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Find video protocols related to scientific articles indexed in Pubmed.
Mechanistic understanding for the greater sensitivity of monkeys to antisense oligonucleotide-mediated complement activation compared with humans.
J. Pharmacol. Exp. Ther.
PUBLISHED: 10-09-2014
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Differences in sensitivity of monkeys and humans to antisense oligonucleotide (ASO)-induced complement alternative pathway (AP) activation were evaluated in monkeys, humans, and in serum using biochemical assays. Transient AP activation was evident in monkeys at higher doses of two 2'-O-methoxyethyl (2'-MOE) ASOs (ISIS 426115 and ISIS 183750). No evidence of AP activation was observed in humans for either ASO, even with plasma ASO concentrations that reached the threshold for activation in monkeys. The absence of complement activation in humans is consistent with a query of the Isis Clinical Safety Database containing 767 subjects. The in vivo difference in sensitivity was confirmed in vitro, as monkey and human serum exposed to increasing concentrations of ASO indicated that monkeys were more sensitive to AP activation with this class of compounds. The mechanistic basis for the greater sensitivity of monkeys to AP activation by 2'-MOE ASO was evaluated using purified human or monkey factor H protein. The binding affinities between a representative 2'-MOE ASO and either purified protein are similar. However, the IC50 of fluid-phase complement inhibition for monkey factor H is about 3-fold greater than that for human protein using either monkey serum or factor H-depleted human serum. Interestingly, there is a sequence variant in the monkey complement factor H gene similar to a single nucleotide polymorphism in humans that is correlated with decreased factor H protein function. These findings show that monkeys are more sensitive to 2'-MOE ASO-mediated complement activation than humans likely because of differences in factor H inhibitory capacity.
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Ceramides mediate cigarette smoke-induced metabolic disruption in mice.
Am. J. Physiol. Endocrinol. Metab.
PUBLISHED: 09-30-2014
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Cigarette smoke exposure increases lung ceramide biosynthesis and alters metabolic function. We hypothesized that ceramides are released from the lung during cigarette smoke exposure and result in elevated skeletal muscle ceramide levels, resulting in insulin resistance and altered mitochondrial respiration. Employing cell and animal models, we explored the effect of cigarette smoke on muscle cell insulin signaling and mitochondrial respiration. Muscle cells were treated with conditioned medium from cigarette smoke extract (CSE)-exposed lung cells, followed by analysis of ceramides and assessment of insulin signaling and mitochondrial function. Mice were exposed to daily cigarette smoke and a high-fat, high-sugar (HFHS) diet with myriocin injections to inhibit ceramide synthesis. Comparisons were conducted between these mice and control animals on standard diets in the absence of smoke exposure and myriocin injections. Muscle cells treated with CSE-exposed conditioned medium were completely unresponsive to insulin stimulation, and mitochondrial respiration was severely blunted. These effects were mitigated when lung cells were treated with the ceramide inhibitor myriocin prior to and during CSE exposure. In mice, daily cigarette smoke exposure and HFHS diet resulted in insulin resistance, which correlated with elevated ceramides. Although myriocin injection was protective against insulin resistance with either smoke or HFHS, it was insufficient to prevent insulin resistance with combined CS and HFHS. However, myriocin injection restored muscle mitochondrial respiration in all treatments. Ceramide inhibition prevents metabolic disruption in muscle cells with smoke exposure and may explain whole body insulin resistance and mitochondrial dysfunction in vivo.
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Acute secondhand smoke-induced pulmonary inflammation is diminished in RAGE knockout mice.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 09-26-2014
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The receptor for advanced glycation end-products (RAGE) has increasingly been demonstrated to be an important modulator of inflammation in cases of pulmonary disease. Published reports involving tobacco smoke exposure have demonstrated increased expression of RAGE, its participation in proinflammatory signaling, and its role in irreversible pulmonary remodeling. The current research evaluated the in vivo effects of short-term secondhand smoke (SHS) exposure in RAGE knockout and control mice compared with identical animals exposed to room air only. Quantitative PCR, immunoblotting, and immunohistochemistry revealed elevated RAGE expression in controls after 4 wk of SHS exposure and an anticipated absence of RAGE expression in RAGE knockout mice regardless of smoke exposure. Ras activation, NF-?B activity, and cytokine elaboration were assessed to characterize the molecular basis of SHS-induced inflammation in the mouse lung. Furthermore, bronchoalveolar lavage fluid was procured from RAGE knockout and control animals for the assessment of inflammatory cells and molecules. As a general theme, inflammation coincident with leukocyte recruitment was induced by SHS exposure and significantly influenced by the availability of RAGE. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to SHS. However, ongoing research is still warranted to fully explain roles for RAGE and other receptors in cells coping with involuntary smoke exposure for prolonged periods of time.
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Australian Idiopathic Pulmonary Fibrosis Registry: vital lessons from a national prospective collaborative project.
Respirology
PUBLISHED: 08-14-2014
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There is little Australian epidemiologic data on idiopathic pulmonary fibrosis (IPF), a relatively uncommon but devastating disease. The vast geographic distances in Australia have been a major impediment for collaborative research into IPF. A collaborative national effort, the Australian IPF Registry, has been formed, launched and is recruiting successfully (n = 359, January 2014). Our experience provides unique insights for others wishing to set up IPF registries and in time for a global IPF registry.
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Antenatal exposure of maternal secondhand smoke (SHS) increases fetal lung expression of RAGE and induces RAGE-mediated pulmonary inflammation.
Respir. Res.
PUBLISHED: 07-30-2014
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Receptors for advanced glycation end-products (RAGE) are immunoglobulin-like pattern recognition receptors abundantly localized to lung epithelium. Our research demonstrated that primary tobacco smoke exposure increases RAGE expression and that RAGE partly mediates pro-inflammatory signaling during exposure. However, the degree to which RAGE influences developing lungs when gestating mice are exposed to secondhand smoke (SHS) has not been determined to date.
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Nanotopographical effects on mesenchymal stem cell morphology and phenotype.
J. Cell. Biochem.
PUBLISHED: 07-24-2014
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There is a rapidly growing body of literature on the effects of topography and critically, nanotopography on cell adhesion, apoptosis and differentiation. Understanding the effects of nanotopography on cell adhesion and morphology and the consequences of cell shape changes in the nucleus, and consequently, gene expression offers new approaches to the elucidation and potential control of stem cell differentiation. In the current study we have used molecular approaches in combination with immunohistology and transcript analysis to understand the role of nanotopography on mesenchymal stem cell morphology and phenotype. Results demonstrate large changes in cell adhesion, nucleus and lamin morphologies in response to the different nanotopographies. Furthermore, these changes relate to alterations in packing of chromosome territories within the interphase nucleus. This, in turn, leads to changes in transcription factor activity and functional (phenotypical) signalling including cell metabolism. Nanotopography provides a useful, non-invasive tool for studying cellular mechanotransduction, gene and protein expression patterns, through effects on cell morphology. The different nanotopographies examined, result in different morphological changes in the cyto- and nucleo-skeleton. We propose that both indirect (biochemical) and direct (mechanical) signalling are important in these early stages of regulating stem cell fate as a consequence of altered metabolic changes and altered phenotype. The current studies provide new insight on cell-surface interactions and enhance our understanding of the modulation of stem cell differentiation with significant potential application in regenerative medicine.
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Genome sequencing of Mycobacterium abscessus isolates from patients in the united states and comparisons to globally diverse clinical strains.
J. Clin. Microbiol.
PUBLISHED: 07-23-2014
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Nontuberculous mycobacterial infections caused by Mycobacterium abscessus are responsible for a range of disease manifestations from pulmonary to skin infections and are notoriously difficult to treat, due to innate resistance to many antibiotics. Previous population studies of clinical M. abscessus isolates utilized multilocus sequence typing or pulsed-field gel electrophoresis, but high-resolution examinations of genetic diversity at the whole-genome level have not been well characterized, particularly among clinical isolates derived in the United States. We performed whole-genome sequencing of 11 clinical M. abscessus isolates derived from eight U.S. patients with pulmonary nontuberculous mycobacterial infections, compared them to 30 globally diverse clinical isolates, and investigated intrapatient genomic diversity and evolution. Phylogenomic analyses revealed a cluster of closely related U.S. and Western European M. abscessus subsp. abscessus isolates that are genetically distinct from other European isolates and all Asian isolates. Large-scale variation analyses suggested genome content differences of 0.3 to 8.3%, relative to the reference strain ATCC 19977(T). Longitudinally sampled isolates showed very few single-nucleotide polymorphisms and correlated genomic deletion patterns, suggesting homogeneous infection populations. Our study explores the genomic diversity of clinical M. abscessus strains from multiple continents and provides insight into the genome plasticity of an opportunistic pathogen.
