Moraxella catarrhalis acquisition, airway inflammation and protease-antiprotease balance in chronic obstructive pulmonary disease

<p>Abstract</p> <p>Background</p> <p><it>Moraxella catarrhalis </it>causes approximately 10% of exacerbations in chronic obstructive pulmonary disease (COPD) and also colonizes the lower airway in stable patients. Little is known about the effects of colonization by <it>M. catarrhalis </it>on airway inflammation and protease-antiprotease balance, and how these changes compare to those seen during exacerbations. Since COPD is a progressive inflammatory disease, elucidating the effects of bacterial colonization and exacerbation on airway inflammation is relevant to understanding disease progression in COPD. Our aims were (1) Analyze changes in airway inflammation in colonization and exacerbation of COPD due to <it>M. catarrhalis</it>; (2) Explore protease-antiprotease balance in colonization and exacerbation due to <it>M. catarrhalis</it>. Our hypothesis were (1) Acquisition of a new strain of M. catarrhalis in COPD increases airway inflammation from baseline and alters the protease-antiprotease balance towards a more proteolytic environment; (2) These changes are greater during exacerbations associated with <it>M. catarrhalis </it>as compared to colonization.</p> <p>Methods</p> <p>Thirty-nine consecutive COPD patients with 76 acquisitions of a new strain of <it>M. catarrhalis </it>over a 6-year period were identified in a prospective study. Seventy-six pre-acquisition sputum supernatant samples, obtained just before acquisition of <it>M catarrhalis</it>, and 76 acquisition samples (34 were associated with exacerbation, 42 with colonization) were analyzed for IL-8, TNF-α, Neutrophil Elastase (NE) and Secretory leukocyte protease inhibitor (SLPI). Changes were compared in paired samples from each patient.</p> <p>Results</p> <p>IL-8, TNF-α and NE were significantly elevated after acquisition of <it>M. catarrhalis</it>, compared to pre-acquisition samples (p =< 0.001 for all three). These changes were present in colonization (p = 0.015 for IL-8; p =< 0.001 for TNF-α and NE) as well as in exacerbation (p =< 0.001 for all three), compared to pre-acquisition levels. SLPI was significantly lower after acquisition (p =< 0.001), in colonization (p =< 0.001) as well as in exacerbation (p = 0.004), compared to pre-acquisition levels. SLPI levels correlated negatively with NE levels (R²= 0.07; p = 0.001).</p> <p>Conclusion</p> <p>Acquisition of <it>M. catarrhalis </it>in COPD causes increased airway inflammation and worsening protease-antiprotease imbalance during exacerbations and also in colonization, even in the absence of increased symptoms. These effects could contribute to progression of airway disease in COPD.</p

The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post-partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome-wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper-polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long-lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility

Neutrophil elastase reduces secretion of secretory leukoproteinase inhibitor (SLPI) by lung epithelial cells: role of charge of the proteinase-inhibitor complex

<p>Abstract</p> <p>Background</p> <p>Secretory leukoproteinase inhibitor (SLPI) is an important inhibitor of neutrophil elastase (NE), a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes <it>in vitro</it>.</p> <p>Methods</p> <p>Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling.</p> <p>Results</p> <p>We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration.</p> <p>Conclusion</p> <p>These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon <it>in vitro</it>, which may correlate with changes observed <it>in vivo</it>.</p

Inhibition of SLPI ameliorates disease activity in experimental autoimmune encephalomyelitis

