Differential Expression Profiles of Oxidative Stress Levels, 8-oxo-dG and 4-HNE, in Barrett's Esophagus Compared to Esophageal Adenocarcinoma.

Barrett's esophagus (BE), a chronic inflammatory condition, is the leading risk factor for esophageal adenocarcinoma (EAC). In inflammation to cancer pathways, oxidative stress profiles have been linked to cancer progression. However, the relevance of oxidative stress profiles along the BE-disease sequence remains to be elucidated. In this study, markers of oxidative stress; DNA adducts (8-oxo-dG) and lipoperoxidation (4-HNE), and markers of proliferation (Ki67) were measured in patient biopsies representing the BE-disease sequence. Differences in expression of these markers in Barrett's patients with cancer-progression and non-progression were examined. Proliferation was reduced in Barrett's specialized intestinal metaplasia (SIM) compared with EAC (p < 0.035). Correcting for cell proliferation levels, a confounding factor, linked to oxidative stress profiles, SIM demonstrated increased levels of 8-oxo-dG and 4-HNE (p < 0.05) compared with EAC. Longitudinal analysis of Barrett's patients demonstrated decreased levels of 8-oxo-dG in SIM cancer progression (p < 0.05). BE is an environment of increased oxidative stress and inflammation. Patients with progressive disease demonstrated reduced oxidative stress levels in 8-oxo-dG. Perhaps these alterations facilitate Barrett's progression, whereas in non-progressive disease, cells follow the rules of increased oxidative stress ultimately triggers cell apoptosis, thereby preventing propagation and survival.

Deoxycholic acid promotes development of gastroesophageal reflux disease and Barrett's oesophagus by modulating integrin-αv trafficking.

The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barrett's oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET-1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α3 , α4, α5 , α6 and αν ). Experimental findings were validated in human explant oesophageal biopsies, a rat model of gastroesophageal reflux disease (GORD) and in patient tissue microarrays. The bile acid deoxycholic acid (DCA) specifically reduced adhesion of HET-1A cells to vitronectin and reduced cell-surface expression of integrin-αν via effects on endocytic recycling processes. Increased expression of integrin-αv was observed in ulcerated tissue in a rat model of GORD and in oesophagitis and Barrett's intestinal metaplasia patient tissue compared to normal squamous epithelium. Increased expression of integrin-αν was observed in QH BO cells compared to HET-1A cells. QH cells were resistant to DCA-mediated loss of adhesion and reduction in cell-surface expression of integrin-αν . We demonstrated that a specific component of the gastric refluxate, DCA, affects the epithelial barrier through modulation of integrin αν expression, providing a novel mechanism for bile acid-mediated erosion of oesophageal squamous epithelium and promotion of BO. Strategies aimed at preventing bile acid-mediated erosion should be considered in the clinical management of patients with GORD.

HLA-DR expression in tumor epithelium is an independent prognostic indicator in esophageal adenocarcinoma patients.

Esophageal adenocarcinoma (EAC) is an aggressive cancer with poor prognosis, and incidence is increasing rapidly in the Western world. Measurement of immune markers has been shown to have prognostic significance in a growing number of cancers, but whether this is true for EAC has yet to be evaluated. This study aimed to characterize HLA-DR expression in the esophagus across the inflammation to cancer progression sequence and to assess the prognostic significance of HLA-DR expression in EAC. Tissue microarrays (TMA) were constructed from esophageal tissue taken from patients at different stages in the cancer progression sequence; normal, esophagitis, Barrett's esophagus (BE), low- and high-grade dysplasia (LGD, HGD) and EAC. HLA-DR expression in tissue epithelium and stroma was assessed by immunohistochemistry. HLA-DR expression increased early in the inflammation to cancer progression sequence; with higher expression detected in esophagitis and BE compared to normal tissue. Patients with low (<50%) HLA-DR expression in the EAC tumor epithelium had significantly worse survival outcomes, compared to those with high expression, in both the tumor core (hazard ratio, HR = 2.178, p = 0.024, n = 70) and leading edge (HR = 2.86, p = 0.013, n = 41). Multivariate analysis demonstrated that low HLA-DR expression in leading edge tumor epithelium was an independent predictor of poor survival, associated with a 2.8-fold increase in disease-associated death (p = 0.023). This study shows that HLA-DR is an independent prognostic marker in EAC tumor epithelium. This may have implications for patient stratification strategies as well as EAC tumor immunology.