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Conditionally induced RAGE expression by proximal airway epithelial cells in transgenic mice causes lung inflammation.
Respir. Res.
PUBLISHED: 06-26-2014
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BackgroundReceptors for advanced glycation end-products (RAGE) are multiligand cell-surface receptors expressed abundantly by distal pulmonary epithelium. Our lab has discovered RAGE-mediated effects in the orchestration of lung inflammation induced by tobacco smoke and environmental pollutants; however, the specific contribution of RAGE to the progression of proximal airway inflammation is still inadequately characterized.Methods and resultsWe generated a Tet-inducible transgenic mouse that conditionally overexpressed RAGE using the club cell (Clara) secretory protein (CCSP) promoter expressed by club (Clara) cells localized to the proximal airway. RAGE was induced for 40 days from weaning (20 days of age) until sacrifice date at 60 days. Immunohistochemistry, immunoblotting, and qPCR revealed significant RAGE up-regulation when compared to non-transgenic controls; however, H&E staining revealed no detectible morphological abnormalities and apoptosis was not enhanced during the 40 days of augmentation. Freshly procured bronchoalveolar lavage fluid (BALF) from CCSP-RAGE TG mice had significantly more total leukocytes and PMNs compared to age-matched control littermates. Furthermore, CCSP-RAGE TG mice expressed significantly more tumor necrosis factor alpha (TNF-¿), interleukin 7 (IL-7), and interleukin 14 (IL-14) in whole lung homogenates compared to controls.ConclusionsThese data support the concept that RAGE up-regulation specifically in lung airways may function in the progression of proximal airway inflammation.
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Increased CD8 T-cell granzyme B in COPD is suppressed by treatment with low-dose azithromycin.
Respirology
PUBLISHED: 05-28-2014
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Corticosteroid resistance in chronic obstructive pulmonary disease (COPD) is a major challenge. We have reported increased bronchial epithelial cell apoptosis and increased airway CD8 T-cell numbers in COPD. Apoptosis can be induced via the serine protease, granzyme B. However, glucocorticosteroids fail to adequately suppress granzyme B production by CD8 T cells. We previously showed that low-dose azithromycin reduced airways inflammation in COPD subjects and we hypothesized that it would also reduce granzyme B production by CD8 T cells.
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Targeting peripheral blood pro-inflammatory cytotoxic lymphocytes by inhibiting CD137 expression: novel potential treatment for COPD.
BMC Pulm Med
PUBLISHED: 04-11-2014
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We have shown that chronic obstructive pulmonary disease (COPD) is associated with increased production of pro-inflammatory cytokines and the cytotoxic mediator, granzyme B by peripheral blood steroid resistant CD28nullCD137 + CD8+ T cells and granzyme B by NKT-like and NK cells. We hypothesized that we could target these pro-inflammatory/cytotoxic lymphocytes by inhibiting co-stimulation through CD137.
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Early administration of infliximab for severe ipilimumab-related diarrhea in a critically ill patient.
Ann Pharmacother
PUBLISHED: 03-20-2014
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To report a case of ipilimumab-associated life-threatening diarrhea responding quickly to a single dose of infliximab.
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Developmental lung expression and transcriptional regulation of claudin-6 by TTF-1, Gata-6, and FoxA2.
Respir. Res.
PUBLISHED: 03-11-2014
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Claudins are transmembrane proteins expressed in tight junctions that prevent paracellular transport of extracellular fluid and a variety of other substances. However, the expression profile of Claudin-6 (Cldn6) in the developing lung has not been characterized.
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Neuroendocrine signaling via the serotonin transporter regulates clearance of apoptotic cells.
J. Biol. Chem.
PUBLISHED: 02-25-2014
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Serotonin (5-hydroxytryptamine; 5-HT) is a CNS neurotransmitter increasingly recognized to exert immunomodulatory effects outside the CNS that contribute to the pathogenesis of autoimmune and chronic inflammatory diseases. 5-HT signals to activate the RhoA/Rho kinase (ROCK) pathway, a pathway known for its ability to regulate phagocytosis. The clearance of apoptotic cells (i.e. efferocytosis) is a key modulator of the immune response that is inhibited by the RhoA/ROCK pathway. Because efferocytosis is defective in many of the same illnesses where 5-HT has been implicated in disease pathogenesis, we hypothesized that 5-HT would suppress efferocytosis via activation of RhoA/ROCK. The effect of 5-HT on efferocytosis was examined in murine peritoneal and human alveolar macrophages, and its mechanisms were investigated using pharmacologic blockade and genetic deletion. 5-HT impaired efferocytosis by murine peritoneal macrophages and human alveolar macrophages. 5-HT increased phosphorylation of myosin phosphatase subunit 1 (Mypt-1), a known ROCK target, and inhibitors of RhoA and ROCK reversed the suppressive effect of 5-HT on efferocytosis. Peritoneal macrophages expressed the 5-HT transporter and 5-HT receptors (R) 2a, 2b, but not 2c. Inhibition of 5-HTR2a and 5-HTR2b had no effect on efferocytosis, but blockade of the 5-HT transporter prevented 5-HT-impaired efferocytosis. Genetic deletion of the 5-HT transporter inhibited 5-HT uptake into peritoneal macrophages, prevented 5-HT-induced phosphorylation of Mypt-1, reversed the inhibitory effect of 5-HT on efferocytosis, and decreased cellular peritoneal inflammation. These results suggest a novel mechanism by which 5-HT might disrupt efferocytosis and contribute to the pathogenesis of autoimmune and chronic inflammatory diseases.
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Histamine-2 receptor antagonists vs proton pump inhibitors on gastrointestinal tract hemorrhage and infectious complications in the intensive care unit.
JAMA Intern Med
PUBLISHED: 02-19-2014
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IMPORTANCE Histamine-2 receptor antagonists (H2RAs) and proton pump inhibitors (PPIs) are commonly used to prevent gastrointestinal tract (GI) hemorrhage in critically ill patients. The stronger acid suppression of PPIs may reduce the rate of bleeding but enhance infectious complications, specifically pneumonia and Clostridium difficile infection (CDI). OBJECTIVE To evaluate the occurrence and risk factors for GI hemorrhage, pneumonia, and CDI in critically ill patients. DESIGN, SETTING, AND PARTICIPANTS A pharmacoepidemiological cohort study was conducted of adult patients requiring mechanical ventilation for 24 hours or more and administered either an H2RA or PPI for 48 hours or more while intubated across 71 hospitals between January 1, 2003, and December 31, 2008. Propensity score-adjusted and propensity-matched multivariate regression models were used to control for confounders. MAIN OUTCOMES AND MEASURES Primary outcomes were secondary diagnoses of International Classification of Diseases, Ninth Revision (ICD-9)-coded GI hemorrhage, pneumonia, and CDI occurring 48 hours or more after initiating invasive ventilation. RESULTS Of 35?312 patients, 13?439 (38.1%) received H2RAs and 21?873 (61.9%) received PPIs. Gastrointestinal hemorrhage (2.1% vs 5.9%; P?<?.001), pneumonia (27% vs 38.6%; P?<?.001), and CDI (2.2% vs 3.8%; P?<?.001) occurred less frequently in the H2RA group. After adjusting for propensity score and covariates, odds ratios of GI hemorrhage (2.24; 95% CI, 1.81-2.76), pneumonia (1.2; 95% CI, 1.03-1.41), and CDI (1.29; 95% CI, 1.04-1.64) were greater with PPIs. Similar results were obtained in the propensity-matched models of 8799 patients in each cohort. CONCLUSIONS AND RELEVANCE Proton pump inhibitors are associated with greater risks of GI hemorrhage, pneumonia, and CDI than H2RAs in mechanically ventilated patients. Numerous other risk factors are apparent. These data warrant confirmation in comparative prospective studies.
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Loss of glucocorticoid receptor from pro-inflammatory T cells after lung transplant.
J. Heart Lung Transplant.
PUBLISHED: 01-21-2014
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Pro-inflammatory cytokines in T and natural killer T (NKT)-like cells increase with time post-transplant in otherwise stable patients, suggesting that some patients become relatively resistant to immunosuppressants such as glucocorticoids (GC). We hypothesized that GC receptor (GCR) would be down-regulated in peripheral blood pro-inflammatory T and NKT-like cells after lung transplantation and loss of GCR would correlate with time post-transplant.
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Management of extravasation injuries: a focused evaluation of noncytotoxic medications.
