Jeremy Allen

TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT)

BackgroundFibroblastic 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.MethodsA549 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.ResultsThe 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.ConclusionOur 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.

Th2 cytokines exert a dominant influence on epithelial cell expression of the major group human rhinovirus receptor, ICAM-1

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.

The omega-3 fatty acid, eicosapentaenoic acid (EPA), prevents the damaging effects of tumour necrosis factor (TNF)-alpha during murine skeletal muscle cell differentiation

BackgroundEicosapentaenoic acid (EPA) is a ώ-3 polyunsaturated fatty acid with anti-inflammatory and anti-cachetic properties that may have potential benefits with regards to skeletal muscle atrophy conditions where inflammation is present. It is also reported that pathologic levels of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α are associated with muscle wasting, exerted through inhibition of myogenic differentiation and enhanced apoptosis. These findings led us to hypothesize that EPA may have a protective effect against skeletal muscle damage induced by the actions of TNF-α.ResultsThe deleterious effects of TNF-α on C2C12 myogenesis were completely inhibited by co-treatment with EPA. Thus, EPA prevented the TNF-mediated loss of MyHC expression and significantly increased myogenic fusion (p < 0.05) and myotube diameter (p < 0.05) indices back to control levels. EPA protective activity was associated with blocking cell death pathways as EPA completely attenuated TNF-mediated increases in caspase-8 activity (p < 0.05) and cellular necrosis (p < 0.05) back to their respective control levels. EPA alone significantly reduced spontaneous apoptosis and necrosis of differentiating myotubes (p < 0.001 and p < 0.05, respectively). A 2 hour pre-treatment with EPA, prior to treatment with TNF alone, gave similar results.ConclusionIn conclusion, EPA has a protective action against the damaging effects of TNF-α on C2C12 myogenesis. These findings support further investigations of EPA as a potential therapeutic agent during skeletal muscle regeneration following injury.

Hepatic stellate cells and parasite-induced liver fibrosis.

Fibrogenesis is a common feature of many diseases where there is severe insult to the liver. The hepatic stellate cell trans-differentiation into a myofibroblast has been identified as an important event in liver fibrogenesis and has been well investigated over the last few years in a number of liver diseases. The trans-differentiation process can be monitored in vitro by evaluation of biomarkers that are characteristic of normal quiescent hepatic stellate cells or activated myofibroblasts. Two major parasitic diseases associated with liver injury and fibrosis are schistosomiasis and echinococcosis. Recent studies have highlighted a role for activated hepatic stellate cells in both murine and human schistosomiasis as well as demonstrating that schistosome antigens are able to regulate this trans-differentiation process. Study of the hepatic stellate cell and its interaction with parasite-derived antigens may be pivotal in our understanding of the pathology associated with schistosomiasis and other parasitic diseases, including echinococcosis, as well as revealing new information on the trans-differentiation process in this cell type.

Schistosoma mansoni: egg-induced downregulation of hepatic stellate cell activation and fibrogenesis.

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<0.001), connective tissue growth factor (p<0.01) and type I collagen (p<0.001) but significantly increased expression of peroxisome proliferator-activated receptor-gamma (p<0.01). Morphologically, HSC exhibited elongated fine cellular processes and reduced size, increased accumulation of lipid droplets and reduced expression and organization of alpha-smooth muscle actin and F-actin stress fibres. Additionally, schistosome eggs prevented the HSC fibrogenic response to exogenous transforming growth factor-beta. In summary, schistosome eggs blocked fibrogenesis in HSC, a finding which may have implications for our understanding of the fibrotic pathology in S. mansoni infections.

PPARγ as a molecular target of EPA anti-inflammatory activity during TNF-α-impaired skeletal muscle cell differentiation

Activated skeletal muscle satellite cells facilitate muscle repair or growth through proliferation, differentiation and fusion into new or existing myotubes. Elevated levels of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) impair this process and are documented to have significant roles in muscle pathology. Recent evidence shows that the ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) can block TNF-mediated suppression of progenitor cell differentiation, but the nature of this activity and its significance for local regulation of inflammation are not known. In the current study, we examined differentiation of the C2C12 myoblast line during treatment with TNF-α and EPA and measured the expression, activation and inhibition of peroxisome proliferator-activated receptor-γ (PPARγ), as several studies have shown its involvement in mediating EPA activity and the inhibition of nuclear factor (NF)-κB inflammatory gene activation. We found that TNF-α treatment increased NF-κB activity and reduced expression and activation of PPARγ, resulting in impaired myotube formation. EPA treatment attenuated these effects of TNF-α and was associated with up-regulation of PPARγ. Furthermore, EPA inhibited TNF-α-mediated transcription and secretion of interleukin (IL)-6, a key target gene of TNF-mediated NF-κB transcriptional activity. Pretreatment with a PPARγ selective antagonist inhibited some of the actions of EPA but was only partially effective in reversing inhibition of IL-6 production. These results show that EPA activity was associated with altered expression and activation of PPARγ, but exerted through both PPARγ-dependent and PPARγ-independent pathways leading to suppression of the proinflammatory cellular microenvironment.

A Role for Peroxisome Proliferator-Activated Receptors in the Immunopathology of Schistosomiasis?

