by forkhead box a2 and positive control by hypoxia inducible factor-1. Mucin secretion is regulated separately from production, principally by extracellular triphosphate nucleotides that bind P2Y2 receptors on the lumenal surface of airway secretory cells, generating intracellular second messengers that activate the exocytic proteins, Munc13-2 and synaptotagmin-2.
Summary
Markedly upregulated production of MUC5AC together with stimulated secretion leads to airflow obstruction in asthma. As MUC5B appears to mediate homeostatic functions, it may be possible to selectively inhibit MUC5AC production without impairing airway function. The precise roles of mucin hypersecretion in asthma symptoms such as dyspnea and cough and in physiologic phenomena such as airway hyperresponsiveness remain to be defined.
doi:10.1097/MCP.0b013e32831da8d3
PMCID: PMC2709596  PMID: 19077699
airway; asthma; mucin; mucous; mucus
9.  Stimulation of Lung Innate Immunity Protects against Lethal Pneumococcal Pneumonia in Mice 
Rationale: The lungs are a common site of serious infection in both healthy and immunocompromised subjects, and the most likely route of delivery of a bioterror agent. Since the airway epithelium shows great structural plasticity in response to inflammatory stimuli, we hypothesized it might also show functional plasticity.
Objectives: To test the inducibility of lung defenses against bacterial challenge.
Methods: Mice were treated with an aerosolized lysate of ultraviolet-killed nontypeable (unencapsulated) Haemophilus influenzae (NTHi), then challenged with a lethal dose of live Streptococcus pneumoniae (Spn) delivered by aerosol.
Measurements and Main Results: Treatment with the NTHi lysate induced complete protection against challenge with a lethal dose of Spn if treatment preceded challenge by 4 to 24 hours. Lesser levels of protection occurred at shorter (83% at 2 h) and longer (83% at 48–72 h) intervals between treatment and challenge. There was also some protection when treatment was given 2 hours after challenge (survival increased from 14 to 57%), but not 24 hours after challenge. Protection did not depend on recruited neutrophils or resident mast cells and alveolar macrophages. Protection was specific to the airway route of infection, correlated in magnitude and time with rapid bacterial killing within the lungs, and was associated with increases of multiple antimicrobial polypeptides in lung lining fluid.
Conclusions: We infer that protection derives from stimulation of local innate immune mechanisms, and that activated lung epithelium is the most likely cellular effector of this response. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value.
doi:10.1164/rccm.200607-1038OC
PMCID: PMC2427056  PMID: 18388354
innate immunity; pneumonia; immunocompromised host; lung epithelium
10.  Haemophilus influenzae Lysate Induces Aspects of the Chronic Obstructive Pulmonary Disease Phenotype 
Nontypeable Haemophilus influenzae (NTHi) commonly colonizes the lower airways of patients with chronic obstructive pulmonary disease (COPD). Whether it contributes to COPD progression is unknown. Here, we determined which aspects of the COPD phenotype can be induced by repetitive exposure to NTHi products. Mice were exposed weekly to an aerosolized NTHi lysate, and inflammation was evaluated by measurement of cells and cytokines in bronchoalveolar lavage fluid (BALF) and immunohistochemical staining; structural changes were evaluated histochemically by periodic acid fluorescent Schiff's reagent, Masson's trichrome, and Picrosirius red staining; mucin gene expression was measured by quantitative RT-PCR; and the role of TNF-α was examined by transgenic airway overexpression and use of an inhibitory antibody. NTHi lysate induced rapid activation of NF-κB in airway cells and increases of inflammatory cytokines and neutrophils in BALF. Repetitive exposure induced infiltration of macrophages, CD8+ T cells, and B cells around airways and blood vessels, and collagen deposition in airway and alveolar walls, but airway mucin staining and gel-forming mucin transcripts were not increased. Transgenic overexpression of TNF-α caused BALF neutrophilia and inflammatory cell infiltration around airways, but not fibrosis, and TNF-α neutralization did not reduce BALF neutrophilia in response to NTHi lysate. In conclusion, NTHi products elicit airway inflammation in mice with a cellular and cytokine profile similar to that in COPD, and cause airway wall fibrosis but not mucous metaplasia. TNF-α is neither required for inflammatory cell recruitment nor sufficient for airway fibrosis. Colonization by NTHi may contribute to the pathogenesis of small airways disease in patients with COPD.
