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Aims\ud Cystic Echinococcosis is characterised by fluid filled hydatid cysts in the liver and lungs. The cysts are surrounded by a host fibrous layer (the pericyst) which acts to isolate the parasite from surrounding tissues. Previous studies in liver cysts have indicated that the parasite may be stimulating fibrosis. The aim of this study was to investigate whether Hydatid Cyst Fluid (HCF) could influence the potential for fibrosis to occur in lung tissue by stimulating epithelial to mesenchymal transition (EMT) in a human lung epithelial cell line.\ud Methods and Results\ud An adenocarcinoma-derived alveolar basal epithelial cell line (A549) was used as a model for human alveolar epithelial cells (AEC II). These were cultured in vitro with HCF (UK sheep origin). Assays to investigate cell proliferation, cell migration and expression of cytoskeletal markers showed that HCF could stimulate changes indicative of EMT, including enhanced cell proliferation and migration; increased expression of mesenchymal cytoskeletal markers (fibronectin and vimentin) accompanied by a down regulation of an epithelial marker (E-cadherin). \ud Conclusions\ud Molecules within hydatid cyst fluid are capable of inducing phenotypic changes in A549 cells indicating that the parasite has the potential to modify lung epithelial cells which could contribute to fibrotic reactions.

Reverse transcription PCR showed that mRNA encoding the neurohormones growth hormone-releasing factor (GRF) and GH, and its receptor GH-R, together with IGF-1 splice variants and IGFBPs are expressed by inflammatory cells found in the normal human airway. Unfractionated BALC moderately express GRF, GH and GH-R, IGFBP-2 to IGFBP-6, and IGFBP-rPl. In addition, BALC preferentially express the class 1 IGF-1Ea splice variant of the IGF-1 gene. A similar pattern of expression occurs in purified AM, except they do not appear to express GH-R. In marked contrast, AM precursor peripheral blood monocytes, do not express neuropeptides or IGF-1 and only express IGFBP-1, -4 and -6 and IGFBP-rP1. These data suggest that normal human inflammatory airway cells possess a powerful array of neurohormones and IGFBPs that are available for modulating local IGF-1 bioavailability in the lung.

Intercellular adhesion molecule (ICAM)-1 is a cell receptor important in both human rhinovirus (HRV) attachment and immune effector cell mobilization. The level of expression of ICAM-1 by epithelial cells (EC) therefore plays a crucial role in the intricate biological phenomena underlying viral binding, host infection and consequent inflammatory events. As T-helper (Th)2 lymphocytes predominate within the asthmatic airway, the influence was evaluated of Th2-associated mediators in the modulation of ICAM-1 expression on uninfected and HRV-infected EC. H292 EC were cultured in vitro, with varying concentrations of interleukin (IL)-4, IL-5, IL-10 and IL-13 for 24 h and then infected with live HRV-14. Surface ICAM-1 expression was assessed by immunocytochemistry. Infection with HRV-14 resulted in a twofold increase in ICAM-1 expression. IL-4, IL-5, IL-10 and IL-13 produced a 2.7-5.1-fold enhancement of ICAM-1 expression of uninfected cells and caused approximately a further twofold increase in infected cells over the expression induced by HRV infection itself. Interferon-gamma in combination with each Th2-associated cytokine only slightly reduced, but did not override, the Th2-induced level of ICAM-1 expression on both uninfected and virus-infected EC. These data suggest that the effects of Th2-associated cytokines on intercellular adhesion molecule-1 expression and recovery of infectious virus are dominant over the effects of the Th1-associated cytokines such as interferon-gamma. Since the airway mucosa in atopic asthma is predominantly infiltrated by Th2 lymphocytes, these results could explain both the increased susceptibility to human rhinovirus infection in asthmatic patients and the associated exacerbation of asthma symptoms.

Pulmonary sarcoidosis involves development of parenchymal granulomata that usually resolve spontaneously; however, it remains unclear what pathogenic mechanisms are responsible for the progression to local or diffuse fibrosis with irreversible lung remodeling that occurs in 20% of patients. Alveolar macrophages have a pivotal role in sarcoidosis, releasing mediators including insulin-like growth factor (IGF)-1, a potent profibrogenic molecule. IGF-1 bioavailability in the lung is dependent on at least six high-affinity IGF-binding proteins (IGFBP), which mainly inhibit IGF-1 action. We have investigated their presence in patients with established stage III sarcoidosis to determine whether IGF-1 and IGFBP contribute to the fibrogenic process in these patients and as such contribute to the (clinical) progression of the disease. The fibroblast mitogenic potential of bronchoalveolar lavage fluid (BALF) was more than 3-fold higher (P < 0.005) in sarcoid patients. Sarcoid BALF-induced activity could be inhibited (P < 0.0005) by neutralizing antibodies to IGF-1. We established the IGFBP profile of BALF with Western ligand analysis and quantified expression of IGFBP-3 by immunoblotting. IGFBP-2 and IGFBP-4 predominate in normal and sarcoid BALF, but IGFBP-3 occurs only as a modified, smaller, 29-kD form, expression of which was raised (P < 0.003) in sarcoid patients. Gene expression of IGF-1 and IGFBP-3 was demonstrated by reverse transcription-polymerase chain reaction in BAL cells. Thus, local production of pro-fibrogenic IGF-1 may be subject to substantial post-translational regulation by associated IGFBP and IGFBP proteases that may contribute to enhanced fibrogenesis in sarcoidosis patients with evidence of progression or (development) of fibrosis.

