Symptom-related sputum microbiota in stable chronic obstructive pulmonary disease.

Background: The role of airway microbiota in COPD is highly debated. Symptomology assessment is vital for the management of clinically stable COPD patients; however, the link between symp toms and the airway microbiome is currently unknown. Purpose: The present study aimed to evaluate the relationship among stable COPD patients. Patients and methods: We conducted pyrosequencing of bacterial 16S rRNA using induced sputum samples in a Han Chinese cohort that included 40 clinically stable COPD patients and 19 healthy controls. Results: Alterations in community composition and core bacte rial taxa (Neisseria subflava, etc.) were observed in patients with severe symptoms compared with controls. The co-occurrence network indicated that the key microbiota enriched in COPD patients showed higher expression in patients with severe symptoms. The association pattern of symptoms with the sputum microbiome was obviously different from that of lung function in COPD patients. Conclusion: These findings broaden our insights into the relationship between the sputum microbiota and the symptom severity in COPD patients, emphasizing the role of symptoms in the airway microbiome, independent of lung function.

Gender Disparities in Academic Practice.

BACKGROUND: In academia, women remain underrepresented. The authors' sought to examine differences in faculty position and professional satisfaction among academic physicians by gender. METHODS: From 2008 to 2012, academic faculty members at a single institution were surveyed (2008, n = 737; 2010, n = 1151; and 2012, n = 971) regarding current position, choice of position, professional satisfaction, and desire for leaving. Logistic regression was performed to compare aspects of professional satisfaction by gender. RESULTS: Men more often held tenure track positions compared with women (2008, 45 percent versus 20 percent; 2010, 47 percent versus 20 percent; and 2012, 49 percent versus 20 percent; p < 0.001). Women were more likely to engage in only clinical activities compared with men (2008, 31 percent versus 18 percent; 2010, 28 percent versus 14 percent; and 2012, 33 percent versus 13 percent; p < 0.001) and less likely to participate in research. Women chose tracks to accommodate work-life balance [2008, OR, 1.9 (95 percent CI, 1.29 to 2.76); 2010, OR, 2.0 (95 percent CI, 1.38 to 2.76); and 2012, OR, 2.1 (95 percent CI, 1.40 to 3.00)], rather than the opportunity of tenure [2008, OR, 0.4 (95 percent CI, 0.23 to 0.75); 2010, OR, 0.5 (95 percent CI, 0.35 to 0.85); and 2012, OR, 0.5 (95 percent CI, 0.29 to 0.76) compared with men. Men reported higher professional satisfaction compared with women (2008, 5.7 versus 5.4, p < 0.009; 2012, 5.3 versus 5.0, p < 0.03). Men were more likely to leave because of leadership opportunities (14.4 percent versus 9.2 percent, p < 0.03) and compensation (14.2 percent versus 9.2 percent, p < 0.03) compared with women. CONCLUSIONS: Women report lower levels of professional satisfaction in academic practice compared with men. Given the increasing pressures of academic practice, efforts to align work-life balance and professional goals could potentially improve faculty satisfaction and retention.

Regulation of alveolar epithelial cell apoptosis and pulmonary fibrosis by coordinate expression of components of the fibrinolytic system.

Alveolar type II (ATII) cell apoptosis and depressed fibrinolysis that promotes alveolar fibrin deposition are associated with acute lung injury (ALI) and the development of pulmonary fibrosis (PF). We therefore sought to determine whether p53-mediated inhibition of urokinase-type plasminogen activator (uPA) and induction of plasminogen activator inhibitor-1 (PAI-1) contribute to ATII cell apoptosis that precedes the development of PF. We also sought to determine whether caveolin-1 scaffolding domain peptide (CSP) reverses these changes to protect against ALI and PF. Tissues as well as isolated ATII cells from the lungs of wild-type (WT) mice with BLM injury show increased apoptosis, p53, and PAI-1, and reciprocal suppression of uPA and uPA receptor (uPAR) protein expression. Treatment of WT mice with CSP reverses these effects and protects ATII cells against bleomycin (BLM)-induced apoptosis whereas CSP fails to attenuate ATII cell apoptosis or decrease p53 or PAI-1 in uPA-deficient mice. These mice demonstrate more severe PF. Thus p53 is increased and inhibits expression of uPA and uPAR while increasing PAI-1, changes that promote ATII cell apoptosis in mice with BLM-induced ALI. We show that CSP, an intervention targeting this pathway, protects the lung epithelium from apoptosis and prevents PF in BLM-induced lung injury via uPA-mediated inhibition of p53 and PAI-1.