doi:10.1165/rcmb.2007-0366OC
PMCID: PMC2396243  PMID: 18096867
pulmonary disease, chronic obstructive; Haemophilus influenzae; bronchiolitis; inflammation; fibrosis
11.  β2-Adrenoceptor Agonists Are Required for Development of the Asthma Phenotype in a Murine Model 
β2-Adrenoceptor (β2AR) agonists are the most effective class of bronchodilators and a mainstay of asthma management. The first potent β2AR agonist discovered and widely used in reversing the airway constriction associated with asthma exacerbation was the endogenous activator of the β2AR, epinephrine. In this study, we demonstrate that activation of the β2AR by epinephrine is paradoxically required for development of the asthma phenotype. In an antigen-driven model, mice sensitized and challenged with ovalbumin showed marked elevations in three cardinal features of the asthma phenotype: inflammatory cells in their bronchoalveolar lavage fluid, mucin over production, and airway hyperresponsiveness. However, genetic depletion of epinephrine using mice lacking the enzyme to synthesize epinephrine, phenylethanolamine N-methyltransferase, or mice that had undergone pharmacological sympathectomy with reserpine to deplete epinephrine, had complete attenuation of these three cardinal features of the asthma phenotype. Furthermore, administration of the long-acting β2AR agonist, formoterol, a drug currently used in asthma treatment, to phenylethanolamine N-methyltransferase–null mice restored the asthma phenotype. We conclude that β2AR agonist–induced activation is needed for pathogenesis of the asthma phenotype. These findings also rule out constitutive signaling by the β2AR as sufficient to drive the asthma phenotype, and may help explain why chronic administration of β2AR agonists, such as formoterol, have been associated with adverse outcomes in asthma. These data further support the hypothesis that chronic asthma management may be better served by treatment with certain “β-blockers.”
doi:10.1165/rcmb.2012-0364OC
PMCID: PMC3604060  PMID: 23204390
β2-adrenoceptor agonists; formoterol; epinephrine; murine model; asthma
12.  Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase 
Molecular Cancer  2013;12:154.
Background
Tumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer.
Results
We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe lung neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. To further study the role of neutrophils in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody. This resulted in a significant reduction in lung tumor number. We further selectively inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2. Similarly, this resulted in suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant tumor reduction. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. We crossed the CC-LR mice with NE knock-out mice, and found that lack of NE significantly inhibits lung cancer development. These were associated with significant reduction in tumor cell proliferation and angiogenesis.
Conclusion
We conclude that lung cancer promotion by inflammation is partly mediated by activation of the IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trials using the IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer.
doi:10.1186/1476-4598-12-154
PMCID: PMC3923587  PMID: 24321240
Neutrophil; Elastase; Lung cancer; Inflammation; CXCR2; K-ras
13.  Assessment of Intracellular Mucin Content In Vivo 
Airway mucus presents a first line of defense against inhaled materials. It also, however, is a significant pathological contributor to chronic lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Thus, gaining a better understanding of the mechanisms of mucus production and secretion is an important goal for improving respiratory health. Mucins, the chief glycoprotein components of airway mucus, are very large polymeric glycoproteins, and measuring their production and secretion in experimental animals present significant technical challenges. Over the past several years, we have developed assays for accurately quantifying mucin production and secretion using histological and biochemical assays. These methods are described here.
doi:10.1007/978-1-61779-513-8_17
PMCID: PMC3751172  PMID: 22259143
airways; asthma; cystic fibrosis; chronic obstructive pulmonary disease; goblet cell; lungs; mouse; mucin; mucous; mucus
14.  Synergistic interactions of TLR2/6 and TLR9 induce a high level of resistance to lung infection in mice 
Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have historically focused on antibiotic development and vaccine-enhanced adaptive immunity. However, we have recently reported that the lungs’ innate defenses can be therapeutically induced by inhalation of a bacterial lysate that protects mice against otherwise lethal pneumonia. Here, we tested in mice the hypothesis that Toll-like receptors (TLRs) are required for this antimicrobial phenomenon, and found that resistance could not be induced in the absence of the TLR signaling adaptor protein MyD88. We then attempted to recapitulate the protection afforded by the bacterial lysate by stimulating the lung epithelium with aerosolized synthetic TLR ligands. While most single or combination treatments yielded no protection, simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent Gram-positive and Gram-negative pathogens. Protection was associated with rapid pathogen killing in the lungs, and pathogen killing could be induced from lung epithelial cells in isolation. Taken together, these data demonstrate the requirement for TLRs in inducible resistance against pneumonia, reveal a remarkable, unanticipated synergistic interaction of TLR2/6 and TLR9, reinforce the emerging evidence supporting the antimicrobial capacity of the lung epithelium, and may provide the basis for a novel clinical therapeutic that can protect patients against pneumonia during periods of peak vulnerability.
