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Acute respiratory distress syndrome (ARDS) is characterized by unrelenting polymorphonuclear neutrophil (PMN) inflammation and vascular permeability. The matrikine proline-glycine-proline (PGP) and acetylated PGP (Ac-PGP) have been shown to induce PMN inflammation and endothelial permeability in vitro and in vivo. In this study, we investigated the presence and role of airway PGP peptides in acute lung injury (ALI)/ARDS. Pseudomonas aeruginosa-derived lipopolysaccharide (LPS) was instilled intratracheally in mice to induce ALI, and increased Ac-PGP with neutrophil inflammation was noted. The PGP inhibitory peptide, arginine-threonine-arginine (RTR), was administered (it) 30 min before or 6 h after LPS injection. Lung injury was evaluated by detecting neutrophil infiltration and permeability changes in the lung. Pre- and posttreatment with RTR significantly inhibited LPS-induced ALI by attenuating lung neutrophil infiltration, pulmonary permeability, and parenchymal inflammation. To evaluate the role of PGP levels in ARDS, minibronchoalveolar lavage was collected from nine ARDS, four cardiogenic edema, and five nonlung disease ventilated patients. PGP levels were measured and correlated with Acute Physiology and Chronic Health Evaluation (APACHE) score, [Formula: see text] to [Formula: see text] (P/F), and ventilator days. PGP levels in subjects with ARDS were significantly higher than cardiogenic edema and nonlung disease ventilated patients. Preliminary examination in both ARDS and non-ARDS populations demonstrated PGP levels significantly correlated with P/F ratio, APACHE score, and duration on ventilator. These results demonstrate an increased burden of PGP peptides in ARDS and suggest the need for future studies in ARDS cohorts to examine correlation with key clinical parameters.

The extracellular matrix (ECM) is a dynamic, bioactive structure critical to organ development, structure and function. Excessive remodeling of the ECM is a hallmark of a variety of inflammatory conditions including vascular disease. Endothelin-1 (ET1) synthesis is understood to promote cardiovascular diseases including acute cardiac transplant rejection; however, the contribution of ECM-derived chemokines (matrikines) to vascular inflammation remains poorly understood. Herein we report that the matrikine acetylated Pro-Gly-Pro (PGP) stimulates vascular inflammation through activation of endothelial CXC Chemokine Receptor 2 (CXCR2) and production of endothelin-1 both in vitro and in vivo. As a proof of hypothesis, we demonstrate that coronary PGP levels associate with both circulating endothelin-1 and acute rejection in cardiac transplant patients (sensitivity of 100% and specificity of 86%). These findings establish PGP as a novel mediator in cardiovascular disease, and implicate bioactive matrix fragments as underappreciated agents potentially active in numerous conditions propagated by progressive vascular inflammation.

Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to chronic obstructive pulmonary disease pathogenesis and is a potential therapeutic target. We sought to determine the acute effects of cigarette smoke on ion transport and the mucociliary transport apparatus, their mechanistic basis, and whether deleterious effects could be reversed with the CFTR potentiator ivacaftor (VX-770). Primary human bronchial epithelial (HBE) cells and human bronchi were exposed to cigarette smoke extract (CSE) and/or ivacaftor. CFTR function and expression were measured in Ussing chambers and by surface biotinylation. CSE-derived acrolein modifications on CFTR were determined by mass spectroscopic analysis of purified protein, and the functional microanatomy of the airway epithelia was measured by 1-μm resolution optical coherence tomography. CSE reduced CFTR-dependent current in HBE cells (P

Cigarette smoking is associated with chronic obstructive pulmonary disease and chronic bronchitis. Acquired ion transport abnormalities, including cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, caused by cigarette smoking have been proposed as potential mechanisms for mucus obstruction in chronic bronchitis. Although e-cigarette use is popular and perceived to be safe, whether it harms the airways via mechanisms altering ion transport remains unclear. In the present study, we sought to determine if e-cigarette vapor, like cigarette smoke, has the potential to induce acquired CFTR dysfunction, and to what degree. Electrophysiological methods demonstrated reduced chloride transport caused by vaporized e-cigarette liquid or vegetable glycerin at various exposures (30 min, 57.2% and 14.4% respectively, vs. control; P

Background Proline–glycine–proline (PGP) is a bioactive fragment of collagen generated by the action of matrix metalloproteinase-9 (MMP-9) and prolylendopeptidase (PE), and capable of eliciting neutrophil chemotaxis and epithelial remodelling. PGP is normally then degraded by leukotriene A4 hydrolase (LTA4H) to limit inflammation and remodelling. This study hypothesized that early and persistent airway neutrophilia in Cystic Fibrosis (CF) may relate to abnormalities in the PGP pathway and sought to understand underlying mechanisms. Methods Broncho-alveolar lavage (BAL) fluid was obtained from 38 CF (9 newborns and 29 older children) and 24 non-CF children. BAL cell differentials and levels of PGP, MMP-9, PE and LTA4H were assessed. Results Whilst PGP was present in all but one of the older CF children tested, it was absent in non-CF controls and the vast majority of CF newborns. BAL levels of MMP-9 and PE were elevated in older children with CF relative to CF newborns and non-CF controls, correlating with airway neutrophilia and supportive of PGP generation. Furthermore, despite extracellular LTA4H commonly being greatly elevated concomitantly with inflammation to promote PGP degradation, this was not the case in CF children, potentially owing to degradation by neutrophil elastase. Conclusions A striking imbalance between PGP-generating and -degrading enzymes enables PGP accumulation in CF children from early life and potentially supports airway neutrophilia., Graphical abstract Unlabelled Image, Highlights • PGP is a collagen-derived pro-neutrophilic mediator. • PGP accumulates in the airways of children with cystic fibrosis. • Imbalance of PGP generation (MMP/PE) vs degradation (LTA4H) enables PGP build-up. • LTA4H is destroyed by neutrophil elastase to prevent PGP degradation. • Infections are key to spiking PGP generation in setting it cannot be degraded.