Hepatocyte growth factor acts as a mitogen and chemoattractant for postnatal subventricular zone-olfactory bulb neurogenesis.

Neural progenitor cells persist throughout life in the forebrain subventricular zone (SVZ). They generate neuroblasts that migrate to the olfactory bulb and differentiate into interneurons, but mechanisms underlying these processes are poorly understood. Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic factor that influences cell motility, proliferation and morphogenesis in neural and non-neural tissues. HGF and its receptor, c-Met, are present in the rodent SVZ-olfactory bulb pathway. Using in vitro neurogenesis assays and in vivo studies of partially HGF-deficient mice, we find that HGF promotes SVZ cell proliferation and progenitor cell maintenance, while slowing differentiation and possibly altering cell fate choices. HGF also acts as a chemoattractant for SVZ neuroblasts in co-culture assays. Decreased HGF signaling induces ectopic SVZ neuroblast migration and alters the timing of migration to the olfactory bulb. These results suggest that HGF influences multiple steps in postnatal forebrain neurogenesis. HGF is a mitogen for SVZ neural progenitors, and regulates their differentiation and olfactory bulb migration.

Activation of urokinase plasminogen activator and its receptor axis is essential for macrophage infiltration in a prostate cancer mouse model.

Macrophages within the tumor microenvironment promote angiogenesis, extracellular matrix breakdown, and tumor cell migration, invasion, and metastasis. Activation of the urokinase plasminogen activator (uPA) and its receptor (uPAR) axis promotes prostate cancer tumorigenicity, invasion, metastasis, and survival within the tumor microenvironment. The link between macrophage infiltration and the uPA/uPAR axis in prostate cancer development has not been established, although it has been reported that uPA plays a critical role inmonocyte and macrophage chemotaxis. In this study, murine prostate cancer RM-1 cells were subcutaneously inoculated into wild-type (WT), uPA(-/-), and uPAR(-/-) mice. Tumor volume was significantly diminished in both uPA(-/-) and uPAR(-/-) mice compared with WT controls. Greater inhibition of tumor volume was also observed in uPA(-/-) mice compared with uPAR(-/-) mice, suggesting the important contribution of stromal-derived uPA to sustain the tumor growth. Immunohistochemical staining revealed that tumors in uPA(-/-) and uPAR(-/-) mice displayed significantly lower proliferative indices, higher apoptotic indices, and less neovascularity compared with the tumors in WT mice. Tumors in uPA(-/-) and uPAR(-/-) mice displayed significantly less macrophage infiltration as demonstrated by F4/80 staining and MAC3(+) cell numbers by flow cytometry compared with the tumors from WT mice. These findings suggest that the uPA/uPAR axis acts in both autocrine and paracrine manners in the tumor microenvironment, and activation of uPA/uPAR axis is essential for macrophage infiltration into prostate tumors.

Induction of tissue factor by urokinase in lung epithelial cells and in the lungs.

RATIONALE: Urokinase-type plasminogen activator (uPA) regulates extracellular proteolysis in lung injury and repair. Although alveolar expression of uPA increases, procoagulant activity predominates. OBJECTIVES: This study was designed to investigate whether uPA alters the expression of tissue factor (TF), the major initiator of the coagulation cascade, in lung epithelial cells (ECs). METHODS: Bronchial, primary airway ECs and C57B6 wild-type, uPA-deficient (uPA(-/-)) mice were exposed to phosphate-buffered saline, uPA, or LPS. Immunohistochemistry, protein, cellular, and molecular techniques were used to assess TF expression and activity. MEASUREMENTS AND MAIN RESULTS: uPA enhanced TF mRNA and protein expression, and TF-dependent coagulation in lung ECs. uPA-induced expression of TF involves both increased synthesis and enhanced stabilization of TF mRNA. uPA catalytic activity had little effect on induction of TF. By contrast, deletion of the uPA receptor binding growth factor domain from uPA markedly attenuated the induction of TF, suggesting that uPA receptor binding is sufficient for TF induction. Lung tissues of uPA-deficient mice expressed less TF protein and mRNA compared with wild-type mice. In addition, intratracheal instillation of mouse uPA increased TF mRNA and protein expression and accelerated coagulation in lung tissues. uPA(-/-) mice exposed to LPS failed to induce TF. CONCLUSIONS: uPA increased TF expression and TF-dependent coagulation in the lungs of mice. We hypothesize that uPA-mediated induction of TF occurs in lung ECs to promote increased fibrin deposition in the airways during acute lung injury.