doi:10.4049/jimmunol.1002122
PMCID: PMC3654378  PMID: 21482737
15.  Pleuropericarditis, obliterative bronchiolitis and lymphocytic interstitial pneumonitis after allogeneic haematopoietic stem cell transplantation 
BMJ Case Reports  2011;2011:bcr1120103488.
Chronic graft-versus-host disease (GVHD) is a common complication of allogeneic haematopoietic cell transplantation, with pulmonary involvement occurring in 5–10% of cases. Obliterative bronchiolitis (OB) is recognised as a diagnostic manifestation of chronic GVHD, whereas lymphocytic interstitial pneumonitis (LIP) has been reported but is not considered diagnostic, and pleuritis is not clearly associated. The authors describe a transplant patient who simultaneously manifested three distinct pulmonary processes: OB, patchy LIP and pleuropericarditis. The onset of these entities along with other manifestations of chronic GVHD, their resolution with increased immunosuppression and their recurrence upon tapering support all three entities as manifestations of GVHD in the lungs.
doi:10.1136/bcr.11.2010.3488
PMCID: PMC3062890  PMID: 22707568
16.  Performance of a Standardized Bronchoalveolar Lavage Protocol in a Comprehensive Cancer Center 
Cancer  2011;117(15):3424-3433.
BACKGROUND
Flexible bronchoscopy with bronchoalveolar lavage (BAL) is performed widely for the diagnosis of pulmonary infections in patients with cancer, but there is no consensus regarding the technical parameters of the lavage procedure in this setting.
METHODS
The authors evaluated the mechanics (instilled and recovered volumes), diagnostic yield, and safety of a standardized BAL protocol in 284 patients with cancer who underwent bronchoscopy for the evaluation of new radiologic infiltrates.
RESULTS
Physician adherence to the BAL protocol was > 90%. The most common protocol deviations were reductions in the saline volume instilled because of actual or anticipated oxyhemoglobin desaturation during the procedure. The mean volume instilled was 121.5 ± 13.9 mL, the mean volume recovered was 68.7 ± 18.1 mL, and the mean ratio of volume instilled to that recovered was 56.7% ± 14.5%. The overall diagnostic yield of BAL was 33.8% and was higher in the nonhematologic malignancy group (42.3% vs 29.4%; P = .021). The diagnostic yield in neutropenic patients was significantly higher than in nonneutropenic patients (41.5% vs 24.6%; P = .019). No major complications were encountered.
CONCLUSIONS
In summary, the diagnostic performance of a standardized BAL protocol was comparable to that of nonprotocolized BAL reported in the literature with few complications. Adherence to a standardized BAL protocol may improve clinical and laboratory comparisons between studies, potentially facilitating research into the diagnosis and management of pneumonia in patients with cancer.
doi:10.1002/cncr.25905
PMCID: PMC3457647  PMID: 21246537
pneumonia; bronchoalveolar lavage; immunocompromise; cancer; diagnosis
17.  Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells 
Biochemical Journal  2012;446(Pt 3):383-394.