Pharmacotherapy
PUBLISHED: 01-13-2014
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Extravasations are common manifestations of iatrogenic injury that occur in patients requiring intravenous delivery of known vesicants. These injuries can contribute substantially to patient morbidity, cost of therapy, and length of stay. Many different mechanisms are behind the tissue damage during extravasation injuries. In general, extravasations consist of four different subtypes of tissue injury: vasoconstriction, osmotic, pH related, and cytotoxic. Recognition of high-risk patients, appropriate cannulation technique, and monitoring of higher risk materials remain the standard of care for the prevention of extravasation injury. Prompt interdisciplinary action is often necessary for the treatment of extravasation injuries. Knowledge of the mechanism of extravasation-induced tissue injury, agents for reversal, and appropriate nonpharmacologic treatment methods is essential. The best therapeutic agent for treatment of vasopressor extravasation is intradermal phentolamine. Topical vasodilators and intradermal terbutaline may provide relief. Intradermal hyaluronidase has been effective for hyperosmotic extravasations, although its use largely depends on the risk of tissue injury and the severity of extravasation. Among the hyperosmotic agents, calcium extravasation is distinctive because it may present as an acute tissue injury or may possess delayed clinical manifestations. Extravasation of acidic or basic materials can produce significant tissue damage. Phenytoin is the prototypical basic drug that causes a clinical manifestation known as purple glove syndrome (PGS). This syndrome is largely managed through preventive and conservative treatment measures. Promethazine is acidic and can cause a devastating extravasation, particularly if administered inadvertently through the arteriolar route. Systemic heparin therapy remains the accepted treatment option for intraarteriolar administration of promethazine. Overall, the evidence for managing extravasations due to noncytotoxic medications is nonexistent or limited to case reports. More research is needed to improve knowledge of patient risk, prompt recognition of the extravasation, and time course for tissue injury, and to develop prevention and treatment strategies for extravasation injuries.
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Primary alveolar macrophages exposed to diesel particulate matter increase RAGE expression and activate RAGE signaling.
Cell Tissue Res.
PUBLISHED: 01-08-2014
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Receptors for advanced glycation end-products (RAGE) are members of the immunoglobulin superfamily of cell-surface receptors implicated in mechanisms of pulmonary inflammation. In the current study, we test the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) exposed to diesel particulate matter (DPM). Quantitative RT-PCR and immunoblotting revealed that RAGE was up-regulated in Raw264.7 cells, an immortalized murine macrophage cell line and primary AMs exposed to DPM for 2 h. Because DPM increased RAGE expression, we exposed Raw264.7 cells and primary AMs isolated from RAGE null and wild-type (WT) mice to DPM prior to the assessment of inflammatory signaling intermediates. DPM led to the activation of Rat sarcoma GTPase (Ras), p38 MAPK and NF-?B in WT AMs and, when compared to WT AMs, these intermediates were diminished in DPM-exposed AMs isolated from RAGE null mice. Furthermore, cytokines implicated in inflammation, including IL-4, IL-12, IL-13 and TNF?, were all significantly decreased in DPM-exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that diesel-induced inflammatory responses by primary AMs are mediated, at least in part, via RAGE signaling mechanisms. Further work may show that RAGE signaling in both alveolar epithelial cells and resident macrophages is a potential target in the treatment of inflammatory lung diseases exacerbated by environmental pollution.
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Oxidative stress decreases functional airway mannose binding lectin in COPD.
PLoS ONE
PUBLISHED: 01-01-2014
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We have previously established that a defect in the ability of alveolar macrophages (AM) to phagocytose apoptotic cells (efferocytosis) and pathogens is a potential therapeutic target in COPD. We further showed that levels of mannose binding lectin (MBL; required for effective macrophage phagocytic function) were reduced in the airways but not circulation of COPD patients. We hypothesized that increased oxidative stress in the airway could be a cause for such disturbances. We therefore studied the effects of oxidation on the structure of the MBL molecule and its functional interactions with macrophages. Oligomeric structure of plasma derived MBL (pdMBL) before and after oxidation (oxMBL) with 2,2'-azobis(2-methylpropionamidine)dihydrochroride (AAPH) was investigated by blue native PAGE. Macrophage function in the presence of pd/oxMBL was assessed by measuring efferocytosis, phagocytosis of non-typeable Haemophilus influenzae (NTHi) and expression of macrophage scavenger receptors. Oxidation disrupted higher order MBL oligomers. This was associated with changed macrophage function evident by a significantly reduced capacity to phagocytose apoptotic cells and NTHi in the presence of oxMBL vs pdMBL (eg, NTHi by 55.9 and 27.0% respectively). Interestingly, oxidation of MBL significantly reduced macrophage phagocytic ability to below control levels. Flow cytometry and immunofluorescence revealed a significant increase in expression of macrophage scavenger receptor (SRA1) in the presence of pdMBL that was abrogated in the presence of oxMBL. We show the pulmonary macrophage dysfunction in COPD may at least partially result from an oxidative stress-induced effect on MBL, and identify a further potential therapeutic strategy for this debilitating disease.
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Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway.
Respirology
PUBLISHED: 08-31-2013
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Natural killer (NK) and natural killer T (NKT)-like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro-inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin.
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Genome Sequence of an Epidemic Isolate of Mycobacterium abscessus subsp. bolletii from Rio de Janeiro, Brazil.
Genome Announc
PUBLISHED: 08-17-2013
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Multiple isolates of Mycobacterium abscessus subsp. bolletii, collectively called BRA100, were associated with outbreaks of postsurgical skin infections across various regions of Brazil from 2003 to 2009. We announce the draft genome sequence of a newly sequenced BRA100 strain, M. abscessus subsp. bolletii CRM-0020, isolated from a patient in Rio de Janeiro, Brazil.
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Conditional over-expression of RAGE by embryonic alveolar epithelium compromises the respiratory membrane and impairs endothelial cell differentiation.
Respir. Res.
PUBLISHED: 08-14-2013
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Receptors for advanced glycation end-products (RAGE) are cell surface receptors prominently expressed by lung epithelium. Previous research demonstrated that over-expression of RAGE by murine alveolar epithelial cells during embryogenesis caused severe lung hypoplasia and neonatal lethality. However, the effects of RAGE over-expression on adjacent matrix and endothelial cells remained unknown.
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Altered sputum granzyme B and granzyme B/proteinase inhibitor-9 in patients with non-eosinophilic asthma.
Respirology
PUBLISHED: 07-02-2013
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The non-eosinophilic phenotype of asthma (NEA) is associated with chronic airway inflammation and airway neutrophilia. An accumulation of apoptotic airway epithelial cells, if not efficiently cleared by efferocytosis, can undergo secondary necrosis, with the potential for inflammation of surrounding tissues. Apoptosis may occur via the T cell granzyme B pathway. The role of granzyme B in NEA is not known. The aim of this study was to investigate production of granzyme B and its inhibitor proteinase inhibitor (PI)-9 by T cells from induced sputum and compare expression between eosinophilic, NEA and healthy controls.
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The expanding family of FERM proteins.
Biochem. J.
PUBLISHED: 05-14-2013
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Our understanding of the FERM (4.1/ezrin/radixin/moesin) protein family has been rapidly expanding in the last few years, with the result that many new physiological functions have been ascribed to these biochemically unique proteins. In the present review, we will discuss a number of new FRMD (FERM domain)-containing proteins that were initially discovered from genome sequencing but are now being established through biochemical and genetic studies to be involved both in normal cellular processes, but are also associated with a variety of human diseases.
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Human tissue in systems medicine.
FEBS J.
PUBLISHED: 04-19-2013
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Histopathology, the examination of an architecturally artefactual, two-dimensional and static image remains a potent tool allowing diagnosis and empirical expectation of prognosis. Considerable optimism exists that the advent of molecular genetic testing and other biomarker strategies will improve or even replace this ancient technology. A number of biomarkers already add considerable value for prediction of whether a treatment will work. In this short review we argue that a systems medicine approach to pathology will not seek to replace traditional pathology, but rather augment it. Systems approaches need to incorporate quantitative morphological, protein, mRNA and DNA data. A significant challenge for clinical implementation of systems pathology is how to optimize information available from tissue, which is frequently sub-optimal in quality and amount, and yet generate useful predictive models that work. The transition of histopathology to systems pathophysiology and the use of multiscale data sets usher in a new era in diagnosis, prognosis and prediction based on the analysis of human tissue.
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Targeting peripheral blood pro-inflammatory CD28null T cells and natural killer T-like cells by inhibiting CD137 expression: possible relevance to treatment of bronchiolitis obliterans syndrome.
J. Heart Lung Transplant.
PUBLISHED: 04-10-2013
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We have shown that bronchiolitis obliterans syndrome (BOS) is associated with attenuated suppression of pro-inflammatory cytokines and granzyme B by steroid-resistant peripheral blood CD28nullCD137+ T cells and natural killer T (NKT)-like cells. We hypothesized that we could target these steroid-resistant lymphocytes by inhibiting costimulation through CD137.
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Association of a functional FAAH polymorphism with methamphetamine-induced symptoms and dependence in a Malaysian population.