Rationale: Cigarette smoking is prevalent in the United States and is the leading cause of preventable diseases. A prominent complication of smoking is an increase in lower respiratory tract infections (LRTIs). Although LRTIs are known to be increased in subjects that smoke, the mechanism(s) by which this occurs is poorly understood. Objectives: Determine how cigarette smoke (CS) reduces reactive oxygen species (ROS) production by the phagocytic NOX2 (NADPH oxidase 2), which is essential for innate immunity in lung macrophages. Methods: NOX2-derived ROS and Rac2 (Ras-related C3 botulinum toxin substrate 2) activity were determined in BAL cells from wild-type and Rac2(−/−) mice exposed to CS or cadmium and in BAL cells from subjects that smoke. Host defense to respiratory pathogens was analyzed in mice infected with Streptococcus pneumoniae. Measurements and Main Results: NOX2-derived ROS in BAL cells was reduced in mice exposed to CS via inhibition of the small GTPase Rac2. These mice had greater bacterial burden and increased mortality compared with air-exposed mice. BAL fluid from CS-exposed mice had increased levels of cadmium, which mediated the effect on Rac2. Similar observations were seen in human subjects that smoke. To support the importance of Rac2 in the macrophage immune response, overexpression of constitutively active Rac2 by lentiviral administration increased NOX2-derived ROS, decreased bacterial burden in lung tissue, and increased survival compared with CS-exposed control mice. Conclusions: These observations suggest that therapies to maintain Rac2 activity in lung macrophages restore host defense against respiratory pathogens and diminish the prevalence of LRTIs in subjects that smoke.

Introduction: Robin sequence (RS) is a congenital disorder resulting in upper airway obstruction (UAO) which can be late-onset or subclinical, leading to risk of under-recognition and associated long-term morbidity. There is currently no consensus about best-practice management of UAO in RS. We aimed to describe current management of RS-related UAO in the United Kingdom and Ireland (UK/I), as a prelude to developing a national clinical guideline. Methods: A surveillance study was conducted throughout UK/I (Jan 2016 - Jan 2017) using a monthly reporting card distributed to 3500 paediatricians/neonatologists and regional cleft teams. For each reported case of RS, a questionnaire was provided to collect detailed clinical data (85% response rate). Results: 153 infants with confirmed RS (52% isolated RS) were identified. 95% were admitted to a neonatal or paediatric unit; specialist respiratory input was sought in 54% cases. 87% had signs of UAO and 66% required an airway adjunct (AA); nasopharyngeal airway (NPA) in 56%, CPAP 27%, endotracheal intubation 11%, tracheostomy 11%. 29% were discharged with NPA after median 21 days. Tracheostomy was associated with non-isolated RS (p=0.002). 52% underwent sleep study (70% oximetry) at median 17 days old, which changed practice in 47% cases (AA required, 32%; AA not required, 15%). Conclusion: UK/I centres favour non-surgical UAO management, mainly with NPA. Surgery is reserved for treatment failure, mainly in non-isolated RS. Our findings differ from those reported by North American and European centres, reinforcing the need for a consensus guideline. Data collection is underway to compare 1-year clinical outcomes between different UAO management approaches.

It is anticipated that bioactive fragments of the extracellular matrix (matrikines) can influence the development and progression of chronic diseases. The enzyme leukotriene A4 hydrolase (LTA4H) mediates opposing proinflammatory and anti-inflammatory activities, through the generation of leukotriene B4 (LTB4) and degradation of proneutrophilic matrikine Pro-Gly-Pro (PGP), respectively. We show that abrogation of LTB4 signaling ameliorated inflammation and airway hyperresponsiveness (AHR) in a murine asthma model, yet global loss of LTA4H exacerbated AHR, despite the absence of LTB4. This exacerbated AHR was attributable to a neutrophil-independent capacity of PGP to promote pathological airway epithelial remodeling. Thus, we demonstrate a disconnect between airway inflammation and AHR and the ability of a matrikine to promote an epithelial remodeling phenotype that negatively affects lung function. Subsequently, we show that substantial quantities of PGP are detectable in the sputum of moderate-severe asthmatics in two distinct cohorts of patients. These studies have implications for our understanding of remodeling phenotypes in asthma and may rationalize the failure of LTA4H inhibitors in the clinic.