SUMMARY Human rhinoviruses (HRV) are a major cause of upper respiratory tract infections in man, and can exacerbate existing pulmonary disease. The major group of HRV attach to ICAM-1, which is expressed on nasal and bronchial epithelial cells. To study the influence of biological mediators on ICAM-1 expression, and consequently HRV attachment and infection, we compared the effects of various cytokines, alone and in combination, on ICAM-1 expression by an uninfected and HRV-infected bronchial epithelial cell line H292. Cytokines known to be released soon after viral infection, such as tumour necrosis factor-alpha (TNF-α), IL-1β and the chemokine IL-8 increase ICAM-1 expression on uninfected cells. Epithelial cells infected with live HRV-14 displayed marked up-regulation of ICAM-1 compared with baseline. TNF-α further enhanced the HRV-induced increase in ICAM-1 expression on epithelial cells, peaking at day 4 after infection, whilst IL-8 exhibited a steady increase in ICAM-1 expression over 14 days. In contrast, IFN-γ, a known Th1 antiviral lymphokine, whilst increasing the level of ICAM-1 on uninfected cells, induced a significant persistent down-regulation of ICAM-1 expression on HRV-infected epithelial cells. With combinations of TNF-α and IFN-γ, ICAM-1 expression on HRV-infected cells was reduced to basal levels. The effects of IFN-γ were paralleled by a reduction in viral titres. Our in vitro model has provided useful insights into the early pathogenic events of HRV infection at the level of the host cell–virus interaction. Our data confirm that biological mediators play a crucial role in the pathogenesis as well as the course of HRV infection which is modulated by the types, and time kinetics of inflammatory cytokines in the immediate microenvironment.

Substantial gains in muscle strength and hypertrophy are clearly associated with the routine performance of resistance training. What is less evident is the optimal timing of the resistance training stimulus to elicit these significant functional and structural skeletal muscle changes. Therefore, this investigation determined the impact of a single bout of resistance training performed either in the morning or evening upon acute anabolic signalling (insulinlike growth factor-binding protein-3 (IGFBP-3), myogenic index and differentiation) and cat-abolic processes (cortisol). Twenty-four male participants (age 21.4±1.9yrs, mass 83.7 ±13.7kg) with no sustained resistance training experience were allocated to a resistance exercise group (REP). Sixteen of the 24 participants were randomly selected to perform an additional non-exercising control group (CP) protocol. REP performed two bouts of resistance exercise (80% 1RM) in the morning (AM: 0800 hrs) and evening (PM: 1800 hrs), with the sessions separated by a minimum of 72 hours. Venous blood was collected immediately prior to, and 5 min after, each resistance exercise and control sessions. Serum cortisol and IGFBP-3 levels, myogenic index, myotube width, were determined at each sampling period. All data are reported as mean ± SEM, statistical significance was set at P0.05). Timing of resistance training regimen in the evening appears to augment some markers of hypertrophic potential, with elevated IGFBP-3, suppressed cortisol and a superior cellular environment. Further investigation, to further elucidate the time course of peak anabolic signalling in morning vs evening training conditions, are timely.

Eggs of Schistosoma mansoni trapped in human liver can lead to fibrosis. Since liver fibrosis requires activation of hepatic stellate cells (HSC) from a quiescent to a myofibroblastic phenotype, we investigated the effects of S. mansoni eggs on this process using in vitro co-cultures with human HSC and evaluated established biomarkers for activation and fibrosis. HSC demonstrate significantly reduced expression of alpha-smooth muscle actin (p

Background Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT. Methods A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA. Results The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes. Conclusion Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon.

Treatment of idiopathic pulmonary fibrosis patients has evolved very slowly; the fundamental approach of corticosteroids alone or in combination with other immunosuppressive agents has had little impact on long-term survival. The continued use of corticosteroids is justified because of the lack of a more effective alternative. Current research indicates that the mechanisms driving idiopathic pulmonary fibrosis reflect abnormal, dysregulated wound healing within the lung, involving increased activity and possibly exaggerated responses by a spectrum of profibrogenic growth factors. An understanding of the roles of these growth factors, and the way in which they modulate events at cellular level, could lead to more targeted therapeutic strategies, improving patients' quality of life and survival.