Pharmacogenomics
PUBLISHED: 04-06-2013
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FAAH is a membrane enzyme that terminates the activity of a large class of endogenous signaling lipids. Recent studies suggest that the FAAH Pro129Thr polymorphism is a common mutation in the FAAH gene that is significantly associated with drug-addictive traits. This study investigated the association of the Pro129Thr polymorphism of the FAAH gene with methamphetamine dependence, methamphetamine-induced psychosis, manic episodes and panic disorder in a Malaysian population.
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Conditional overexpression of receptors for advanced glycation end-products in the adult murine lung causes airspace enlargement and induces inflammation.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 03-26-2013
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Receptors for advanced glycation end-products (RAGE) are multiligand surface receptors detected abundantly in pulmonary tissue. Our previous work revealed increased RAGE expression in cells and lungs exposed to tobacco smoke and RAGE-mediated cytokine expression via proinflammatory mechanisms involving NF-?B. RAGE expression is elevated in various pathological states, including chronic obstructive pulmonary disease; however, precise contributions of RAGE to the progression of emphysema and pulmonary inflammation in the adult lung are unknown. In the current study, we generated a RAGE transgenic (RAGE TG) mouse and conditionally induced adult alveolar epithelium to overexpress RAGE. RAGE was induced after the period of alveologenesis, from weaning (20 d of age) until animals were killed at 50, 80, and 110 days (representing 30, 60, and 90 d of RAGE overexpression). Hematoxylin and eosin staining and mean chord length revealed incremental dilation of alveolar spaces as RAGE overexpression persisted. TUNEL staining and electron microscopy confirmed increased apoptosis and blebbing of alveolar epithelium in lungs from RAGE TG mice when compared with control mice. Immunohistochemistry for matrix metalloproteinase 9 revealed an overall increase in matrix metalloproteinase 9, which correlated with decreased elastin expression in RAGE TG mice. Furthermore, RAGE TG mice manifested significant inflammation measured by elevated bronchoalveolar lavage protein, leukocyte infiltration, and secreted cytokines. These data support the concept that innovative transgenic mice that overexpress RAGE may model pulmonary inflammation and alveolar destabilization independent of tobacco smoke and validate RAGE signaling as a target pathway in the prevention or attenuation of smoke-related inflammatory lung diseases.
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Cooccurrence of free-living amoebae and nontuberculous Mycobacteria in hospital water networks, and preferential growth of Mycobacterium avium in Acanthamoeba lenticulata.
Appl. Environ. Microbiol.
PUBLISHED: 03-08-2013
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The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.
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The drug efflux pump Pgp1 in pro-inflammatory lymphocytes is a target for novel treatment strategies in COPD.
Respir. Res.
PUBLISHED: 02-19-2013
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BACKGROUND: Pro-inflammatory/cytotoxic T cells (IFNgamma, TNFalpha, granzyme B+) are increased in the peripheral circulation in COPD. NKT-like and NK cells are effector lymphocytes that we have also shown to be major sources of pro-inflammatory cytokines and granzymes. P-glycoprotein 1 (Pgp1) is a transmembrane efflux pump well characterised in drug resistant cancer cells. We hypothesized that Pgp1 would be increased in peripheral blood T, NKT-like and NK cells in patients with COPD, and that this would be accompanied by increased expression of IFNgamma, TNFalpha and granzyme B. We further hypothesized that treatment with cyclosporine A, a Pgp1 inhibitor, would render cells more sensitive to treatment with corticosteroids. METHODS: Pgp1, granzyme B, IFNgamma and TNFalpha expression were measured in peripheral blood T, NK and NKT-like cells from COPD patients and control subjects (+/- cyclosporine A and prednisolone) following in vitro stimulation and results correlated with uptake of efflux dye Calcein-AM using flow cytometry. RESULTS: There was increased Pgp1 expression by peripheral blood T, NKT-like and NK cells co-expressing IFNgamma, TNFalpha and granzyme B in COPD patients compared with controls (eg %IFNgamma/Pgp1 T, NKT-like, NK for COPD (Control): 25(6), 54(27), 39(23)). There was an inverse correlation between Pgp1 expression and Calcein-AM uptake. Treatment with 2.5 ng/ml cylosporin A and10-6 M prednisolone resulted in synergistic inhibition of pro-inflammatory cytokines in Pgp1 + cells (p < 0.05 for all). CONCLUSIONS: Treatment strategies that target Pgp1 in T, NKT-like and NK cells may reduce systemic inflammatory mediators in COPD and improve patient morbidity.
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Catabolic flexibility of mammalian-associated lactobacilli.
Microb. Cell Fact.
PUBLISHED: 02-14-2013
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Metabolic flexibility may be generally defined as "the capacity for the organism to adapt fuel oxidation to fuel availability". The metabolic diversification strategies used by individual bacteria vary greatly from the use of novel or acquired enzymes to the use of plasmid-localised genes and transporters. In this review, we describe the ability of lactobacilli to utilise a variety of carbon sources from their current or new environments in order to grow and survive. The genus Lactobacillus now includes more than 150 species, many with adaptive capabilities, broad metabolic capacity and species/strain variance. They are therefore, an informative example of a cell factory capable of adapting to new niches with differing nutritional landscapes. Indeed, lactobacilli naturally colonise and grow in a wide variety of environmental niches which include the roots and foliage of plants, silage, various fermented foods and beverages, the human vagina and the mammalian gastrointestinal tract (GIT; including the mouth, stomach, small intestine and large intestine). Here we primarily describe the metabolic flexibility of some lactobacilli isolated from the mammalian gastrointestinal tract, and we also describe some of the food-associated species with a proven ability to adapt to the GIT. As examples this review concentrates on the following species - Lb. plantarum, Lb. acidophilus, Lb. ruminis, Lb. salivarius, Lb. reuteri and Lb. sakei, to highlight the diversity and inter-relationships between the catabolic nature of species within the genus.
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Mycobacterium abscessus induces a limited pattern of neutrophil activation that promotes pathogen survival.
PLoS ONE
PUBLISHED: 01-21-2013
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Mycobacterium abscessus is a rapidly growing mycobacterium increasingly detected in the neutrophil-rich environment of inflamed tissues, including the cystic fibrosis airway. Studies of the immune reaction to M. abscessus have focused primarily on macrophages and epithelial cells, but little is known regarding the neutrophil response despite the predominantly neutrophillic inflammation typical of these infections. In the current study, human neutrophils released less superoxide anion in response to M. abscessus than to Staphylococcus aureus, a pathogen that shares common sites of infection. Exposure to M. abscessus induced neutrophil-specific chemokine and proinflammatory cytokine genes. Although secretion of these protein products was confirmed, the quantity of cytokines released, and both the number and level of gene induction, was reduced compared to S. aureus. Neutrophils mediated killing of M. abscessus, but phagocytosis was reduced when compared to S. aureus, and extracellular DNA was detected in response to both bacteria, consistent with extracellular trap formation. In addition, M. abscessus did not alter cell death compared to unstimulated cells, while S. aureus enhanced necrosis and inhibited apoptosis. However, neutrophils augment M. abscessus biofilm formation. The response of neutrophils to M. abscessus suggests that the mycobacterium exploits neutrophil-rich settings to promote its survival and that the overall neutrophil response was reduced compared to S. aureus. These studies add to our understanding of M. abscessus virulence and suggest potential targets of therapy.
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Lectins offer new perspectives in the development of macrophage-targeted therapies for COPD/emphysema.
PLoS ONE
PUBLISHED: 01-07-2013
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We have previously shown that the defective ability of alveolar macrophages (AM) to phagocytose apoptotic cells (efferocytosis) in chronic obstructive pulmonary disease/emphysema (COPD) could be therapeutically improved using the C-type lectin, mannose binding lectin (MBL), although the exact mechanisms underlying this effect are unknown. An S-type lectin, galectin-3, is also known to regulate macrophage phenotype and function, via interaction with its receptor CD98. We hypothesized that defective expression of galectin/CD98 would be associated with defective efferocytosis in COPD and that mechanisms would include effects on cytoskeletal remodeling and macrophage phenotype and glutathione (GSH) availability. Galectin-3 was measured by ELISA in BAL from controls, smokers and current/ex-smokers with COPD. CD98 was measured on AM using flow cytometry. We assessed the effects of galectin-3 on efferocytosis, CD98, GSH, actin polymerisation, rac activation, and the involvement of PI3K (using ?-actin probing and wortmannin inhibition) in vitro using human AM and/or MH-S macrophage cell line. Significant decreases in BAL galectin-3 and AM CD98 were observed in BAL from both current- and ex-smoker COPD subjects vs controls. Galectin 3 increased efferocytosis via an increase in active GTP bound Rac1. This was confirmed with ?-actin probing and the role of PI3K was confirmed using wortmannin inhibition. The increased efferocytosis was associated with increases in available glutathione and expression of CD98. We provide evidence for a role of airway lectins in the failed efferocytosis in COPD, supporting their further investigation as potential macrophage-targeted therapies.