The neutrophil chemoattractant proline-glycine-proline (PGP) is generated from collagen by matrix metalloproteinase-8/9 (MMP-8/9) and prolyl endopeptidase (PE), and it is concomitantly degraded by extracellular leukotriene A4 hydrolase (LTA4H) to limit neutrophilia. Components of cigarette smoke can acetylate PGP, yielding a species (AcPGP) that is resistant to LTA4H-mediated degradation and can, thus, support a sustained neutrophilia. In this study, we sought to elucidate if an antiinflammatory system existed to degrade AcPGP that is analogous to the PGP-LTA4H axis. We demonstrate that AcPGP is degraded through a previously unidentified action of the enzyme angiotensin-converting enzyme (ACE). Pulmonary ACE is elevated during episodes of acute inflammation, as a consequence of enhanced vascular permeability, to ensure the efficient degradation of AcPGP. Conversely, we suggest that this pathway is aberrant in chronic obstructive pulmonary disease (COPD) enabling the accumulation of AcPGP. Consequently, we identify a potentially novel protective role for AcPGP in limiting pulmonary fibrosis and suggest the pathogenic function attributed to ACE in idiopathic pulmonary fibrosis (IPF) to be a consequence of overzealous AcPGP degradation. Thus, AcPGP seemingly has very divergent roles: it is pathogenic in its capacity to drive neutrophilic inflammation and matrix degradation in the context of COPD, but it is protective in its capacity to limit fibrosis in IPF., ACE degrades the collagen-derived matrikine, acetylate proline–glycine–proline, to limit pulmonary inflammation and promote repair.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US. The majority of COPD patients have symptoms of chronic bronchitis, which lacks specific therapies. A major impediment to therapeutic development has been the absence of animal models that recapitulate key clinical and pathologic features of human disease. Ferrets are well suited for the investigation of the significance of respiratory diseases, given prior data indicating similarities to human airway physiology and submucosal gland distribution. Here, we exposed ferrets to chronic cigarette smoke and found them to approximate complex clinical features of human COPD. Unlike mice, which develop solely emphysema, smoke-exposed ferrets exhibited markedly higher numbers of early-morning spontaneous coughs and sporadic infectious exacerbations as well as a higher level of airway obstruction accompanied by goblet cell metaplasia/hyperplasia and increased mucus expression in small airways, indicative of chronic bronchitis and bronchiolitis. Overall, we demonstrate the first COPD animal model exhibiting clinical and pathologic features of chronic bronchitis to our knowledge, providing a key advance that will greatly facilitate the preclinical development of novel treatments for this disease.

COPD is a major cause of mortality worldwide, mainly caused by cigarette smoking and driven by an excessive neutrophilic response. The matrikine ac-pro-gly-pro (acPGP) is derived from collagen and is chemotactic to neutrophils by acting on CXC receptor 2 (CXCR2). The PGP neutralizing peptide L-arg-thr-arg (RTR) was used in a 23 weeks cigarette smoke exposure (CSE) murine model. Mice started the RTR treatment after 10 weeks of CSE and continued the treatment together with the CSE during the last 13 weeks. RTR significantly inhibited neutrophil and macrophage influx into the lungs as seen in the broncho alveolar lavage fluid(Fig 1). Furthermore, mice receiving RTR during the last 13 weeks did not develop an emphysema phenotype and less right ventricular hypertrophy , compared to controls. Finally, in vitro experiments using primary human bronchial epithelial (HBE) cells from healthy individuals or COPD patients were carried out. Pretreatment of the cells with cigarette smoke extract and stimulated with acPGP resulted in a higher CXCR2 protein signal in the COPD cells. Also, acPGP facilitated the release of MMP-9 and interleukine-8 from primary HBE cells, thereby highlighting a new mechanism for acPGP to augment neutrophilic inflammation. Our data strongly underscore an important role for acPGP in the development of the emphysema and epithelial cell dysfunction in COPD.

Matrix metalloprotease-9 (MMP-9) plays a role in progression of cystic fibrosis, and doxycycline can reduce MMP-9 in vitro . Here, we explore the effect of doxycycline during cystic fibrosis exacerbation treatment on MMP-9 related readouts and clinical end-points. This randomised, double-blind, placebo-controlled study enrolled hospitalised patients with cystic fibrosis undergoing exacerbation. In total, 20 participants were given doxycycline and 19 participants were given placebo over an 8-day period during hospitalisation. Biospecimens were collected at the beginning and the end of the study period. Primary end-points were total MMP-9 levels in the sputum and safety/tolerability. Secondary end-points included change in lung function, time to next exacerbation, and markers of MMP-9-related protease activity (active MMP-9 and TIMP-1). Nonparametric testing was used for within-group and between-group analyses. Doxycycline was well tolerated, with no treatment discontinuations or serious adverse events. Doxycycline reduced total sputum MMP-9 levels by 63.2% (p

Background Premature rupture of membranes and preterm delivery are associated with Ureaplasma infection. We hypothesized that Ureaplasma induced extracellular collagen fragmentation results in production of the tripeptide PGP (proline-glycine-proline), a neutrophil chemoattractant. PGP release from collagen requires matrix metalloproteases (MMP-8/MMP-9) along with a serine protease, prolyl endopeptidase (PE). Methods Ureaplasma culture negative amniotic fluid (indicated preterm birth, n=8; spontaneous preterm birth, n=8) and Ureaplasma positive amniotic fluid (spontaneous preterm birth, n=8) were analyzed by electro-spray ionization-liquid chromatography tandem mass spectrometry for PGP, and for MMP-9 by zymography. PE was evaluated in lysates of U. parvum serovar 3 (Up3) and U. urealyticum serovar 10 (Uu10) by western blotting and activity assay. Results PGP and MMP-9 were increased in amniotic fluid from spontaneous preterm birth with positive Ureaplasma cultures, but not with indicated preterm birth or spontaneous preterm birth with negative Ureaplasma cultures. Human neutrophils co-cultured with Ureaplasma strains showed increased MMP-9 activity. PE presence and activity were noted with both Ureaplasma strains. Conclusions Ureaplasma spp. carry the protease necessary for PGP release, and PGP and MMP-9 are increased in amniotic fluid during Ureaplasma infection, suggesting Ureaplasma spp. induced collagen fragmentation contributes to preterm rupture of membranes and neutrophil influx causing chorioamnionitis.