Current and emerging drugs for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic, progressive and often fatal form of interstitial lung disease. It is characterized by injury with loss of lung epithelial cells and abnormal tissue repair, resulting in abnormal accumulation of fibroblasts and myofibroblasts, deposition of extracellular matrix and distortion of lung architecture, leading to respiratory failure. This lethal lung disorder continues to pose major clinical challenges as an effective therapeutic regimen has yet to be developed. In this report, therapeutic strategies are reviewed, including the use of antifibrotic agents, inhibition of cytokines, leukotrienes and cytokines receptors, and molecular targeting of specific signaling pathways during fibrotic processes and angiogenesis. This article examines the body of evidence supporting present therapies and reviews the newer agents being tested in patients with idiopathic pulmonary fibrosis.

Mactinin treatment promotes wound-healing-associated inflammation in urokinase knockout mice.

Mactinin, a 31 kDa fragment from the amino-terminal end of alpha-actinin, is chemotactic for monocytes and can promote monocyte/macrophage maturation. Macrophages are essential for wound healing, in which they play key roles in debridement, angiogenesis, fibroblast proliferation, and collagen metabolism. We have previously determined that urokinase is necessary to form mactinin from extracellular alpha-actinin, which may be present at sites of inflammation as a result of cell movement. Thus, urokinase knockout mice are unable to form mactinin and therefore are an ideal model to study mactinin's effects on wound healing. Saline- and mactinin-treated wounds were analyzed in a subcutaneous sponge wound model in both wild-type and urokinase knockout mice. The wounded urokinase knockout mice had markedly decreased leukocyte infiltration compared with wounded wild-type mice. In addition, production of the proinflammatory cytokine, interleukin-12, and of collagen was also decreased in knockouts. Treatment of knockout mice with mactinin resulted in leukocyte infiltration numbers, interleukin-12 levels, and hydroxyproline measurements similar to those in wild-type mice. The results suggest that impaired wound healing in urokinase-deficient mice can be restored by administration of mactinin.

Induction of p53 by urokinase in lung epithelial cells.

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. The plasminogen/plasmin system includes the uPA, its receptor, and its inhibitor (plasminogen activator inhibitor-1). Interactions between these molecules regulate cellular proteolysis as well as adhesion, cellular proliferation, and migration, processes germane to the pathogenesis of lung injury and neoplasia. In previous studies, we found that uPA regulates cell surface fibrinolysis by regulating its own expression as well as that of the uPA receptor and plasminogen activator inhibitor-1. In this study, we found that uPA alters expression of the tumor suppressor protein p53 in Beas2B airway epithelial cells in both a time- and concentration-dependent manner. These effects do not require uPA catalytic activity because the amino-terminal fragment of uPA lacking catalytic activity was as potent as two chain active uPA. Single chain uPA also enhanced p53 expression to the same extent as intact two chain active uPA and the amino-terminal fragment. Pretreatment of cells with anti-beta1 integrin antibody blocked uPA-induced p53 expression. uPA-induced p53 expression occurs without increased p53 mRNA expression. However, uPA induced oncoprotein MDM2 in a concentration-dependent manner. uPA-induced p53 expression does not require activation of tyrosine kinases. Inactivation of protein-tyrosine phosphatase SHP-2 inhibits both basal and uPA-induced p53 expression. Plasmin did not alter uPA-mediated p53 expression. The induction of p53 expression by exposure of lung epithelial cells to uPA is a newly recognized pathway by which urokinase may influence the proliferation of lung epithelial cells. This pathway could regulate pathophysiologic alterations of p53 expression in the setting of lung inflammation or neoplasia.

Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator.

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA-/-) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1). developed a novel methodology to detect pericellular proteolytic function, (2). demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3). demonstrated its usefulness in real-time studies of cell migration, and (4). showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.

Urokinase-deficient mice fail to generate a type 2 immune response following schistosomal antigen challenge.