<p>Abstract</p> <p>Background</p> <p>The secretory leukocyte protease inhibitor (SLPI) exerts wide ranging effects on inflammatory pathways and is upregulated in EAE but the biological role of SLPI in EAE, an animal model of multiple sclerosis is unknown</p> <p>Methods</p> <p>To investigate the pathophysiological effects of SLPI within EAE, we induced SLPI-neutralizing antibodies in mice and rats to determine the clinical severity of the disease. In addition we studied the effects of SLPI on the anti-inflammatory cytokine TGF-β.</p> <p>Results</p> <p>The induction of SLPI neutralizing antibodies resulted in a milder disease course in mouse and rat EAE. SLPI neutralization was associated with increased serum levels of TGF-β and increased numbers of FoxP3+ CD4+ T cells in lymph nodes. <it>In vitro</it>, the addition of SLPI significantly decreased the number of functional FoxP3+ CD25^hiCD4+ regulatory T cells in cultures of naive human CD4+ T cells. Adding recombinant TGF-β to SLPI-treated human T cell cultures neutralized SLPI's inhibitory effect on regulatory T cell differentiation.</p> <p>Conclusion</p> <p>In EAE, SLPI exerts potent pro-inflammatory actions by modulation of T-cell activity and its neutralization may be beneficial for the disease.</p

Glucocorticosteroids Differentially Regulate MMP-9 and Neutrophil Elastase in COPD

Background: Chronic Obstructive Pulmonary Disease (COPD) is currently the fifth leading cause of death worldwide. Neutrophilic inflammation is prominent, worsened during infective exacerbations and is refractory to glucocorticosteroids (GCs). Deregulated neutrophilic inflammation can cause excessive matrix degradation through proteinase release. Gelatinase and azurophilic granules within neutrophils are a major source of matrix metalloproteinase (MMP)-9 and neutrophil elastase (NE), respectively, which are elevated in COPD. Methods: Secreted MMP-9 and NE activity in BALF were stratified according to GOLD severity stages. The regulation of secreted NE and MMP-9 in isolated blood neutrophils was investigated using a pharmacological approach. In vivo release of MMP-9 and NE in mice exposed to cigarette smoke (CS) and/or the TLR agonist lipopolysaccharide (LPS) in the presence of dexamethasone (Dex) was investigated. Results: Neutrophil activation as assessed by NE release was increased in severe COPD (36-fold, GOLD II vs. IV). MMP-9 levels (8-fold) and activity (21-fold) were also elevated in severe COPD, and this activity was strongly associated with BALF neutrophils (r = 0.92, p &lt; 0.001), but not macrophages (r = 0.48, p = 0.13). In vitro, release of NE and MMP-9 from fMLP stimulated blood neutrophils was insensitive to Dex and attenuated by the PI3K inhibitor, wortmannin. In vivo, GC resistant neutrophil activation (NE release) was only seen in mice exposed to CS and LPS. In addition, GC refractory MMP-9 expression was only associated with neutrophil activation. Conclusions: As neutrophils become activated with increasing COPD severity, they become an important source of NE and MMP-9 activity, which secrete proteinases independently of TIMPs. Furthermore, as NE and MMP-9 release was resistant to GC, targeting of the PI3K pathway may offer an alternative pathway to combating this proteinase imbalance in severe COPD

Transcriptome profiling of immune responses to cardiomyopathy syndrome (CMS) in Atlantic salmon

<p>Abstract</p> <p>Background</p> <p>Cardiomyopathy syndrome (CMS) is a disease associated with severe myocarditis primarily in adult farmed Atlantic salmon (<it>Salmo salar </it>L.), caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV) with structural similarities to the <it>Totiviridae </it>family. Here we present the first characterisation of host immune responses to CMS assessed by microarray transcriptome profiling.</p> <p>Results</p> <p>Unvaccinated farmed Atlantic salmon post-smolts were infected by intraperitoneal injection of PMCV and developed cardiac pathology consistent with CMS. From analysis of heart samples at several time points and different tissues at early and clinical stages by oligonucleotide microarrays (SIQ2.0 chip), six gene sets representing a broad range of immune responses were identified, showing significant temporal and spatial regulation. Histopathological examination of cardiac tissue showed myocardial lesions from 6 weeks post infection (wpi) that peaked at 8-9 wpi and was followed by a recovery. Viral RNA was detected in all organs from 4 wpi suggesting a broad tissue tropism. High correlation between viral load and cardiac histopathology score suggested that cytopathic effect of infection was a major determinant of the myocardial changes. Strong and systemic induction of antiviral and IFN-dependent genes from 2 wpi that levelled off during infection, was followed by a biphasic activation of pathways for B cells and MHC antigen presentation, both peaking at clinical pathology. This was preceded by a distinct cardiac activation of complement at 6 wpi, suggesting a complement-dependent activation of humoral Ab-responses. Peak of cardiac pathology and viral load coincided with cardiac-specific upregulation of T cell response genes and splenic induction of complement genes. Preceding the reduction in viral load and pathology, these responses were probably important for viral clearance and recovery.</p> <p>Conclusions</p> <p>By comparative analysis of gene expression, histology and viral load, the temporal and spatial regulation of immune responses were characterised and novel immune genes identified, ultimately leading to a more complete understanding of host-virus responses and pathology and protection in Atlantic salmon during CMS.</p