Airway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by ~50%. Homozygous mutant mice were not viable, but heterozygotes showed a ~50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxis was also reduced by ~50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identify Munc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.
doi:10.1042/BJ20120057
PMCID: PMC3430001  PMID: 22694344
exocytosis; mast cell; mucin; mucus; Munc18; secretion; AB-PAS, Alcian Blue/periodic acid/Schiff reagent; bHLH, basic helix–loop–helix; CCSP, Clara cell secretory protein; Clca3, chloride channel, calcium-activated, family member 3; CRE, cAMP-response element; DNP, 2,4-dinitrophenol; FBS, fetal bovine serum; FcϵRIα, high-affinity IgE receptor, α subunit; FRT, flippase recognition target; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRE, glucocorticoid-response element; HA, haemagglutinin; HSA, human serum albumin; HRP, horseradish peroxidase; IL-3, interleukin-3; INR, initiator; ISH, in situ hybridization; MC, mast cell; mBMMC, mouse bone-marrow-derived MC; mClca3, mouse Clca3; MFI, mean fluorescent intensity; mtCC, mouse transformed Clara cell; NK, natural killer; OCT, optimal cutting temperature compound; PAFS, periodic acid/fluorescent Schiff reagent; PBST, PBS containing 0.05% Tween 20; PGD2, prostaglandin D2; PGK, phosphoglucokinase; SCF, stem cell factor; SM, Sec1/Munc18; SNAP, soluble N-ethylmaleimide-sensitive factor-attachment protein; SNARE, SNAP receptor; Stxbp2, syntaxin-binding protein 2; TK, thymidine kinase; TNFα, tumour necrosis factor α; WT, wild-type; YFP, yellow fluorescent protein
18.  A Common MUC5B Promoter Polymorphism and Pulmonary Fibrosis 
The New England Journal of Medicine  2011;364(16):1503-1512.
BACKGROUND
The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk.
METHODS
Using a genomewide linkage scan, we detected linkage between idiopathic interstitial pneumonia and a 3.4-Mb region of chromosome 11p15 in 82 families. We then evaluated genetic variation in this region in gel-forming mucin genes expressed in the lung among 83 subjects with familial interstitial pneumonia, 492 subjects with idiopathic pulmonary fibrosis, and 322 controls. MUC5B expression was assessed in lung tissue.
RESULTS
Linkage and fine mapping were used to identify a region of interest on the p-terminus of chromosome 11 that included gel-forming mucin genes. The minor-allele of the single-nucleotide polymorphism (SNP) rs35705950, located 3 kb upstream of the MUC5B transcription start site, was present at a frequency of 34% among subjects with familial interstitial pneumonia, 38% among subjects with idiopathic pulmonary fibrosis, and 9% among controls (allelic association with familial interstitial pneumonia, P = 1.2×10−15; allelic association with idiopathic pulmonary fibrosis, P = 2.5×10−37). The odds ratios for disease among subjects who were heterozygous and those who were homozygous for the minor allele of this SNP were 6.8 (95% confidence interval [CI], 3.9 to 12.0) and 20.8 (95% CI, 3.8 to 113.7), respectively, for familial interstitial pneumonia and 9.0 (95% CI, 6.2 to 13.1) and 21.8 (95% CI, 5.1 to 93.5), respectively, for idiopathic pulmonary fibrosis. MUC5B expression in the lung was 14.1 times as high in subjects who had idiopathic pulmonary fibrosis as in those who did not (P<0.001). The variant allele of rs35705950 was associated with up-regulation in MUC5B expression in the lung in unaffected subjects (expression was 37.4 times as high as in unaffected subjects homozygous for the wild-type allele, P<0.001). MUC5B protein was expressed in lesions of idiopathic pulmonary fibrosis.
CONCLUSIONS
A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis. Our findings suggest that dys-regulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis. (Funded by the National Heart, Lung, and Blood Institute and others.)
doi:10.1056/NEJMoa1013660
PMCID: PMC3379886  PMID: 21506741
19.  Synergistic TLR2/6 and TLR9 Activation Protects Mice against Lethal Influenza Pneumonia 
PLoS ONE  2012;7(1):e30596.