Rationale Tumor necrosis factor-alpha (TNF-α) is a potent pro-inflammatory mediator and its expression is up-regulated in chronic obstructive pulmonary disease (COPD). Tristetraprolin (TTP) is implicated in regulation of TNF-α expression; however, whether TTP is involved in cigarette smoke-induced TNF-α expression has not been determined. Methods TTP expression was examined by western blot analysis in murine alveolar macrophages and alveolar epithelial cells challenged without or with cigarette smoke extract (CSE). TNF-α mRNA stability, and the decay of TNF-α mRNA, were determined by real-time quantitative RT-PCR. TNF-α protein levels were examined at the same time in these cells. To identify the molecular mechanism involved, a construct expressing the human beta-globin reporter mRNA containing the TNF-α 3’-untranslated region was generated to characterize the TTP targeted site within TNF-α mRNA. Results CSE induced TTP down-regulation in alveolar macrophages and alveolar epithelial cells. Reduced TTP expression resulted in significantly increased TNF-α mRNA stability. Importantly, increased TNF-α mRNA stability due to impaired TTP function resulted in significantly increased TNF-α levels in these cells. Forced TTP expression abrogated the increased TNF-α mRNA stability and expression induced by CSE. By using the globin reporter construct containing TNF-α mRNA 3’-untranslated region, the data indicate that TTP directly targets the adenine- and uridine-rich region (ARE) of TNF-α mRNA and negatively regulates TNF-α expression at the post-transcriptional level. Conclusion The data demonstrate that cigarette smoke exposure reduces TTP expression and impairs TTP function, resulting in significantly increased TNF-α mRNA stability and excessive TNF-α expression in alveolar macrophages and epithelial cells. The data suggest that TTP is a novel post-transcriptional regulator and limits excessive TNF-α expression and inflammatory response induced by cigarette smoke.

Members of the heme peroxidase family play an important role in host defense. Myeloperoxidase (MPO) is expressed in phagocytes and is the only animal heme peroxidase previously reported to be capable of using chloride ion as a substrate to form the highly microbicidal species hypochlorous acid (HOCl) at neutral pH. Despite the potent bacterial killing activity of HOCl, individuals who fail to express MPO typically show only a modest increase in some fungal infections. This may point to the existence of redundant host defense mechanisms. Vascular peroxidase 1 (VPO1) is newly discovered member of the heme peroxidase family. VPO1 is expressed in cells of the cardiovascular system and is secreted into the bloodstream. In the present study, we investigate whether VPO1 is capable of generating HOCl and its role in host defense. Like MPO, VPO1 in the presence of H 2 O 2 and chloride generates HOCl. VPO1-dependent HOCl generation was demonstrated by chlorination of taurine and tyrosine using mass spectrometry. In addition, the VPO1/H 2 O 2 /Cl − system can cause the chlorination of monochlorodimedone and the oxidation of 5-thio-2-nitrobenzoic acid. Purified VPO1 and VPO1 in plasma mediate bacterial killing that is dependent on chloride and H 2 O 2 ; killing is inhibited by peroxidase inhibitors and by the H 2 O 2 scavenger catalase. In the presence of erythrocytes, bacterial killing by VPO1 is slightly reduced. Thus, VPO1, in addition to MPO, is the second member of the heme peroxidase family capable of generating HOCl under physiological conditions. VPO1 is likely to participate in host defense, with bactericidal activity mediated through the generation of HOCl.

Smoke Gets in Your Lungs Chronic obstructive pulmonary disease (COPD) is a leading cause of death in the United States, primarily caused by cigarette smoking. The chronic inflammation that leads to tissue damage and organ dysfunction in COPD is mediated in large part by neutrophils, a type of granulocytic immune cell. Snelgrove et al. (p. 90 , published online 2 September; see the Perspective by Barnes ) now provide an explanation for why neutrophils stick around in the lung during COPD. The neutrophil chemoattractant Pro-Gly-Pro (PGP) is a biomarker for COPD and promotes neutrophil accumulation. The enzyme leukotriene A 4 hydrolase degrades PGP in mice, and its activity was reduced by cigarette smoke both in vivo and in vitro. In contrast, during acute influenza infection in mice, leukotriene A 4 hydrolase functioned normally, allowing for PGP degradation and the resolution of inflammation. Thus, in COPD, cigarette smoking may lead to the accumulation PGP—which, in turn, could keep neutrophils in the lung to drive inflammation and subsequent lung damage and dysfunction.