Antimicrobial proteins and polypeptides in pulmonary innate defence

Inspired air contains a myriad of potential pathogens, pollutants and inflammatory stimuli. In the normal lung, these pathogens are rarely problematic. This is because the epithelial lining fluid in the lung is rich in many innate immunity proteins and peptides that provide a powerful anti-microbial screen. These defensive proteins have anti-bacterial, anti- viral and in some cases, even anti-fungal properties. Their antimicrobial effects are as diverse as inhibition of biofilm formation and prevention of viral replication. The innate immunity proteins and peptides also play key immunomodulatory roles. They are involved in many key processes such as opsonisation facilitating phagocytosis of bacteria and viruses by macrophages and monocytes. They act as important mediators in inflammatory pathways and are capable of binding bacterial endotoxins and CPG motifs. They can also influence expression of adhesion molecules as well as acting as powerful anti-oxidants and anti-proteases. Exciting new antimicrobial and immunomodulatory functions are being elucidated for existing proteins that were previously thought to be of lesser importance. The potential therapeutic applications of these proteins and peptides in combating infection and preventing inflammation are the subject of ongoing research that holds much promise for the future

Regulation of Progranulin Expression in Human Microglia and Proteolysis of Progranulin by Matrix Metalloproteinase-12 (MMP-12)

Background: The essential role of progranulin (PGRN) as a neurotrophic factor has been demonstrated by the discovery that haploinsufficiency due to GRN gene mutations causes frontotemporal lobar dementia. In addition to neurons, microglia in vivo express PGRN, but little is known about the regulation of PGRN expression by microglia. Goal: In the current study, we examined the regulation of expression and function of PGRN, its proteolytic enzyme macrophage elastase (MMP-12), as well as the inhibitor of PGRN proteolysis, secretory leukocyte protease inhibitor (SLPI), in human CNS cells. Methods: Cultures of primary human microglia and astrocytes were stimulated with the TLR ligands (LPS or poly IC), Th1 cytokines (IL-1/IFNc), or Th2 cytokines (IL-4, IL-13). Results were analyzed by Q-PCR, immunoblotting or ELISA. The roles of MMP-12 and SLPI in PGRN cleavage were also examined. Results: Unstimulated microglia produced nanogram levels of PGRN, and PGRN release from microglia was suppressed by the TLR ligands or IL-1/IFNc, but increased by IL-4 or IL-13. Unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of SLPI, none were detected in microglial cultures. We also identified MMP-12 as a PGRN proteolytic enzyme, and SLPI as an inhibitor of MMP-12-induced PGRN proteolysis. Experiments employing PGRN siRNA demonstrated that microglial PGRN was involved in the cytokine and chemokine production following TLR3/4 activation