Insulin-like growth factor-1 (IGF-1) is a highly mitogenic polypeptide detectable in human lung. Using competitive reverse transcriptase/polymerase chain reaction (RT-PCR), expression of four IGF-1 transcripts was examined in bronchoalveolar lavage cells (BALC) from normal subjects, idiopathic pulmonary fibrosis (IPF), stage I/II (no fibrosis), and stage III/IV (confirmed fibrosis) pulmonary sarcoidosis patients, and fibroblast strains isolated from normal and IPF lungs. Transcripts studied were Class 1 and Class 2 (exons 1 or 2, respectively) with IGF-1Eb or IGF-1Ea (exons 5 or 6, respectively). Total IGF-1 expression was downregulated in BALC of both patients with IPF (p0.01) and patients with sarcoidosis (p0.04) compared with healthy subjects. In contrast, both constitutive (p0.003) and transforming growth factor-beta (TGF-beta)- induced (p0.02) IGF-1 expression was higher in fibrotic, compared with normal, fibroblasts. These changes were associated with differential expression of IGF-1 splice variants. Healthy subjects and sarcoidosis patients without fibrosis showed similar expression of Class 1/Class 2 and IGF-1Ea/IGF-1Eb. However, patients with fibrosis demonstrated discordant, increased relative abundance of Class 1 transcripts (p0.01). In parallel, all fibrosis patients failed to express Class 2, IGF-1Eb forms and sarcoidosis patients with fibrosis did not express the Class 1, IGF-1Eb variant either. Fibrotic fibroblasts expressed higher constitutive levels of Class 1, IGF-1Ea transcripts compared with normal fibroblasts. Class 2, IGF-1Eb forms were moderately expressed by fibroblasts only after stimulation with TGF-beta, which also further increased levels of Class 1, IGF-1Ea transcripts. Our findings suggest that transition from a healthy to a fibrotic phenotype occurs in association with a changing pattern of IGF-1 mRNA heterogeneity and leads us to hypothesize a potential role for specific IGF-1 variants in fibrogenesis.

SUMMARYSince clinical experimental studies indicate that upper respiratory tract viral infections may exacerbate acute asthma symptoms in atopic/asthmatic individuals, we have investigated the expression and modulation of ICAM-1 on human nasal epithelial cells (HNEC) from normal and atopic subjects. ICAM-1 is the attachment molecule for the majority of serotypes of human rhinovirus (HRV), including HRV-14, and is also critical for the migration and activation of immune effector cells. Basal ICAM-1 expression was significantly higher in HNEC obtained by brushings from atopic compared with non-atopic subjects (P = 0.031), and was also significantly increased on atopic HNEC harvested in season compared with out of season (P

Connective tissue growth factor is a recently described chemoattractant and fibroblast mitogen which, because of sequence homology and weak binding to insulin-like growth factor (IGF)-1, has been proposed as the eighth member of the IGF binding protein (IGFBP) superfamily, named IGFBP-related protein 2 (IGFBP-rP2). Previous studies have implicated IGFBP-rP2 in a number of heterogeneous fibrotic pathologies, including renal fibrosis, dermal scleroderma, and bleomycin-induced pulmonary fibrosis in mice. Because profibrogenic cytokines may be produced by inflammatory cells, we developed a multiplex competitive reverse transcription/polymerase chain reaction to quantify IGFBP-rP2 transcripts in bronchoalveolar lavage cells from healthy subjects and patients with idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis. IGFBP-rP2 messenger RNA expression was enhanced10-fold (P0.003) in patients with IPF;40-fold (P0.006) in stage I/II sarcoidosis patients, and90-fold (P0.005) in stage III/IV sarcoidosis patients by comparison with healthy nonsmoking control subjects. We suggest these increases are predominantly associated with lymphocyte- and neutrophil-driven IGFBP-rP2 production. These findings, together with previous reports implicating other IGFBPs in the pathogenesis of pulmonary fibrosis, suggest that the complex network of IGFBPs within the human lung is an important determinant of the outcome of the fibroproliferative response to injury.

In the lower atmosphere ozone is a toxic and an unwanted oxidising pollutant causing injury to the airway epithelial cells by lipid peroxidation to yield products such as phospholipid hydroperoxides (PLHP). Measurements of PLHP, which are primary oxidation products, may reflect an early susceptibility of the target cell to oxidative stress. Biphasic cultures of bronchial epithelial cells (BEAS-2B) were exposed to ozone at environmentally relevant concentrations (0.1-1.0 ppm) for 4 and 12 h. Detection of PLHP was made using a novel technique based on fourier transform infrared spectroscopy (FTIR) in combination with high performance thin-layer chromatography (HPTLC). Six phospholipids were identified on the HPTLC plate; lysophosphatidylcholine (LPC), sphingomyelin (SM), phosphatidylcholine (PC), lysophosphatidylethanolamine (LPE), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). From the FTIR spectra, O-O stretching of hydroperoxides was identified in the range 890-820cm(-1). Multivariate data analysis revealed a positive correlation (r = 0.99 for 4 h exposure and r = 0.98 for 12h exposure) between ozone exposure levels and the region of the FTIR-spectrum comprising the main wavelengths for hydroperoxides. These data support this alternative, versatile and novel spectroscopic approach for the early detection of ozone-mediated damage in human airway epithelial cells.