BACKGROUND AND PURPOSEThe lung adenocarcinoma cell line, A549, undergoes epithelial-mesenchymal cell transition (EMT) in response to TGF-b. Glucocorticoids do not prevent the EMT response, but TGF-b induced resistance to the cytokine-regulatory action of glucocorticoids. We sought to characterize the impairment of glucocorticoid response in A549 cells. EXPERIMENTAL APPROACHA549 cells were exposed to TGF-b for up to 96 h before glucocorticoid treatment and challenge with IL-1a to assess glucocorticoid regulation of IL-6 and CXCL8 production. Nuclear localization of the glucocorticoid receptor a (GRa) was ascertained by immunofluorescence and Western blotting. Transactivation of the glucocorticoid response element (GRE) was measured with a transfected GRE-secreted human placental alkaline phosphatase reporter. KEY RESULTSTGF-b (40-400 pM) reduced the maximum inhibitory effect of dexamethasone on IL-1a-induced IL-6 and CXCL8 production. The impaired glucocorticoid response was detected with 4 h of TGF-b (40 pM) exposure (and 4 h IL-1a to induce CXCL8 expression) and therefore was not secondary to EMT, a process that requires longer incubation periods and higher concentrations of TGF-b. TGF-b also impaired dexamethasone regulation of granulocyte-macrophage colony-stimulating factor in thrombin-stimulated BEAS-2B epithelial cells. Impaired regulation of CXCL8 was associated with markedly reduced GRE transactivation and reduced induction of mRNA for IkBa, the glucocorticoid-inducible leucine zipper and the epithelial sodium channel (SCNN1A). The expression, cellular levels and nuclear localization of GRa were reduced by TGF-b. CONCLUSIONS AND IMPLICATIONSWe have identified mechanisms underlying the impairment of responses to glucocorticoids by TGF-b in the A549 and BEAS-2B cell lines. AbbreviationsEMT, epithelial-mesenchymal transition; GRa, glucocorticoid receptor a; GRE, glucocorticoid response element

BackgroundWe have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.MethodsGC transactivation was measured using a Glucocorticoid Response Element (GRE)–Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα–Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant.ResultsTGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.ConclusionsOur results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.

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Here, we investigated the prevalence of headache among adults in Jordan. The study was conducted from January 2007 to November 2008. A sample of 4,836 participants were permitted to complete a self-conducted screening questionnaire. As much as 82.3% of participants complained from headache at least once per year. 36.1% were tension-type headache and 59% of the participants had other family members who suffered from headache. Headaches affected everyday activities in 51.6% of the participants; 82.7% of participants did not seek medical attention for their headaches. Among those who used analgesics (75.6%), acetaminophen was the most common (91.43%). In conclusion, headache and overuse of analgesics were prevalent in a significant part of the society. Thus, there is a need to educate the public to ensure safe practices and to make the use and selling of analgesics more stringent.

Introduction Novel tobacco products require independent research to assess their safety. This study assessed the current literature for trials comparing levels of biomarkers of exposure (BoE) between conventional cigarettes (CC) and heat-not-burn (HNB) devices. Methods Ten databases were searched using terms including: “heat not burn,” “iqos,” “teeps,” “mrtp,” “tobacco heating,” and “glo” between January 1, 2010 and August 13, 2019. Randomized controlled trials (RCTs) assessing comparative BoE levels in humans using either CC or novel HNB devices were eligible. BoE were tabulated, and differences between the intervention and control groups were analyzed and combined using a random-effects meta-analysis. Results Ten nonblinded, RCTs were eligible, involving a total of 1766 participants. Studies regularly reported on 12 BoE (including nicotine). HNB devices assessed included the “IQOS” and “glo” devices and “precursor” (being developed) HNB devices. In comparison to CC, all 12 BoEs assessed were significantly lower for participants assigned to an HNB device. In comparison to smoking abstinence, HNB devices were statistically equivalent for eight BoEs and significantly elevated for four BoEs. Conclusions This review found that the potential for harm to humans is reduced when using HNB devices compared to CC as indicated by significant reductions in BoE levels. Whilst these results support tobacco manufacturer claims of improved safety, the small number of studies included, limited range of BoE assessed, and involvement of the tobacco industry necessitate further independent research to confirm the HNB devices as being a safer alternative to CC. Implications This study supports claims made by tobacco manufacturers on the improved safety of HNB tobacco devices in comparison to CC. These novel devices lead to reduced exposure to key biomarkers, which are linked to the health consequences attributed to tobacco use. This has strong implications for international public health as well as further research and policy development relating to the safety aspects and legalities of novel tobacco products.