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Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that theHAreceptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/ Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS [ABSTRACT FROM AUTHOR]

Background: IL-17A and IL-19 are highly expressed in chronic inflammatory diseases, such as psoriasis and asthma. IL-19 plays a significant role in the enhancement of TH2 cytokine secretion in allergic diseases, but its cellular source in asthmatic patients remains unknown. Objective: Our aims were to determine whether the epithelium is a major source of airway mucosal IL-19 and to elucidate the mechanism of gene expression regulation. Methods: Immunofluorescent staining was used to determine IL-19 protein expression in tracheal tissue sections of various airway diseases. Well-differentiated primary human bronchial epithelial cultures and a corresponding cell line were used as in vitro models to study gene regulation. Results: We found significantly higher IL-19 expression in airway epithelia of asthmatic patients than in epithelia of patients with other diseases. Using a cytokine panel, we demonstrated the upregulation of IL-19 expression in cultures by two TH2 cytokines, IL-4 and IL-13, in addition to the previously found TH17 cytokine IL-17A. Moreover, cotreatment of IL-17A and IL-4/IL-13 synergistically upregulated IL-19 expression. Using siRNA and chemical inhibitor approaches, we demonstrated a transcriptional regulation of IL-19 by nuclear factor κB and signal transducer and activator of transcription (STAT) 6. The addition of IL-13 to IL-17A stimulation triggers a shift from nuclear factor κB–dependent transcriptional regulation to one that is STAT6 based. Using chromatin immunoprecipitation assays, we demonstrated the presence of STAT6-binding elements in the IL-19 promoter region. Conclusion: We propose that an IL-17A– and IL-13–induced synergism in IL-19 stimulation in airway epithelia occurs through a STAT6-dependent pathway. [Copyright &y& Elsevier]

Abstract: In this paper, we present a methodology for solving the Public Transport Network Design Problem (PTNDP) and describe its application in the context of the Design Study developed in order to propose a new structure for the transit system of the city of Santiago, Chile. Firstly, we briefly define the PTNDP as a multilevel programming problem and discuss the solution method implemented. Then, the application of this methodology to the Santiago transit system is presented, and the main results obtained are analyzed. The new restructured system, based on a hierarchy of specialized services that complement and coordinate their operations and using an integrated fare scheme, is compared with an optimized version (optimal frequencies) of the current one, a set of direct services, mainly based on the operation of independent itineraries, without fare integration. The most important conclusions are the following: (a) the private operating costs and the social costs of the restructured system, using higher standard buses, are considerably lower than the costs of the current system; (b) these cost reductions allow government authorities to introduce an important number of modernizing measures without subsidies and fare increases. [Copyright &y& Elsevier]

• FXII, HK, prekallikrein, sgp120 and C1-INH are cleaved during cold activation. • FXII and kallikrein increase their function with cold activation, while C1-INH decreases. • Contact system is activated in HAEnCI or in estrogen samples when incubated in cold. • FXII is necessary for cold induced contact system activation. • Kallikrein appears to play a major role in cold activation. Hereditary angioedema (HAE) attacks are caused by excessive activation of the contact system. Understanding how the contact system is activated in HAE, especially in patients with normal C1 inhibitor (HAEnCI), is essential to effectively treat this disease. Contact system activation involves the cleavage of several proteins including Factor XII (FXII), high molecular weight kininogen (HK), prekallikrein, sgp120 (ITIH4) and C1 inhibitor (C1-INH) before the subsequent generation of bradykinin that mediates HAE. In this study, we evaluated the fragmentation and enzymatic activity of contact system proteins in HAEnCI plasma samples before and after contact system activation induced by incubation in the cold. Our results show that in contrast to normal plasma, cold activation induced contact system activation in the majority of the HAEnCI patient samples we tested, in which each contact system protein exhibited fragmentation, FXII and kallikrein enzymatic activity increased, and C1-INH functional activity decreased. HAEnCI samples with low FXII concentrations or functional activity were not affected by cold activation. One HAEnCI sample with a plasminogen gene mutation activated the fibrinolytic system, as shown by an increase in concentration of plasma D dimers. Our results suggest that cold activation seems to be initiated by the cleavage of prekallikrein, and that it needs FXII in order to occur. Reported to be susceptible to excessive contact system activation after incubation in the cold, we further applied this system of study to the evaluation of plasma from women undergoing estrogen treatment. Similar to plasma from HAEnCI patients, excessive contact system activation was demonstrated. [ABSTRACT FROM AUTHOR]

• sgp120 has been identified as ITIH4, can be cleaved by kallikrein, and can be used to study contact system activation. • sgp120 fragmentation and spontaneous contact system activation is present in the plasma of a majority of HAE-C1inh patients. • sgp120 fragmentation and contact system activation occur in a majority of HAE-nl-C1inh samples after cold incubation. • The loss of C1 inhibitor function and C1 inhibitor fragmentation occur in HAE-nl-C1inh samples after cold incubation. • sgp120 (ITIH4) may be a potential biomarker for the laboratory diagnosis of HAE-nl-C1inh. Mutations in Factor XII, plasminogen gene, angiopoietin-1 gene and kininogen 1 gene have been found in some patients with hereditary angioedema with normal C1 inhibitor (HAE-nl-C1inh), but the underlying disease mechanisms remain unclear. Additionally, there are no accepted biomarkers for this disease. Because the contact system has been implicated in hereditary angioedema with C1 inhibitor deficiency (HAE-C1inh), we studied the fragmentation patterns of serum glycoprotein 120 (sgp120), a protein that is highly susceptible to cleavage by kallikrein, in 31 HAE-C1inh and 13 HAE-nl-C1inh patient plasma samples. Compared to normal controls, the majority of plasma samples from patients with HAE-C1inh contained fragmented sgp120. These samples also showed increased kallikrein amidolytic activity indicating spontaneous contact system activation. In contrast, most samples from HAE-nl-C1inh patients exhibited intact sgp120. However, if these samples were incubated at 4 °C in plastic, significant sgp120 fragmentation and spontaneous contact system activation were observed. Concurrently, there was C1 inhibitor fragmentation that generated the nonfunctional 94 kD fragment and a reduction in C1 inhibitor function. Normal samples did not show sgp120 or C1 inhibitor fragmentation after incubation. We sequenced sgp120 and found it to be identical to inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4). These results suggest that sgp120 or ITIH4 is cleaved when the contact system is activated and that this cleavage could be used as a biomarker in patients with HAE-nl-C1inh. [ABSTRACT FROM AUTHOR]

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALl), treated with PI-specific phospholipase C (PIPLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported HA degradation was mediated by reactive oxygen species (ROS) in human airways. Here we show that ROS increase Hyal2 expression and activity in NHBE cells and that the p38MAPK signaling pathway is involved in this effect. Hyal2 induction confirmed by using small interfering RNA (siRNA) expressing lentivirus. These in vitro findings correlated in vivo with smokers, where increased Hyal2 immunoreactivity in the epithelium was associated with augmented levels of HA and the appearance of low molecular mass HA species in bronchial secretions. summary, this work provides evidence that ROS induce Hyal2, suggesting that Hyal2 is likely responsible for the sustained HA fragmentation in the airway lumen observed in inflammatory conditions associated with oxidative stress. [ABSTRACT FROM AUTHOR]