High-mobility group box 1 (HMGB1) is a potent inflammatory mediator elevated in sepsis and rheumatoid arthritis, although its role in cystic fibrosis (CF) lung disease is unknown.To determine whether HMGB1 contributes to CF lung inflammation, including neutrophil chemotaxis and lung matrix degradation.We used sputum and serum from subjects with CF and a Scnn1b-transgenic (Scnn1b-Tg) mouse model that overexpresses beta-epithelial Na(+) channel in airways and mimics the CF phenotype, including lung inflammation. Human secretions and murine bronchoalveolar lavage fluid (BALF) was assayed for HMGB1 by Western blot and ELISA. Neutrophil chemotaxis was measured in vitro after incubation with human neutrophils. The collagen fragment proline-glycine-proline (PGP) was measured by tandem mass spectroscopy.HMGB1 was detected in CF sputum at higher levels than secretions from normal individuals. Scnn1b-Tg mice had elevated levels of HMGB1 by Western blot and ELISA. We demonstrated that dose-dependent chemotaxis of human neutrophils stimulated by purified HMGB1 was partially dependent on CXC chemokine receptors and that this could be duplicated in CF sputum and BALF from Scnn1b-Tg mice. Neutralization by anti-HMGB1 antibody, in both the sputum and BALF-reduced chemotaxis, which suggested that HMGB1 contributed to the chemotactic properties of these samples. Intratracheal administration of purified HMGB1 induced neutrophil influx into the airways of mice and promoted the release of PGP. PGP was also elevated in Scnn1b-Tg mice and CF serum.HMGB1 expression contributes to pulmonary inflammation and lung matrix degradation in CF airway disease and deserves further investigation as a biomarker and potential therapeutic target.

Matrix metalloproteinase (MMP)-8 and MMP-9 play several roles in inflammation, including degradation of extracellular matrix (ECM) components and regulation of cytokine activity. To determine the roles of MMP-8 and MMP-9 in a neutrophil-dependent inflammatory response, we used a murine model of corneal inflammation in which LPS is injected into the corneal stroma. In contrast to wild-type mice, we found that i) lipopolysaccharide (LPS)-injected CXCR2(-/-) corneas had impaired neutrophil infiltration and did not express either MMP-8 or MMP-9; ii) neutrophil migration through the central cornea was impaired in Mmp8(-/-), but not Mmp9(-/-), mice; iii) neutrophil migration was inhibited in collagenase-resistant mice; iv) the chemotactic Pro-Gly-Pro (PGP) tripeptide that binds CXCR2 was decreased in CXCR2(-/-) mice; v) PGP production was impaired in Mmp8(-/-) corneas; and vi) neutralizing anti-PGP antibody did not inhibit neutrophil infiltration in Mmp8(-/-) mice. We found no effects of MMP-8 on LPS-induced CXC chemokine (LIX, or CXCL5)-induced neutrophil recruitment or on LPS-induced CXC chemokine production. Together, these studies indicate that neutrophils contribute to the production of both MMP-8 and MMP-9 in LPS-injected corneas and that MMP-8 regulates neutrophil migration through the dense collagenous ECM of the corneal stroma by generating chemotactic PGP during inflammation.

Chronic neutrophilic inflammation is a manifestation of a variety of lung diseases including cystic fibrosis (CF). There is increasing evidence that fragments of extracellular matrix proteins, such as collagen and elastin, play an important role in inflammatory cell recruitment to the lung in animal models of airway inflammation. Unfortunately, the association of these peptides with human disease and the identification of therapeutic targets directed toward these inflammatory pathways have remained elusive. In this study, we demonstrate that a novel extracellular matrix-derived neutrophil chemoattractant, proline-glycine-proline (PGP), acts through CXC receptors 1 and 2 on neutrophils, similar to N-acetylated proline-glycine-proline (N-α-PGP). We describe the specific multistep proteolytic pathway involved in PGP generation from collagen, involving matrix metalloproteases 8 and 9 and prolyl endopeptidase, a serine protease for which we identify a novel role in inflammation. PGP generation correlates closely with airway neutrophil counts after administration of proteases in vivo. Using CF as a model, we show that CF sputum has elevated levels of PGP peptides and that PGP levels decline during the course of CF inpatient therapy for acute pulmonary exacerbation, pointing to its role as a novel biomarker for this disease. Finally, we demonstrate that CF secretions are capable of generating PGP from collagen ex vivo and that this generation is significantly attenuated by the use of inhibitors directed toward matrix metalloprotease 8, matrix metalloprotease 9, or prolyl endopeptidase. These experiments highlight unique protease interactions with structural proteins regulating innate immunity and support a role for these peptides as novel biomarkers and therapeutic targets for chronic, neutrophilic lung diseases.