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Label-free segmentation of Co-cultured cells on a nanotopographical gradient.
Nano Lett.
PUBLISHED: 01-02-2013
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The function and fate of cells is influenced by many different factors, one of which is surface topography of the support culture substrate. Systematic studies of nanotopography and cell response have typically been limited to single cell types and a small set of topographical variations. Here, we show a radical expansion of experimental throughput using automated detection, measurement, and classification of co-cultured cells on a nanopillar array where feature height changes continuously from planar to 250 nm over 9 mm. Individual cells are identified and characterized by more than 200 descriptors, which are used to construct a set of rules for label-free segmentation into individual cell types. Using this approach we can achieve label-free segmentation with 84% confidence across large image data sets and suggest optimized surface parameters for nanostructuring of implant devices such as vascular stents.
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Inflammatory markers associated with osteoarthritis after destabilization surgery in young mice with and without Receptor for Advanced Glycation End-products (RAGE).
Front Physiol
PUBLISHED: 01-01-2013
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HtrA1, Ddr-2, and Mmp-13 are reliable biomarkers for osteoarthritis (OA), yet the exact mechanism for the upregulation of HtrA-1 is unknown. Some have shown that chondrocyte hypertrophy is associated with early indicators of inflammation including TGF-? and the Receptor for Advanced Glycation End-products (RAGE). To examine the correlation of inflammation with the expression of biomarkers in OA, we performed right knee destabilization surgery on 4-week-old-wild type and RAGE knock-out (KO) mice. We assayed for HtrA-1, TGF-?1, Mmp-13, and Ddr-2 in articular cartilage at 3, 7, 14, and 28 days post-surgery by immunohistochemistry on left and right knee joints. RAGE KO and wild type mice both showed staining for key OA biomarkers. However, RAGE KO mice were significantly protected against OA compared to controls. We observed a difference in the total number of chondrocytes and percentage of chondrocytes staining positive for OA biomarkers between RAGE KO and control mice. The percentage of cells staining for OA biomarkers correlated with severity of cartilage degradation. Our results indicate that the absence of RAGE did protect against the development of advanced OA. We conclude that HtrA-1 plays a role in lowering TGF-?1 expression in the process of making articular cartilage vulnerable to damage associated with OA progression.
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Defective lung macrophage function in lung cancer ± chronic obstructive pulmonary disease (COPD/emphysema)-mediated by cancer cell production of PGE2?
PLoS ONE
PUBLISHED: 01-01-2013
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In chronic obstructive pulmonary disease (COPD/emphysema) we have shown a reduced ability of lung and alveolar (AM) macrophages to phagocytose apoptotic cells (defective efferocytosis), associated with evidence of secondary cellular necrosis and a resultant inflammatory response in the airway. It is unknown whether this defect is present in cancer (no COPD) and if so, whether this results from soluble mediators produced by cancer cells. We investigated efferocytosis in AM (26 controls, 15 healthy smokers, 37 COPD, 20 COPD+ non small cell lung cancer (NSCLC) and 8 patients with NSCLC without COPD) and tumor and tumor-free lung tissue macrophages (21 NSCLC with/13 without COPD). To investigate the effects of soluble mediators produced by lung cancer cells we then treated AM or U937 macrophages with cancer cell line supernatant and assessed their efferocytosis ability. We qualitatively identified Arachidonic Acid (AA) metabolites in cancer cells by LC-ESI-MSMS, and assessed the effects of COX inhibition (using indomethacin) on efferocytosis. Decreased efferocytosis was noted in all cancer/COPD groups in all compartments. Conditioned media from cancer cell cultures decreased the efferocytosis ability of both AM and U937 macrophages with the most pronounced effects occurring with supernatant from SCLC (an aggressive lung cancer type). AA metabolites identified in cancer cells included PGE2. The inhibitory effect of PGE2 on efferocytosis, and the involvement of the COX-2 pathway were shown. Efferocytosis is decreased in COPD/emphysema and lung cancer; the latter at least partially a result of inhibition by soluble mediators produced by cancer cells that include PGE2.
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Willin, an upstream component of the hippo signaling pathway, orchestrates mammalian peripheral nerve fibroblasts.
PLoS ONE
PUBLISHED: 01-01-2013
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Willin/FRMD6 was first identified in the rat sciatic nerve, which is composed of neurons, Schwann cells, and fibroblasts. Willin is an upstream component of the Hippo signaling pathway, which results in the inactivation of the transcriptional co-activator YAP through Ser127 phosphorylation. This in turn suppresses the expression of genes involved in cell growth, proliferation and cancer development ensuring the control of organ size, cell contact inhibition and apoptosis. Here we show that in the mammalian sciatic nerve, Willin is predominantly expressed in fibroblasts and that Willin expression activates the Hippo signaling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These results show that Willin modulates sciatic nerve fibroblast activity indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.
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Expression of mutant BMPR-II in pulmonary endothelial cells promotes apoptosis and a release of factors that stimulate proliferation of pulmonary arterial smooth muscle cells.
Pulm Circ
PUBLISHED: 10-29-2011
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Mutations in the bone morphogenetic protein type II receptor gene (BMPR-II) are the major cause of heritable pulmonary arterial hypertension (PAH). Although both endothelial and smooth muscle cell BMPR-II dysfunction have been seen to contribute to pulmonary hypertension in vivo, little is known about the impact of BMPR-II mutation on the interaction between these two important cell types. We employed adenoviral vectors to overexpress wild type or mutant (kinase-deficient mutation, D485G) BMPR-II in human pulmonary arterial endothelial cells (PAECs). PAECs transfected with mutant BMPR-II demonstrated increased susceptibility to apoptosis. Conditioned media from PAECs transfected with mutant BMPR-II increased the proliferation of pulmonary arterial smooth muscle cells (PASMCs), when compared with conditioned media from PAECs transfected with wild-type BMPR-II. PAECs transfected with mutant BMPR-II released higher levels of TGF-?(1) and FGF2 into the conditioned media than the wild-type BMPR-II-transfected cells. Conditioned media from PAECs expressing mutant BMPR-II also showed increased activation of luciferase activity in a TGF-? bioassay. The increased proliferation observed in PASMCs exposed to conditioned media from PAECs expressing mutant BMPR-II was inhibited by neutralizing the antibodies to TGF-?1, or small molecule inhibitors of ALK-5 (SD208) or FGFR1 (SU5402). We conclude that mutation in BMPR-II increases susceptibility to apoptosis of PAECs and leads to secretion of growth factors that stimulate the proliferation of PASMCs. These processes may contribute to the remodeling of pulmonary arteries observed in patients with familial or heritable PAH.
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Bioactive peptides from muscle sources: meat and fish.
Nutrients
PUBLISHED: 07-07-2011
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Bioactive peptides have been identified in a range of foods, including plant, milk and muscle, e.g., beef, chicken, pork and fish muscle proteins. Bioactive peptides from food proteins offer major potential for incorporation into functional foods and nutraceuticals. The aim of this paper is to present an outline of the bioactive peptides identified in the muscle protein of meat to date, with a focus on muscle protein from domestic animals and fish. The majority of research on bioactives from meat sources has focused on angiotensin-1-converting enzyme (ACE) inhibitory and antioxidant peptides.
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Preparation of aqueous core/silica shell microcapsules.
J Colloid Interface Sci
PUBLISHED: 07-03-2011
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Water core/silica shell microcapsules are prepared via the hydrolysis and subsequent polycondensation of tetraethoxysilane in a surfactant stabilised water-in-oil emulsion. The relationship between preparative conditions, including pH and silane concentration, has been related to final particle structure. Furthermore, the nature of the catalyst has been found to affect the mechanism by which the shells are formed, with an interfacial polymerisation proposed for ammonium hydroxide catalysed synthesis in agreement with previous reports and a new colloidosome assembly process for sodium hydroxide catalysis. In both cases shell aging processes are observed to continue beyond initial shell formation suggesting that trans-shell diffusion of reactants may be feasible, or that rapid hydrolysis is required in order to load high concentrations of the reactants into the internal phase before significant shell formation.
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Up-regulation of receptors for advanced glycation end-products by alveolar epithelium influences cytodifferentiation and causes severe lung hypoplasia.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 06-17-2011
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Receptors for advanced glycation end-products (RAGE) are cell-surface receptors expressed by pulmonary tissue that influence alveolar type (AT) II-ATI transition required for normal alveolar formation. However, the precise contribution of RAGE in interactions between pulmonary epithelium and splanchnic mesenchyme during lung organogenesis remains uncertain. To test the hypothesis that RAGE misexpression adversely affects lung morphogenesis, conditional transgenic mice were generated that overexpress RAGE. Mice that overexpress RAGE throughout embryogenesis experienced 100% mortality and significant lung hypoplasia coincident with large, vacuous areas in the periphery when compared with normal airway and alveolar architecture observed in control mouse lungs. Flow cytometry and immunohistochemistry employing cell-specific markers for distal (forkhead box protein A2) and respiratory (thyroid transcription factor-1) epithelium, ATII cells (pro-surfactant protein-C), and ATI cells (T1-?) demonstrated anomalies in key epithelial cell populations resulting from RAGE up-regulation. These results reveal that precise regulation of RAGE expression is required during lung formation. Furthermore, abundant RAGE results in profound alterations in epithelial cell differentiation that culminate in severe respiratory distress and perinatal lethality.