Activated lymphocytes express urokinase-type plasminogen activator (uPA). Previous work suggests that uPA modulates T-lymphocyte responses. Mice deficient in uPA (uPA(-/-)) fail to generate type 1 (T1) immune responses during infection with Cryptococcus neoformans. Failure to generate either a T1 or a T2 immune response is not predictive of defects in the alternative response. Conversely, down-regulation of one type of immune response may result in inappropriate overactivation of the other. It is not known whether the immune defect in uPA(-/-) mice affects only T1 responses or whether T2 responses are also impaired. Impairment of both T1 and T2 responses would suggest a global T-cell defect in the absence of uPA. To determine the role of uPA in T2 immune responses, wild-type (WT) and uPA(-/-) mice were primed and challenged with schistosomal egg antigen (SEA). This elicits strong polarization to T2 immune responses in immunocompetent mice. The challenged WT mice developed delayed-type hypersensitivity (DTH) to SEA; high levels of serum immunoglobulin E (IgE); a strong T2 cytokine phenotype with markedly elevated levels of interleukin-4 (IL-4), IL-5, and IL-13; and eosinophil-rich pulmonary granulomas. uPA(-/-) mice failed to develop DTH to SEA; did not polarize Ig production to IgE; did not produce high levels of IL-4, IL-5, or IL-13; and had markedly reduced numbers of granuloma-associated eosinophils. uPA(-/-) mice fail to generate polarized T2 immune responses to a T2-inducing pathogen. These findings, in conjunction with our previous work, demonstrate that mice deficient in uPA have profoundly impaired immunity involving both T1 and T2 polarization and are largely immunologically unresponsive.

Urokinase-type plasminogen activator potentiates lipopolysaccharide-induced neutrophil activation.

Urokinase plasminogen activator (uPA) is a serine protease that catalyzes the conversion of plasminogen to plasmin. Although increased circulating levels of uPA are present in endotoxemia and sepsis, conditions in which activated neutrophils contribute to the development of acute organ dysfunction, the ability of uPA to participate directly in LPS-induced neutrophil activation has not been examined. In the present experiments, we show that uPA can enhance activation of neutrophils exposed to submaximal stimulatory doses of LPS. In particular, uPA increased LPS-induced activation of intracellular signaling pathways, including Akt and c-Jun N-terminal kinase, nuclear translocation of the transcriptional regulatory factor NF-kappa B, and expression of proinflammatory cytokines, including IL-1 beta, macrophage-inflammatory protein-2, and TNF-alpha. There was no effect of uPA on LPS-induced activation of p38 mitogen-activated protein kinase in neutrophils. Transgenic mice unable to produce uPA (uPA(-/-)) were protected from endotoxemia-induced lung injury, as determined by development of lung edema, pulmonary neutrophil accumulation, lung IL-1 beta, macrophage-inflammatory protein-2, and TNF-alpha cytokine levels. These results demonstrate that uPA can potentiate LPS-induced neutrophil responses and also suggest that such effects are sufficiently important in vivo to play a major contributory role in neutrophil-mediated inflammatory responses, such as the development of acute lung injury.

Urokinase is required for the formation of mactinin, an alpha-actinin fragment that promotes monocyte/macrophage maturation.

We have previously shown that lysates from HL-60 myeloid leukemia cells or from peripheral blood monocytes are able to degrade alpha-actinin to form a 31-kDa amino-terminal fragment with monocyte/macrophage maturation promoting activity. In contrast, intact alpha-actinin, which is a 100-kDa actin-binding protein, has no differentiating activity. The aim of this study was to investigate the enzyme responsible for the degradation of alpha-actinin to form this fragment, named mactinin. The ability of cell lysates to degrade [125I]alpha-actinin in the presence of various enzyme inhibitors, including inhibitors of metalloproteinases, cysteine proteinases, and serine proteases, was measured. Phenylmethylsulfonyl fluoride (PMSF) was the only inhibitor able to prevent formation of mactinin by cell lysate degradation of alpha-actinin, suggesting that a serine protease is responsible for the digestion. Of the various serine proteases tested (thrombin, plasmin, and urokinase), only urokinase was able to produce a 31-kDa band. The urokinase-generated 31-kDa band promoted maturation in HL-60 cells. Amiloride, a specific inhibitor of urokinase, inhibited production of the 31-kDa alpha-actinin fragment by HL-60 cell lysates. For in vivo tests, inflammatory fluid (from bronchoalvelolar lavage) was collected from uPA (urokinase) knockout mice and their wild-type counterparts after intratracheal challenge with Pneumocystis carinii. Although most (6 of 8) wild-type mice had mactinin in their inflammatory fluid samples, none (0 of 8) of the uPA knockout mice had mactinin present (P<0.01). These results demonstrate that urokinase is necessary and sufficient for the formation of the monocyte/macrophage maturation promoting fragment, mactinin, in vitro and in vivo. These findings support the role of urokinase in the regulation of monocyte/macrophage functions, such as that occurring in inflammatory reactions.