Peroxisome proliferator-activated receptors (PPARs) have been demonstrated to have a role in immune regulation. In general, they are anti-inflammatory and promote Th2 type responses, and they are associated with the alternative activation of macrophages. Interestingly, helminth infections, such as the schistosome blood flukes that cause schistosomiasis, are characterised by a Th2 response and the accumulation of alternative activated macrophages. This would suggest that at some level, PPARs could have a role in the modulation of the immune response in schistosomiasis. This paper discusses possible areas where PPARs could have a role in this disease.

The differential hormonal milieu of morning versus evening, may have an impact on muscle hypertrophic potential

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 (insulin-like growth factor-binding protein-3 (IGFBP-3), myogenic index and differentiation) and catabolic 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 P≤0.05. As expected a significant reduction in evening cortisol concentration was observed at pre (AM: 98.4±10.5, PM: 49.8±4.4 ng/ml, P<0.001) and post (AM: 98.0±9.0, PM: 52.7±6.0 ng/ml, P<0.001) exercise. Interestingly, individual cortisol differences pre vs post exercise indicate a time-of-day effect (AM difference: -2±2.6%, PM difference: 14.0±6.7%, P = 0.03). A time-of-day related elevation in serum IGFBP-3 (AM: 3274.9 ± 345.2, PM: 3605.1 ± 367.5, p = 0.032) was also evident. Pre exercise myogenic index (AM: 8.0±0.6%, PM: 16.8±1.1%) and myotube width (AM: 48.0±3.0, PM: 71.6±1.9 μm) were significantly elevated (P<0.001) in the evening. Post exercise myogenic index was greater AM (11.5±1.6%) compared with PM (4.6±0.9%). No difference was observed in myotube width (AM: 48.5±1.5, PM: 47.8±1.8 μm) (P>0.05). 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.

Does hydatid cyst fluid from Echinococcus granulosus cysts have any effect on cells involved in fibrosis in cystic echinococcosis

Aims: Due to lack of available studies on the intestinal parasitic infection in children in Aswan Governorate, the present study is planned to monitor and associated risk factors of the intestinal parasitic parasites among urban and rural primary school children in Aswan Governorate. Study Design: A cross-sectional descriptive study. Place and Duration of Study: Tis study was carried out in Aswan Governorate during the period from October 2015 to March 2016. Methodology: A total of 300 stool samples were collected from children were included in the study. Teir ages arranged from 6 to 12 years, 170 of them were males 130 were females and examined by direct wet mount and formal ether concentration techniques and also stained with different stain for parasitic infections. Medical Sociodemographic data were collected by using a standardized questionnaire. Results: Te overall prevalence of intestinal parasitic infections was 31%; Single infection in 26% and mixed infection in 5%. Te most predominant protozoal parasites found was Entamoeba histolytica 8.3% followed by Giardia lamblia 3.7% and Cryptosporidium parvum 1.7% and commonest helminthic infection was Oxyuris 6.6% followed by H. nana 3% Ascaris lumbricoides 1%. Mixed infection was in form of Entrobius vermicularis plus Entamoeba histolytica in 23.4%, Entrobius vermicularis plus Giardia lamblia in 17.6%, Entrobius vermicularis plus Cryptospordium parvum in 11.8%, Entamoeba histolytica plus Hymenolepis nana in 11.85%, Ascaris lumbricoides plus Entamaobea histolytica in 17.6%, Giardia lamblia plus Entamoeba histolytica in 11.8%. Parasitic infection was more prevalent in rural areas 60% than urban 30%. Parasitic infection was more prevalent in boys 53.8% than girls 46.2%. Tere was signifcant increase of recurrent abdominal pain, dysentery, vomiting, diarrhea, peri-anal itching and pallor in infected students when compared to non infected students. Conclusion: Te present study highlighted on prevalence of intestinal parasitic infection in children in Aswan Governorate, Egypt and provided that, the intestinal parasitic infections are a public health problem and there is an urgent need for efforts towards ensuring adequate control of intestinal parasites in Aswan Governorate and preventive measured to be re-established especially in children of school age.Cystic echinococcosis is characterized by the presence of slow growing hydatid cysts, usually in the liver or lungs. Survival of the parasite is based on an interaction of the host immune system and a range of parasite immune-evasive strategies. Fibrosis in the tissues surrounding the cysts can be seen as a host protective response isolating the parasite and restricting its growth or from another perspective fibrosis may be protective for the parasite by providing a barrier to more effective immunological responses. In this study the adenocarcinomic human alveolar basal epithelial cell line (A549) was used as model system. This cell line can be involved in fibrosis as cells can transform into mesenchymal cells and differentiate later to fibroblasts and/or myofibroblasts which can ultimately secrete collagen. Cells were initially cultured in vitro in RPMI-1640 medium containing 1-10% hydatid cyst fluid (HCF). The possible effect of the parasite extracts on cell migration was investigated using a wound healing assay. The ability of HCF components to modify cell surface markers of mesenchymal transition was also investigated by fluorescence microscopy. Results showed that there was a dose-dependent increase in cell growth in the presence of cyst fluid after 5 days of culture. The migratory response of cells was also enhanced by the presence of HCF. Both the enhanced growth and migratory activity were still evident when the HCF had been boiled indicating that the components responsible were thermostable. Semi-purified extracts of a major HCF component, antigen B showed a similar high stimulatory effect similar to that of HCF. The fluorescence microscopy showed a significant expression in the fibronectin and E-cadherin cell markers in cells treated with HCF. These results indicate that components within HCF have a stimulatory effect in the possible enhancement of fibrosis.