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Immunohistochemical detection and regulation of ?5 nicotinic acetylcholine receptor (nAChR) subunits by FoxA2 during mouse lung organogenesis.
Respir. Res.
PUBLISHED: 05-13-2011
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?5 nicotinic acetylcholine receptor (nAChR) subunits structurally stabilize functional nAChRs in many non-neuronal tissue types. The expression of ?5 nAChR subunits and cell-specific markers were assessed during lung morphogenesis by co-localizing immunohistochemistry from embryonic day (E) 13.5 to post natal day (PN) 20. Transcriptional control of ?5 nAChR expression by FoxA2 and GATA-6 was determined by reporter gene assays.
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Expansion of the surgical Apgar score across all surgical subspecialties as a means to predict postoperative mortality.
Anesthesiology
PUBLISHED: 04-20-2011
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A surgical scoring system, akin to the obstetricians Apgar score, has been developed to assess postoperative risk. To date, evaluation of this scoring system has been limited to general and vascular services. The authors attempt to externally validate and expand the Surgical Apgar Score across a wide breadth of surgical subspecialties.
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Prevalence of habitual snoring in children and occurrence of peri-operative adverse events.
Eur J Anaesthesiol
PUBLISHED: 04-19-2011
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To describe the prevalence of habitual snoring and examine its association with peri-operative adverse events in children undergoing elective non-cardiac surgery--a relationship that has not been previously characterised.
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Hsa-miR-375 is differentially expressed during breast lobular neoplasia and promotes loss of mammary acinar polarity.
J. Pathol.
PUBLISHED: 04-13-2011
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Invasive lobular carcinoma (ILC) of the breast, characterized by loss of E-cadherin expression, accounts for 5-15% of invasive breast cancers and it is believed to arise via a linear histological progression. Genomic studies have identified a clonal relationship between ILC and concurrent lobular carcinoma in situ (LCIS) lesions, suggesting that LCIS may be a precursor lesion. It has been shown that an LCIS diagnosis confers a 15-20% risk of progression to ILC over a lifetime. Currently no molecular test or markers can identify LCIS lesions likely to progress to ILC. Since microRNA (miRNA) expression changes have been detected in a number of other cancer types, we explored whether their dysregulation might be detected during progression from LCIS to ILC. Using the Illumina miRNA profiling platform, designed for simultaneous analysis of 470 mature miRNAs, we analysed the profiles of archived normal breast epithelium, LCIS lesions found alone, LCIS lesions concurrent with ILC, and the concurrent ILCs as a model of linear histological progression towards ILC. We identified two sets of differentially expressed miRNAs, the first set highly expressed in normal epithelium, including hsa-miR-224, -139, -10b, -450, 140, and -365, and the second set up-regulated during lobular neoplasia progression, including hsa-miR-375, -203, -425-5p, -183, -565, and -182. Using quantitative RT-PCR, we validated a trend of increasing expression for hsa-miR-375, hsa-miR-182, and hsa-miR-183 correlating with ILC progression. As we detected increased expression of hsa-miR-375 in LCIS lesions synchronous with ILC, we sought to determine whether hsa-miR-375 might induce phenotypes reminiscent of lobular neoplasia by expressing it in the MCF-10A 3D culture model of mammary acinar morphogenesis. Increased expression of hsa-miR-375 resulted in loss of cellular organization and acquisition of a hyperplastic phenotype. These data suggest that dysregulated miRNA expression contributes to lobular neoplastic progression.
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Association of neck circumference with perioperative adverse respiratory events in children.
Pediatrics
PUBLISHED: 04-04-2011
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The purpose of this investigation was to examine the association of neck circumference (NC) with perioperative respiratory adverse events in children undergoing elective noncardiac surgery, a relationship that has not been previously characterized.
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?-catenin dosage is a critical determinant of tracheal basal cell fate determination.
Am. J. Pathol.
PUBLISHED: 03-22-2011
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The purpose of this study was to determine whether ?-catenin regulates basal cell fate determination in the mouse trachea. Analysis of TOPGal transgene reporter activity and Wnt/?-catenin pathway gene expression suggested a role for ?-catenin in basal cell proliferation and differentiation after naphthalene-mediated Clara-like and ciliated cell depletion. However, these basal cell activities occurred simultaneously, limiting precise determination of the role(s) played by ?-catenin. This issue was overcome by analysis of ?-catenin signaling in tracheal air-liquid interface cultures. The cultures could be divided into two phases: basal cell proliferation and basal cell differentiation. A role for ?-catenin in basal cell proliferation was indicated by activation of the TOPGal transgene on proliferation days 3 to 5 and by transient expression of Myc (alias c-myc). Another peak of TOPGal transgene activity was detected on differentiation days 2 to 10 and was associated with the expression of Axin 2. These results suggest a role for ?-catenin in basal to ciliated and basal to Clara-like cell differentiation. Genetic stabilization of ?-catenin in basal cells shortened the period of basal cell proliferation but had a minor effect on this process. Persistent ?-catenin signaling regulated basal cell fate by driving the generation of ciliated cells and preventing the production of Clara-like cells.
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Neuropsychiatric and cognitive profile of patients with DSM-IV delirium referred to an old age psychiatry consultation-liaison service.
Int Psychogeriatr
PUBLISHED: 01-21-2011
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The phenomenology of delirium is understudied, including how the symptom profile varies across populations. The aim of this study was to explore phenomenology occurring in patients with delirium referred to an old age psychiatry consultation-liaison setting and compare with delirium occurring in palliative care patients.
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Gene therapy for pulmonary hypertension: prospects and challenges.
Expert Opin Biol Ther
PUBLISHED: 01-12-2011
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Recent evidence shows that pulmonary arterial hypertension (PAH) remains a fatal disease despite the introduction of new pharmacological treatments. New options are therefore needed and gene therapy approaches are a rational consideration based on emerging understanding of the genetic basis of PAH.
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Decreased efferocytosis and mannose binding lectin in the airway in bronchiolitis obliterans syndrome.
J. Heart Lung Transplant.
PUBLISHED: 01-04-2011
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Mannose binding lectin (MBL) is a key mediator of both innate immunity and efferocytosis (phagocytosis of apoptotic cells) in the airway. Defective efferocytosis results in a net increase in apoptotic material that can undergo secondary necrosis, leading to tissue damage and chronic inflammation. We have shown reduced MBL and efferocytosis in other chronic inflammatory lung diseases; we therefore hypothesized that reduced MBL and efferocytosis in the airways may be a determinant of bronchiolitis obliterans syndrome (BOS) after lung transplantation.
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Viruses in pharmaceutical research: pulmonary vascular disease.
Mol. Pharm.
PUBLISHED: 12-22-2010
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The management and understanding of pulmonary arterial hypertension (PAH) has undergone something of a revolution in the last 10 years, with new pharmacological agents entering routine clinical practice and significantly improving outcomes. Nevertheless many patients ultimately progress, and additional new treatment approaches are needed. There is now greater understanding of the molecular and genetic basis for the development of PAH, specifically in regard to the role of bone morphogenetic protein receptor 2 (BMPR2) signaling and related pathways. The challenge is to determine whether these new discoveries can be exploited for new therapies. In this article the role of viruses as tools for gene delivery for pulmonary vascular disease is discussed. Gene delivery of BMPR2 has now been shown to ameliorate the development and progression of PAH in animal models, thereby identifying this approach as a therapeutic target.