Lower respiratory tract infections caused by influenza A continue to exact unacceptable worldwide mortality, and recent epidemics have emphasized the importance of preventative and containment strategies. We have previously reported that induction of the lungs' intrinsic defenses by aerosolized treatments can protect mice against otherwise lethal challenges with influenza A virus. More recently, we identified a combination of Toll like receptor (TLR) agonists that can be aerosolized to protect mice against bacterial pneumonia. Here, we tested whether this combination of synthetic TLR agonists could enhance the survival of mice infected with influenza A/HK/8/68 (H3N2) or A/California/04/2009 (H1N1) influenza A viruses. We report that the TLR treatment enhanced survival whether given before or after the infectious challenge, and that protection tended to correlate with reductions in viral titer 4 d after infection. Surprisingly, protection was not associated with induction of interferon gene expression. Together, these studies suggest that synergistic TLR interactions can protect against influenza virus infections by mechanisms that may provide the basis for novel therapeutics.
doi:10.1371/journal.pone.0030596
PMCID: PMC3267724  PMID: 22299046
20.  Enhancement of lung tumorigenesis in a Gprc5a Knockout mouse by chronic extrinsic airway inflammation 
Molecular Cancer  2012;11:4.
Background
Although cigarette smoking is the principal cause of lung carcinogenesis, chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, has been identified as an independent risk factor for lung cancer. Bacterial colonization, particularly with non-typeable Haemophilus influenzae (NTHi), has been implicated as a cause of airway inflammation in COPD besides cigarette smoke. Accordingly, we hypothesized that lung cancer promotion may occur in a chronic inflammatory environment in the absence of concurrent carcinogen exposure.
Results
Herein, we investigated the effects of bacterial-induced COPD-like inflammation and tobacco carcinogen-enhanced tumorigenesis/inflammation in the retinoic acid inducible G protein coupled receptor knock out mouse model (Gprc5a-/- mouse) characterized by late-onset, low multiplicity tumor formation. Three-month-old Gprc5a-/- mice received 4 intraperitoneal injections of the tobacco-specific carcinogen, NNK, followed by weekly exposure to aerosolized NTHi lysate for 6 months. The numbers of inflammatory cells in the lungs and levels of several inflammatory mediators were increased in Gprc5a-/- mice treated with NTHi alone, and even more so in mice pretreated with NNK followed by NTHi. The incidence of spontaneous lung lesions in the Gprc5a-/- mice was low, but NTHi exposure led to enhanced development of hyperplastic lesions. Gprc5a-/- mice exposed to NNK alone developed multiple lung tumors, while NTHi exposure increased the number of hyperplastic foci 6-fold and the tumor multiplicity 2-fold. This was associated with increased microvessel density and HIF-1α expression.
Conclusion
We conclude that chronic extrinsic lung inflammation induced by bacteria alone or in combination with NNK enhances lung tumorigenesis in Gprc5a-/- mice.
doi:10.1186/1476-4598-11-4
PMCID: PMC3281775  PMID: 22239913
lung cancer; inflammation; COPD; Gpcr5a; NTHi
21.  Interleukin 6 But Not T Helper 2 Cytokines Promotes Lung Carcinogenesis 
Several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer compared to smokers without COPD. We have shown a causal role for COPD-like airway inflammation in lung cancer promotion in the CCSPCre/LSL–K-rasG12D mouse model (CC-LR). In contrast, existing epidemiologic data do not suggest any definite association between allergic airway inflammation and lung cancer. To test this, CC-LR mice were sensitized to ovalbumin (OVA) then challenged with an OVA aerosol weekly for eight weeks. This resulted in eosinophilic lung inflammation associated with increased levels of T helper 2 cytokines and mucous metaplasia of airway epithelium, similar to what is seen in asthma patients. However, this type of inflammation did not result in a significant difference in lung surface tumor number (49 ± 9 in OVA vs 52 ± 5 in control), in contrast to a 3.2-fold increase with COPD-like inflammation. Gene expression analysis of NTHi-treated lungs showed up-regulation of a different profile of inflammatory genes, including interleukin 6 (IL-6), compared to OVA-treated lungs. Therefore, to determine the causal role of cytokines that mediate COPD-like inflammation in lung carcinogenesis, we genetically ablated IL-6 in CC-LR mice. This not only inhibited intrinsic lung cancer development (1.7-fold), but also inhibited the promoting effect of extrinsic COPD-like airway inflammation (2.6-fold). We conclude that there is a clear specificity for the nature of inflammation in lung cancer promotion, and IL-6 has an essential role in lung cancer promotion.
doi:10.1158/1940-6207.CAPR-10-0180
PMCID: PMC3058282  PMID: 21098042
lung cancer; inflammation; COPD; asthma; IL-6
22.  Muc5ac: a critical component mediating the rejection of enteric nematodes 
The mucin Muc5ac is essential for the expulsion of Trichuris muris and other gut-dwelling nematodes.