A striking feature of pulmonary infection with the Gram-negative intracellular bacterium Francisella tularensis, a category A biological threat agent, is an intense accumulation of inflammatory cells, particularly neutrophils and macrophages, at sites of bacterial replication. Given the essential role played by host matrix metalloproteinases (MMPs) in modulating leukocyte recruitment and the potentially indiscriminate destructive capacity of these cells, we investigated whether MMP-9, an important member of this protease family released by neutrophils and activated macrophages, plays a role in the pathogenesis of respiratory tularemia. We found that F. tularensis induced expression of MMP-9 in FVB/NJ mice and that the action of this protease is associated with higher bacterial burdens in pulmonary and extrapulmonary tissues, development of more extensive histopathology predominated by neutrophils, and increased morbidity and mortality compared with mice lacking MMP-9 (MMP-9−/−). Moreover, MMP-9−/− mice were able to resolve infection with either the virulence-attenuated type B (live vaccine strain) or the highly virulent type A (SchuS4) strain of F. tularensis. Disease resolution was accompanied by diminished leukocyte recruitment and reductions in both bacterial burden and proinflammatory cytokine production. Notably, neutrophilic infiltrates were significantly reduced in MMP-9−/− mice, owing perhaps to limited release of Pro-Gly-Pro, a potent neutrophil chemotactic tripeptide released from extracellular matrix through the action of MMP-9. Collectively, these results suggest that MMP-9 activity plays a central role in modulating the clinical course and severity of respiratory tularemia and identifies MMPs as novel targets for therapeutic intervention as a means of modulating neutrophil recruitment.

Roflumilast is a therapeutic agent in the treatment of chronic obstructive pulmonary disease (COPD). It has antiinflammatory effects; however, it is not known whether it can affect a biologic pathway implicated in COPD pathogenesis and progression. The self-propagating acetyl-proline-glycine-proline (AcPGP) pathway is a novel means of neutrophilic inflammation that is pathologic in the development of COPD. AcPGP is produced by extracellular matrix collagen breakdown with prolyl endopeptidase and leukotriene A4 hydrolase serving as the enzymes responsible for its production and degradation, respectively.We hypothesized that roflumilast would decrease AcPGP, halting the feed-forward cycle of inflammation.We conducted a single-center, placebo-controlled, randomized study investigating 12 weeks of roflumilast treatment added to current therapy in moderate-to-severe COPD with chronic bronchitis. Subjects underwent sputum and blood analyses, pulmonary function testing, exercise tolerance, and quality-of-life assessment at 0, 4, and 12 weeks.Twenty-seven patients were enrolled in the intention-to-treat analysis. Roflumilast treatment decreased sputum AcPGP by more than 50% (P 0.01) and prolyl endopeptidase by 46% (P = 0.02), without significant improvement in leukotriene A4 hydrolase activity compared with placebo. Roflumilast also reduces other inflammatory markers. There were no significant changes in lung function, quality of life, or exercise tolerance between roflumilast- and placebo-treated groups.Roflumilast reduces pulmonary inflammation through decreasing prolyl endopeptidase activity and AcPGP. As expected for lower AcPGP levels, markers of neutrophilic inflammation are blunted. Inhibiting this self-propagating pathway lessens the overall inflammatory burden, which may alter the natural history of COPD, including the risk of exacerbation. Clinical trial registered with www.clinicaltrials.gov (NCT 01572948).

Bioactive matrix fragments (matrikines) have been identified in a myriad of disorders, but their impact on the evolution of airway inflammation has not been demonstrated. We recently described a pathway where the matrikine and neutrophil chemoattractant proline–glycine–proline (PGP) could be degraded by the enzyme leukotriene A4 hydrolase (LTA4H). LTA4H classically functions in the generation of pro-inflammatory leukotriene B4, thus LTA4H exhibits opposing pro- and anti-inflammatory activities. The physiological significance of this secondary anti-inflammatory activity remains unknown. Here we show, using readily resolving pulmonary inflammation models, that loss of this secondary activity leads to more pronounced and sustained inflammation and illness owing to PGP accumulation. PGP elicits an exacerbated neutrophilic inflammation and protease imbalance that further degrades the extracellular matrix, generating fragments that perpetuate inflammation. This highlights a critical role for the secondary anti-inflammatory activity of LTA4H and thus has consequences for the generation of global LTA4H inhibitors currently being developed., Proteases degrade extracellular matrix during inflammation, releasing peptides that can recruit neutrophils. Here the authors show that degradation of such bioactive peptide by the enzyme leukotriene A4 hydrolase is critical to limit pulmonary inflammation during bacterial infection in mice.

Rationale: Chronic neutrophilic inflammation is a hallmark in the pathogenesis of chronic obstructive pulmonary disease (COPD) and persists after cigarette smoking has stopped. Mechanisms involved in this ongoing inflammatory response have not been delineated. Objectives: We investigated changes to the leukotriene A4 hydrolase (LTA4H)–proline-glycine-proline (PGP) pathway and chronic inflammation in the development of COPD. Methods: A/J mice were exposed to air or cigarette smoke for 22 weeks followed by bronchoalveolar lavage and lung and cardiac tissue analysis. Two human cohorts were used to analyze changes to the LTA4H–PGP pathway in never smokers, control smokers, COPD smokers, and COPD former smokers. PGP/AcPGP and LTA4H aminopeptidase activity were detected by mass spectroscopy, LTA4H amounts were detected by ELISA, and acrolein was detected by Western blot. Measurements and Main Results: Mice exposed to cigarette smoke developed emphysema with increased PGP, neutrophilic inflammation, and selective inhibition of LTA4H aminopeptidase, which ordinarily degrades PGP. We recapitulated these findings in smokers with and without COPD. PGP and AcPGP are closely associated with cigarette smoke use. Once chronic inflammation is established, changes to LTA4H aminopeptidase remain, even in the absence of ongoing cigarette use. Acrolein modifies LTA4H and inhibits aminopeptidase activity to the same extent as cigarette smoke. Conclusions: These results demonstrate a novel pathway of aberrant regulation of PGP/AcPGP, suggesting this inflammatory pathway may be intimately involved in disease progression in the absence of ongoing cigarette smoke exposure. We highlight a mechanism by which acrolein potentiates neutrophilic inflammation through selective inhibition of LTA4H aminopeptidase activity. Clinical trial registered with www.clinicaltrials.gov (NCT 00292552).