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Receptor for advanced glycation end-products signals through Ras during tobacco smoke-induced pulmonary inflammation.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 12-03-2010
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We previously demonstrated up-regulation of the receptor for advanced glycation end-products (RAGE) and its ligands by cigarette smoke extract (CSE) in rat R3/1 cells, a type I-like alveolar epithelial cell line. However, RAGE-mediated intracellular signaling pathways that lead to pulmonary inflammation remained unclear. Using ELISAs, we demonstrate that alveolar epithelial cell lines exposed to 25% CSE for 2 hours induce the activation of Ras, a small GTPase that functions as a molecular switch in the control of several intracellular signaling networks. Conversely, cells treated with siRNA for RAGE (siRAGE) resulted in decreased Ras activation. Furthermore, Ras was significantly diminished in lungs from RAGE null mice exposed to chronic tobacco smoke when compared with smoke-exposed wild-type mice. The use of a luciferase reporter containing NF-?B binding sites also demonstrated elevated NF-?B activation in R3/1 cells after CSE stimulation and decreased NF-?B activation in cells transfected with siRAGE before CSE exposure. ELISA revealed an increase in the secretion of IL-1? and CCL5 by R3/1 cells, two cytokines induced by NF-?B and associated with leukocyte chemotaxis. Furthermore, real-time RT-PCR and ELISAs revealed decreased cytokine secretion in RAGE null mouse lung exposed to tobacco smoke compared with lungs from smoke-exposed wild-type animals. These results support the conclusion that CSE-induced RAGE expression functions in pathways that involve Ras-mediated NF-?B activation and cytokine elaboration. This RAGE-Ras-NF-?B axis likely contributes to inflammation associated with several smoking-related inflammatory lung diseases.
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Analog signal multiplexing for PSAPD-based PET detectors: simulation and experimental validation.
Phys Med Biol
PUBLISHED: 11-16-2010
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A 1 mm(3) resolution clinical positron emission tomography (PET) system employing 4608 position-sensitive avalanche photodiodes (PSAPDs) is under development. This paper describes a detector multiplexing technique that simplifies the readout electronics and reduces the density of the circuit board design. The multiplexing scheme was validated using a simulation framework that models the PSAPDs and front-end multiplexing circuits to predict the signal-to-noise ratio and flood histogram performance. Two independent experimental setups measured the energy resolution, time resolution, crystal identification ability and count rate both with and without multiplexing. With multiplexing, there was no significant degradation in energy resolution, time resolution and count rate. There was a relative 6.9 ± 1.0% and 9.4 ± 1.0% degradation in the figure of merit that characterizes the crystal identification ability observed in the measured and simulated ceramic-mounted PSAPD module flood histograms, respectively.
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Convex optimization of coincidence time resolution for a high-resolution PET system.
IEEE Trans Med Imaging
PUBLISHED: 09-27-2010
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We are developing a dual panel breast-dedicated positron emission tomography (PET) system using LSO scintillators coupled to position sensitive avalanche photodiodes (PSAPD). The charge output is amplified and read using NOVA RENA-3 ASICs. This paper shows that the coincidence timing resolution of the RENA-3 ASIC can be improved using certain list-mode calibrations. We treat the calibration problem as a convex optimization problem and use the RENA-3s analog-based timing system to correct the measured data for time dispersion effects from correlated noise, PSAPD signal delays and varying signal amplitudes. The direct solution to the optimization problem involves a matrix inversion that grows order (n(3)) with the number of parameters. An iterative method using single-coordinate descent to approximate the inversion grows order (n). The inversion does not need to run to convergence, since any gains at high iteration number will be low compared to noise amplification. The system calibration method is demonstrated with measured pulser data as well as with two LSO-PSAPD detectors in electronic coincidence. After applying the algorithm, the 511 keV photopeak paired coincidence time resolution from the LSO-PSAPD detectors under study improved by 57%, from the raw value of 16.3 ±0.07 ns full-width at half-maximum (FWHM) to 6.92 ±0.02 ns FWHM ( 11.52 ±0.05 ns to 4.89 ±0.02 ns for unpaired photons).
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A novel approach to the assessment of lymphocytic bronchiolitis after lung transplantation--transbronchial brush.
J. Heart Lung Transplant.
PUBLISHED: 08-27-2010
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Lymphocytic bronchiolitis (LB) is the strongest risk factor for subsequent allograft loss due to bronchiolitis obliterative syndrome (BOS); however, it is poorly assessed by transbronchial biopsy (TBBx) because of sampling error, interpretation error and the presence of non-alloimmune airway inflammation. We hypothesized that flow cytometric evaluation of bronchiolar brushings (transbronchial brush, TBBr) may be a better approach.
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Cloning and expression of a mureinolytic enzyme from the mycobacteriophage TM4.
FEMS Microbiol. Lett.
PUBLISHED: 08-19-2010
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In this study, we describe the characterization, cloning, expression and purification of the lysin A gene of the mycobacteriophage TM4. The gene TM4_gp29 (gp29) is a 1644-bp gene that codes for a 58.6-kDa protein and contains peptidoglycan recognition protein, Zn-binding and amidase catalytic domains. The gene was cloned into Escherichia coli using the His-Tag pQE60 vector. After affinity chromatography-mediated purification, the protein was concentrated and visualized using sodium dodecyl sulphate polyacrylamide gel electrophoresis. Evidence of peptidoglycan-degrading activity was observed initially by a chloroform assay and later by conventional zymogram analysis.
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TTF-1 regulates ?5 nicotinic acetylcholine receptor (nAChR) subunits in proximal and distal lung epithelium.
Respir. Res.
PUBLISHED: 07-29-2010
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Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels comprised of five similar subunits that influence signal transduction and cell turnover. ?5 is a structural subunit detected in many non-neuronal tissues; however, its function during pulmonary development is unknown.
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Cigarette smoke-induced changes to alveolar macrophage phenotype and function are improved by treatment with procysteine.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 07-01-2010
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Defective efferocytosis may perpetuate inflammation in smokers with or without chronic obstructive pulmonary disease (COPD). Macrophages may phenotypically polarize to classically activated M1 (proinflammatory; regulation of antigen presentation) or alternatively activated M2 (poor antigen presentation; improved efferocytosis) markers. In bronchoalveolar lavage (BAL)-derived macrophages from control subjects and smoker/ex-smoker COPD subjects, we investigated M1 markers (antigen-presenting major histocompatibility complex [MHC] Classes I and II), complement receptors (CRs), the high-affinity Fc receptor involved with immunoglobulin binding for phagocytosis (Fc-gamma receptor, Fc?R1), M2 markers (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin [DC-SIGN] and arginase), and macrophage function (efferocytosis and proinflammatory cytokine production in response to LPS). The availability of glutathione (GSH) in BAL was assessed, because GSH is essential for both M1 function and efferocytosis. We used a murine model to investigate macrophage phenotype/function further in response to cigarette smoke. In lung tissue (disaggregated) and BAL, we investigated CRs, the available GSH, arginase, and efferocytosis. We further investigated the therapeutic effects of an oral administration of a GSH precursor, cysteine l-2-oxothiazolidine-4-carboxylic acid (procysteine). Significantly decreased efferocytosis, available GSH, and M1 antigen-presenting molecules were evident in both COPD groups, with increased DC-SIGN and production of proinflammatory cytokines. Increased CR-3 was evident in the current-smoker COPD group. In smoke-exposed mice, we found decreased efferocytosis (BAL and tissue) and available GSH, and increased arginase, CR-3, and CR-4. Treatment with procysteine significantly increased GSH, efferocytosis (BAL: control group, 26.2%; smoke-exposed group, 17.66%; procysteine + smoke-exposed group, 27.8%; tissue: control group, 35.9%; smoke-exposed group, 21.6%; procysteine + smoke-exposed group, 34.5%), and decreased CR-4 in lung tissue. Macrophages in COPD are of a mixed phenotype and function. The increased efferocytosis and availability of GSH in response to procysteine indicates that this treatment may be useful as adjunct therapy for improving macrophage function in COPD and in susceptible smokers.
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Computational modeling and prototyping of a pediatric airway management instrument.
Anesth. Analg.
PUBLISHED: 06-25-2010
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Anterior retraction of the tongue is used to enhance upper airway patency during pediatric fiberoptic intubation. This can be achieved by the use of Magill forceps as a tongue retractor, but lingual grip can become unsteady and traumatic. Our objective was to modify this instrument using computer-aided engineering for the purpose of stable tongue retraction.
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Lymphocytic bronchiolitis is associated with inadequate suppression of blood T-cell granzyme B, IFN-gamma, and TNF-alpha.
Transplantation
PUBLISHED: 06-19-2010
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Lymphocytic bronchiolitis (LB) has been shown to be an important factor for the subsequent development of obliterative bronchiolitis (OB). We have previously shown that OB, which limits long-term survival after lung transplantation, is associated with lack of suppression of peripheral blood T-cell granzyme B, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha. However, the role of these proinflammatory mediators in LB is unknown. We hypothesized that these proinflammatory mediators may also be increased during LB episodes despite standard immunosuppression regimens.
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The physiologic and behavioral effects of oral and intranasal midazolam in pediatric dental patients.
Pediatr Dent
PUBLISHED: 06-19-2010
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The purpose of this study was to compare the safety and effectiveness of oral and intranasal midazolam in healthy children by evaluating their physiological and behavioral responses.
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Diesel particulate matter induces receptor for advanced glycation end-products (RAGE) expression in pulmonary epithelial cells, and RAGE signaling influences NF-?B-mediated inflammation.
Environ. Health Perspect.