De novo expression of Muc5ac, a mucin not normally expressed in the intestinal tract, is induced in the cecum of mice resistant to Trichuris muris infection. In this study, we investigated the role of Muc5ac, which is detected shortly before worm expulsion and is associated with the production of interleukin-13 (IL-13), in resistance to this nematode. Muc5ac-deficient mice were incapable of expelling T. muris from the intestine and harbored long-term chronic infections, despite developing strong TH2 responses. Muc5ac-deficient mice had elevated levels of IL-13 and, surprisingly, an increase in the TH1 cytokine IFN-γ. Because TH1 inflammation is thought to favor chronic nematode infection, IFN-γ was neutralized in vivo, resulting in an even stronger TH2-type immune response. Nevertheless, despite a more robust TH2 effector response, the Muc5ac-deficient mice remained highly susceptible to chronic T. muris infection. Importantly, human MUC5AC had a direct detrimental effect on nematode vitality. Moreover, the absence of Muc5ac caused a significant delay in the expulsion of two other gut-dwelling nematodes (Trichinella spiralis and Nippostrongylus brasiliensis). Thus, for the first time, we identify a single mucin, Muc5ac, as a direct and critical mediator of resistance during intestinal nematode infection.
doi:10.1084/jem.20102057
PMCID: PMC3092342  PMID: 21502330
23.  β-Adrenoceptor Inverse Agonists in Asthma 
Current opinion in pharmacology  2010;10(3):254-259.
β2-adrenoceptor agonists are very effective bronchodilators and play a major role in every stage of asthma management. However, their chronic, regular use is associated with detrimental effects including an increase in asthma-related deaths. Conversely, recent data suggest that certain β-blockers, specifically β-adrenoceptor inverse agonists, may be useful in the chronic treatment of asthma. Here we review the data for this observation and the signaling pathways that may be involved. The data suggest that β2-adrenoceptor signaling is required to produce maximal airway inflammation and hyperresponsiveness, and the signaling pathway responsible for these effects is likely the non-canonical β-arrestin-2 pathway. Therefore, β-adrenoceptor inverse agonists may produce their beneficial chronic effects by inhibiting constitutive or ligand-induced activation of this pathway. Both lung parenchymal and hematopoietic cells appear to be involved in mediating the beneficial effects of β-adrenoceptor inverse agonists.
doi:10.1016/j.coph.2010.03.002
PMCID: PMC2905467  PMID: 20399707
asthma; β-adrenoceptor agonist; airway inflammation; β-blocker; β-adrenoceptor inverse agonist
24.  Helicobacter pylori Bacteremia with Sepsis Syndrome ▿  
Journal of Clinical Microbiology  2010;48(12):4661-4663.
A 65-year-old woman with a history of gastric bleeding, breast cancer, antineoplastic chemotherapy, and prednisone use presented with a fever, chest pain, a dry cough, hypotension, and prominent pulmonary bronchovascular markings. She was treated with piperacillin-tazobactam and azithromycin and rapidly improved. Six days later, the blood culture grew a pleomorphic Gram-negative bacillus. Initial subculture failed, but the organism was identified as Helicobacter pylori by sequencing the 16S rRNA gene. The bacterium eventually grew on brucella agar upon extended incubation.
doi:10.1128/JCM.01481-10
PMCID: PMC3008481  PMID: 20861342
25.  Nontypeable Haemophilus influenzae in chronic obstructive pulmonary disease and lung cancer 
Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death in the world by 2020. It is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases, most commonly cigarette smoke. Among smokers with COPD, even following withdrawal of cigarette smoke, inflammation persists and lung function continues to deteriorate. One possible explanation is that bacterial colonization of smoke-damaged airways, most commonly with nontypeable Haemophilus influenzae (NTHi), perpetuates airway injury and inflammation. Furthermore, COPD has also been identified as an independent risk factor for lung cancer irrespective of concomitant cigarette smoke exposure. In this article, we review the role of NTHi in airway inflammation that may lead to COPD progression and lung cancer promotion.
doi:10.2147/COPD.S15417
PMCID: PMC3048087  PMID: 21407824
COPD; NTHi; inflammation

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