Objective Proline–glycine–proline (PGP) has been shown to have chemotactic effects on neutrophils via CXCR2 in several lung diseases. PGP is derived from collagen by the combined action of matrix metalloproteinase (MMP) 8 and/or MMP9 and prolyl endopeptidase (PE). We investigated the role of PGP in inflammatory bowel disease (IBD). Design In intestinal tissue from patients with IBD and mice with dextran sodium sulfate (DSS)-induced colitis, MMP8, MMP9 and PE were evaluated by ELISA, immunoblot and immunohistochemistry. Peripheral blood polymorphonuclear cell (PMN) supernatants were also analysed accordingly and incubated with collagen to assess PGP generation ex vivo. PGP levels were measured by mass spectrometry, and PGP neutralisation was achieved with a PGP antagonist and PGP antibodies. Results In the intestine of patients with IBD, MMP8 and MMP9 levels were elevated, while PE was expressed at similar levels to control tissue. PGP levels were increased in intestinal tissue of patients with IBD. Similar results were obtained in intestine from DSS-treated mice. PMN supernatants from patients with IBD were far more capable of generating PGP from collagen ex vivo than healthy controls. Furthermore, PGP neutralisation during DSS-induced colitis led to a significant reduction in neutrophil infiltration in the intestine. Conclusions The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of patients with IBD and mice with DSS-induced colitis. PGP neutralisation in DSS-treated mice showed the importance of PGP-guided neutrophilic infiltration in the intestine and indicates a vicious circle in neutrophilic inflammation in IBD.

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.

Multiple sclerosis (MS) is clinically heterogeneous and has an uncertain natural history. A high priority for more effective treatment of MS is an objective and feasible laboratory test for predicting the disease's course and response to treatments. Urinary myelin basic protein (MBP)-like material (MBPLM), so designated because it is immunoreactive as a cryptic epitope in peptide 83-89 of the human MBP molecule of 170 amino acids, is present in normal adults, remains normal in relapsing-remitting, but increases in progressive MS. In the present investigation, MBPLM was purified from urine and characterized. p-Cresol sulfate is the major component of urinary MBPLM. This conclusion is based on the following: (1) MBPLM and p-cresol sulfate both have a mass of 187 on negative scans by electrospray ionization mass spectrometry, the same fragments on tandem mass spectrometry of 80 (SO(-)(3)) and 107 (methylphenol), and similar profiles on multiple reaction monitoring; (2) (1)H and (13)C nuclear magnetic resonance spectroscopy revealed identical spectra for MBPLM and p-cresol sulfate; (3) purified p-cresol sulfate reacted in parallel with MBP peptide 83-89 in the same radioimmunoassay for MBPLM; and (4) p-cresol sulfate has the same behavior on preparative HPLC columns as urinary MBPLM. The unexpected immunochemical degeneracy permitting a cross-reaction between p-cresol sulfate and a peptide of an encephalitogenic myelin protein is postulated to be based on shared conformational features. The mechanisms by which urinary p-cresol sulfate, possibly derived from tyrosine-SO(4), reflects progressive worsening that is disabling in MS are unknown.

Diets enriched in soy foods containing a high concentration of isoflavonoids are associated with a decrease in the incidence of several chronic inflammatory diseases. Studies with experimental models of diseases, such as atherosclerosis, suggest that these effects can be ascribed to the biological properties of the isoflavones. Since the isoflavones and tyrosine have structural similarities and modifications to tyrosine by inflammatory oxidants such as hypochlorous acid (HOCl) and peroxynitrite (ONOO(-)) have been recently recognized, we hypothesized that the isoflavones also react with HOCl and ONOO(-). Using an in vitro approach, we demonstrate in the present study that the isoflavones genistein, daidzein, and biochanin-A can be chlorinated and nitrated by these oxidants. These reactions were investigated using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. In the reaction with HOCl, both mono- and dichlorinated derivatives of genistein and biochanin-A are formed, whereas with daidzein only a monochlorinated derivative was detected. The reaction between genistein or daidzein and ONOO(-) yielded a mononitrated product. However, no nitrated product was detected with biochanin-A. Furthermore, the reaction between genistein and sodium nitrite and HOCl yielded a chloronitrogenistein derivative, as well as a dichloronitrogenistein derivative. These results indicate that the ability of the isoflavones to react with these oxidant species depends on their structure and suggest that they could be formed under conditions where these reactive species are generated under pathological conditions.