Treatment of Infected Hip Arthroplasty

The clinical outcomes of a consecutive series of deep total joint infections treated with a prosthesis retaining protocol were reviewed. The treatment of deep periprosthetic joint infections is challenging. In recent years, two-stage exchange arthroplasty has emerged as the gold standard for successful elimination of infection. With success rates averaging 82% to 96%, this treatment method has both the highest and most consistent rate of infection eradication. Another alternative in the treatment of the deep periprosthetic infection is the single-stage exchange arthroplasty. Successful eradication of infection after single-stage exchange arthroplasty has been reported to average from 60% to 83% after total hip infections. While both the single and two-stage exchange arthroplasty are viable treatment options, they are associated with negative factors such as they are time consuming, expensive, and may entail a 6- to 12-week period with a minimally functioning extremity after prosthesis removal. This paper reports the general principles of management, the treatment of acute infection occurring in the postoperative period or later, and the treatment of chronic infection by exchange arthroplasty or resection arthroplasty

A basal lithostrotian titanosaur (Dinosauria: Sauropoda) with a complete skull: Implications for the evolution and paleobiology of titanosauria

We describe Sarmientosaurus musacchioi gen. et sp. nov., a titanosaurian sauropod dinosaur from the Upper Cretaceous (Cenomanian - Turonian) Lower Member of the Bajo Barreal Formation of southern Chubut Province in central Patagonia, Argentina. The holotypic and only known specimen consists of an articulated, virtually complete skull and part of the cranial and middle cervical series. Sarmientosaurus exhibits the following distinctive features that we interpret as autapomorphies: (1) maximum diameter of orbit nearly 40% rostrocaudal length of cranium; (2) complex maxilla - lacrimal articulation, in which the lacrimal clasps the ascending ramus of the maxilla; (3) medial edge of caudal sector of maxillary ascending ramus bordering bony nasal aperture with low but distinct ridge; (4) ´tongue-like´ ventral process of quadratojugal that overlaps quadrate caudally; (5) separate foramina for all three branches of the trigeminal nerve; (6) absence of median venous canal connecting infundibular region to ventral part of brainstem; (7) subvertical premaxillary, procumbent maxillary, and recumbent dentary teeth; (8) cervical vertebrae with ´strut-like´ centroprezygapophyseal laminae; (9) extremely elongate and slender ossified tendon positioned ventrolateral to cervical vertebrae and ribs. The cranial endocast of Sarmientosaurus preserves some of the most complete information obtained to date regarding the brain and sensory systems of sauropods. Phylogenetic analysis recovers the new taxon as a basal member of Lithostrotia, as the most plesiomorphic titanosaurian to be preserved with a complete skull. Sarmientosaurus provides a wealth of new cranial evidence that reaffirms the close relationship of titanosaurs to Brachiosauridae. Moreover, the presence of the relatively derived lithostrotian Tapuiasaurus in Aptian deposits indicates that the new Patagonian genus represents a ´ghost lineage´ with a comparatively plesiomorphic craniodental form, the evolutionary history of which is missing for at least 13 million years of the Cretaceous. The skull anatomy of Sarmientosaurus suggests that multiple titanosaurian species with dissimilar cranial structures coexisted in the early Late Cretaceous of southern South America. Furthermore, the new taxon possesses a number of distinctive morphologies - such as the ossified cervical tendon, extremely pneumatized cervical vertebrae, and a habitually downward- facing snout - that have rarely, if ever, been documented in other titanosaurs, thus broadening our understanding of the anatomical diversity of this remarkable sauropod clade. The latter two features were convergently acquired by at least one penecontemporaneous diplodocoid, and may represent mutual specializations for consuming low-growing vegetation.Fil: Martínez, Rubén Darío. Universidad Nacional de la Patagonia; ArgentinaFil: Lamanna, Matthew C.. Carnegie Museum Of Natural History; Estados UnidosFil: Novas, Fernando Emilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "bernardino Rivadavia"; ArgentinaFil: Ridgely, Ryan C.. Ohio University College Of Osteopathic Medicine; Estados UnidosFil: Casal, Gabriel. Universidad Nacional de la Patagonia; ArgentinaFil: Martínez, Javier E.. Hospital Regional de Comodoro Rivadavia; ArgentinaFil: Vita, Javier R.. Resonancia Magnética Borelli; ArgentinaFil: Witmer, Lawrence M.. Ohio University College Of Osteopathic Medicine; Estados Unido