PUBLISHED: 06-03-2010
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Receptors for advanced glycation end-products (RAGE) are cell-surface receptors expressed by alveolar type I (ATI) epithelial cells and are implicated in mechanisms of alveolar development and sustained pulmonary inflammation.
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Tracheal Basal cells: a facultative progenitor cell pool.
Am. J. Pathol.
PUBLISHED: 06-03-2010
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Analysis of lineage relationships in the naphthalene-injured tracheal epithelium demonstrated that two multipotential keratin 14-expressing cells (K14ECs) function as progenitors for Clara and ciliated cells. These K14EC were distinguished by their self-renewal capacity and were hypothesized to reside at the stem and transit amplifying tiers of a tissue-specific stem cell hierarchy. In this study, we used gene expression and histomorphometric analysis of the steady-state and naphthalene-injured trachea to evaluate the predictions of this model. We found that the steady-state tracheal epithelium is maintained by two progenitor cell pools, secretory and basal cells, and the latter progenitor pool is further divided into two subsets, keratin 14-negative and -positive. After naphthalene-mediated depletion of the secretory and ciliated cell types, the two basal cell pools coordinate to restore the epithelium. Both basal cell types up-regulate keratin 14 and generate a broadly distributed, abundant, and highly mitotic cell pool. Furthermore, basal cell proliferation is associated with generation of differentiated Clara and ciliated cells. The uniform distribution of basal cell progenitors and of their differentiated progeny leads us to propose that the hierarchical organization of tracheal reparative cells be revised to include a facultative basal cell progenitor pool.
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Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 04-07-2010
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While heparin has been used almost exclusively as a blood anticoagulant, important literature demonstrates that it also has broad anti-inflammatory activity. Herein, using low anti-coagulant 2-O,3-O-desulfated heparin (ODSH), we demonstrate that most of the anti-inflammatory pharmacology of heparin is unrelated to anticoagulant activity. ODSH has low affinity for anti-thrombin III, low anti-Xa, and anti-IIa anticoagulant activities and does not activate Hageman factor (factor XII). Unlike heparin, ODSH does not interact with heparin-platelet factor-4 antibodies present in patients with heparin-induced thrombocytopenia and even suppresses platelet activation in the presence of activating concentrations of heparin. Like heparin, ODSH inhibits complement activation, binding to the leukocyte adhesion molecule P-selectin, and the leukocyte cationic granular proteins azurocidin, human leukocyte elastase, and cathepsin G. In addition, ODSH and heparin disrupt Mac-1 (CD11b/CD18)-mediated leukocyte adhesion to the receptor for advanced glycation end products (RAGE) and inhibit ligation of RAGE by its many proinflammatory ligands, including the advanced glycation end-product carboxymethyl lysine-bovine serum albumin, the nuclear protein high mobility group box protein-1 (HMGB-1), and S100 calgranulins. In mice, ODSH is more effective than heparin in reducing selectin-mediated lung metastasis from melanoma and inhibits RAGE-mediated airway inflammation from intratracheal HMGB-1. In humans, 50% inhibitory concentrations of ODSH for these anti-inflammatory activities can be achieved in the blood without anticoagulation. These results demonstrate that the anticoagulant activity of heparin is distinct from its anti-inflammatory actions and indicate that 2-O and 3-O sulfate groups can be removed to reduce anticoagulant activity of heparin without impairing its anti-inflammatory pharmacology.
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Synovial fluid proteins differentiate between the subtypes of juvenile idiopathic arthritis.
Arthritis Rheum.
PUBLISHED: 03-12-2010
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Juvenile idiopathic arthritis (JIA) is a heterogeneous group of inflammatory diseases, and no clinically useful prognostic markers to predict disease outcome in children with JIA are currently available. Synovial fluid likely reflects the proteins present in the inflamed synovium. The purpose of this study was to delineate the synovial fluid proteome and determine whether protein expression differs in the different subtypes of JIA.
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A non-linear three-dimensional model for quantifying microbubble dynamics.
J. Acoust. Soc. Am.
PUBLISHED: 02-09-2010
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A three-dimensional non-linear model for simulating microbubble response to acoustic insonation is presented. A 1 mum radius microbubble stimulated using positive and inverted 2.4 MHz pulses produced radius-time curves that matched (error <10%) with the experimental observation. A bound 2.3 mum radius microbubble insonated using 2.25 MHz 6 cycle pulse was observed to oscillate with max/min oscillations 45% lower than that of the free microbubble, this correlated ( approximately 10% error) with the observations of Garbin et al. [Appl. Phys. Lett. 90, 114103 (2007)]. The adherent microbubble oscillated asymmetrically in the plan view and symmetrically in the elevation view, consistent with the previous experimental results.
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6-benzyladenine metabolism during reinvigoration of mature Pinus radiata buds in vitro.
Tree Physiol.
PUBLISHED: 02-08-2010
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Maturation or phase change is a serious challenge in the deployment of superior trees of Pinus radiata D. Don because of the difficulties associated with propagation of cuttings from mature trees. We used an in vitro system to study 6-benzyladenine (BA)-induced reinvigoration of the fascicle meristems of mature buds during in vitro culture. Anatomical examinations revealed that BA inhibited the development of secondary needle primordia and rejuvenated the fascicle meristems of the mature bud to produce primary needles, which are characteristic of the juvenile phase in P. radiata. Without BA supplement in the culture media, fascicle primordia continued developing secondary needles and quiescent fascicle meristems. BA metabolite analysis showed that the novel cytokinin pathway reported previously in P. radiata (H. Zhang, K.J. Horgan, P.H.S. Reynolds, G.E. Norris and P.E. Jameson. 2001. Novel cytokinins: The predominant forms in mature buds of Pinus radiata. Physiol. Plant. 112: 127-134) was mirrored in vitro, with BA converted into a variety of metabolites including 6-benzylamino-9-glucopyranosylribosyl-purine and its novel phosphorylated form, 6-benzylamino-9-glucopyranosylribosyl-purine. The culture of mature buds in the presence of BA caused a reduction in the level of endogenous cytokinins, suggesting a direct action of BA itself. Similar correlations are noted between levels of certain metabolites and the maturation status of buds from field-grown trees and buds in culture, indicating that this in vitro system may be a good model for studying the processes of maturation and reinvigoration.
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How do pediatric anesthesiologists define intraoperative hypotension?
Paediatr Anaesth
PUBLISHED: 10-01-2009
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Although blood pressure (BP) monitoring is a recommended standard of care by the ASA, and pediatric anesthesiologists routinely monitor the BP of their patients and when appropriate treat deviations from normal, there is no robust definition of hypotension in any of the pediatric anesthesia texts or journals. Consequently, what constitutes hypotension in pediatric anesthesia is currently unknown. We designed a questionnaire-based survey of pediatric anesthesiologists to determine the BP ranges and thresholds used to define intraoperative hypotension (IOH).
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Bronchiolitis obliterans syndrome is associated with absence of suppression of peripheral blood Th1 proinflammatory cytokines.
Transplantation
PUBLISHED: 07-23-2009
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Bronchiolitis obliterans syndrome (BOS) is the single most important factor that limits long-term survival after lung transplantation. T cells are a major cell type involved in acute graft rejection; however, the role of these cells in BOS is unknown. There have been no previous studies of cytokine production by T cells from blood and bronchoalveolar lavage (BAL) and by intraepithelial T cells from bronchial brushings during BOS, and we hypothesized that proinflammatory cytokines may be increased during BOS despite standard immunosuppression regimes.
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Using the healthcare matrix with interns and medical students as a tool to effect change.
South. Med. J.
PUBLISHED: 07-14-2009
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The Healthcare Matrix is a tool developed at Vanderbilt University Medical Center which assesses the care of patients using the Accreditation Council for Graduate Medical Education (ACGME) competencies and the Institute of Medicine (IOM) Aims for Improvement. House staff have been using this tool since 2004 as a means of learning the competencies in the daily care of patients. As the residents fill in the cells of the Matrix, the opportunities for improvement become evident. Anesthesia interns were introduced to the Matrix at bimonthly meetings to analyze a real case of complex and/or flawed patient management. Each resident completed his/her own Matrix and then the group selected one Matrix as their improvement project. This article will present one Matrix case, how quality improvement (QI) tools and methods were utilized and what resulting improvements were made. The analyzed case revealed numerous flaws with nonstandardized handoffs, incorrect and insufficient documentation, and unclear roles/responsibilities. An ideal process flowchart was developed highlighting improved handoffs and the need for a new admitting policy to the intensive care unit (ICU). The Healthcare Matrix is an effective tool for teaching the ACGME competencies in patient care. The resequencing of the competencies in the Matrix makes it easy to identify where improvements are needed. When taught the tools and methods of quality improvement, even interns can make a significant contribution to the improvement of patient care.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.