We report the detailed solution structure of the 7.2 kDa protein CsE-I, a beta-neurotoxin from the New World scorpion Centruroides sculpturatus Ewing. This toxin binds to sodium channels, but unlike the alpha-neurotoxins, shifts the voltage of activation toward more negative potentials causing the membrane to fire spontaneously. Sequence-specific proton NMR assignments were made using 600 MHz 2D-NMR data. Distance geometry and dynamical simulated annealing refinements were performed using experimental distance and torsion angle constraints from NOESY and pH-COSY data. A family of 40 structures without constraint violations was generated, and an energy-minimized average structure was computed. The backbone conformation of the CsE-I toxin shows similar secondary structural features as the prototypical alpha-neurotoxin, CsE-v3, and is characterized by a short 2(1/2)-turn alpha-helix and a 3-strand antiparallel beta-sheet, both held together by disulfide bridges. The RMSD for the backbone atoms between CsE-I and CsE-v3 is 1.48 A. Despite this similarity in the overall backbone folding, the these two proteins show some important differences in the primary structure (sequence) and electrostatic potential surfaces. Our studies provide a basis for unravelling the role of these differences in relation to the known differences in the receptor sites on the voltage sensitive sodium channel for the alpha- and beta-neurotoxins.

Rationale: Several extrapulmonary disorders have been linked to cigarette smoking. Smoking is reported to cause cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the airway, and is also associated with pancreatitis, male infertility, and cachexia, features characteristic of cystic fibrosis and suggestive of an etiological role for CFTR. Objectives: To study the effect of cigarette smoke on extrapulmonary CFTR function. Methods: Demographics, spirometry, exercise tolerance, symptom questionnaires, CFTR genetics, and sweat chloride analysis were obtained in smokers with and without chronic obstructive pulmonary disease (COPD). CFTR activity was measured by nasal potential difference in mice and by Ussing chamber electrophysiology in vitro. Serum acrolein levels were estimated with mass spectroscopy. Measurements and Main Results: Healthy smokers (29.45 ± 13.90 mEq), smokers with COPD (31.89 ± 13.9 mEq), and former smokers with COPD (25.07 ± 10.92 mEq) had elevated sweat chloride levels compared with normal control subjects (14.5 ± 7.77 mEq), indicating reduced CFTR activity in a nonrespiratory organ. Intestinal current measurements also demonstrated a 65% decrease in CFTR function in smokers compared with never smokers. CFTR activity was decreased by 68% in normal human bronchial epithelial cells exposed to plasma from smokers, suggesting that one or more circulating agents could confer CFTR dysfunction. Cigarette smoke–exposed mice had decreased CFTR activity in intestinal epithelium (84.3 and 45%, after 5 and 17 wk, respectively). Acrolein, a component of cigarette smoke, was higher in smokers, blocked CFTR by inhibiting channel gating, and was attenuated by antioxidant N-acetylcysteine, a known scavenger of acrolein. Conclusions: Smoking causes systemic CFTR dysfunction. Acrolein present in cigarette smoke mediates CFTR defects in extrapulmonary tissues in smokers.

Background: Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD). In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs) 8 and 9 and prolyl endopeptidase (PE). Here we investigated whether cigarette smoke extract (CSE) stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP.Methodology/Principal Findings: Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE.Conclusions: This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE-or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.

Each year thousands of people trip, stumble, and/or fall on stairs. This paper reviews prominent literature on stairway safety, and discusses findings from a preliminary survey of 40 in-depth stair accident cases. Variables reviewed and discussed include personal/demographic, external, and dimensional. From these findings and those of other researchers, it is hypothesized that the greatest problem with accident stairways is not individual (i.e., user) or external variables, but dimensional inconsistency inherent in some stairways. Areas for future research are also discussed.

The purpose of this integrative literature review is to improve anticipatory guidance in early childhood by reviewing the influence of optimism bias on parents' views about safety and beliefs about their children's risk for unintentional injuries. This article reviews the theory of optimism bias and recent research utilizing optimism bias to explain parental health-related behaviors. The three articles in this literature review find a link between optimism bias and parents' failure to implement safety behaviors. Currently, there is no tool to measure a parent's level of optimism bias concerning the risk of unintentional injury to his or her child. It is important for primary care providers to try and identify optimism bias in parents and address it as a barrier to implementation of safety recommendations. More research should be dedicated to developing screening tools to identify optimism bias in parents and interventions to help them accept their children's vulnerability.

Several chronic lung diseases have been linked to cigarette smoking (Chronic Obstructive Pulmonary Disease (COPD), and cancer are associated with increased tobacco use). We recently described a collagen fragment, proline-glycine-proline (PGP), chemotactic for neutrophils, that appears to play a role in COPD, cystic fibrosis, and bronchiolitis obliterans syndrome. PGP can exist in either its native or acetylated form (NAcPGP), although the mechanism of N-terminal-acetylation remains unknown. This work investigates the possibility that cigarette smoke (CS) and its components acetylate PGP, describing a possible mechanism for some of the chronic inflammation seen in tobacco-associated disease. CSE and CSC (3.56 and 12.38 ng/ml NAcPGP respectively, p less than 0.01) and its components (acrolein, acetaldehyde, and methyl glyoxal) acetylated PGP (0.51, 1.03, and 0.23 ng/ml NAcPGP, p less than 0.01). Both N-acetyl-cysteine and carbocysteine (scavengers of reactive aldehydes) blocked chemical acetylation of PGP by CS (100 percent and 97 percent inhibition, respectively, p less than 0.01). NAcPGP is more chemoattractive to neutrophils, and less susceptible to degradation by Leukotriene-A4-Hydrolase (detected in the lung). These experiments propose a mechanism for the increased neutrophil recruitment seen in smoking-associated lung diseases.