High-mobility group box protein 1: a novel mediator of inflammatory-induced renal epithelial-mesenchymal transition.

BACKGROUND: high-mobility group box protein 1 (HMGB-1) is a chromatin-binding protein that bends DNA, thereby facilitating gene transcription. HMGB-1 has also been observed as an extracellular secreted protein in serum of patients with sepsis and has putative intracellular signalling effects regulating the production of interleukin-1 and tumour necrosis factor in a number of inflammatory conditions. METHODS: we established a model of immune-mediated epithelial-mesenchymal transition (EMT) in human proximal tubular epithelial cells (PTECs). PTECs were cultured with conditioned medium containing supernatant from activated peripheral blood mononuclear cells (aPBMCs). The model was characterized using phenotypic and transcriptomic approaches and suppression subtractive hybridisation was performed to identify differentially regulated genes. RESULTS: activation of PBMCs resulted in increased secretion of HMGB-1. In addition, treatment of PTECs with aPBMC-conditioned medium resulted in significant upregulation of HMGB-1 in PTECs. Direct treatment of PTECs with recombinant human HMGB-1 induced alterations in epithelial morphology consistent with EMT including reduced E-cadherin expression, increased α-smooth muscle actin expression and enhanced cell migration. HMGB-1 effects were mediated at least in part by the receptor for advanced glycation end products and through induction of transforming growth factor-ß(1) secretion from PTECs. CONCLUSIONS: these results suggest that HMGB-1 is a key mediator of immune-mediated EMT of PTECs and a potentially important signalling molecule in the development of renal fibrosis.

Understanding and Attitudes toward Cancer Clinical Trials among Patients with a Cancer Diagnosis: National Study through Cancer Trials Ireland.

Cancer clinical trials (CCTs) are critical to translation and development of better therapies to improve outcomes. CCTs require adequate patient involvement but accrual rates are low globally. Several known barriers impede participation and knowing how subpopulations differ in understanding of CCTs can foster targeted approaches to aid accrual and advance cancer treatments. We conducted the first nationwide survey of 1089 patients attending 14 Irish cancer centres, assessing understanding of fundamental concepts in CCT methodology and factors that influence participation, to help tailor patient support for accrual to CCTs. Two-thirds (66%) of patients reported never having been offered a CCT and only 5% of those not offered asked to participate. Misunderstanding of clinical equipoise was prevalent. There were differences in understanding of randomisation of treatment by age (p < 0.0001), ethnicity (p = 0.035) and marital status (p = 0.013), and 58% of patients and 61% previous CCT participants thought that their doctor would ensure better treatment in CCTs. Females were slightly more risk averse. Males indicated a greater willingness to participate in novel drug trials (p = 0.001, p = 0.003). The study identified disparities in several demographics; older, widowed, living in provincial small towns and fewer years-educated patients had generally poorer understanding of CCTs, highlighting requirements for targeted support in these groups.

Altered T Cell Migratory Capacity in the Progression from Barrett Oesophagus to Oesophageal Adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is an inflammation-driven cancer with poor prognosis and incidence is increasing rapidly. OAC arises from gastro-oesophageal reflux disease (GORD) and reflux-induced Barrett oesophagus (BO). The role of T cells in this disease progression is not yet fully understood. We have previously demonstrated higher proportions of pro-tumour Th2 cells in BO tissue, implicating them in its pathogenesis. While a Th2 immune profile is thought to underlie the metaplastic transformation in BO and promote OAC development, our studies suggest that the abundance of Th2 cells in BO tissue is likely to occur through altered T cell recruitment. This study examined the chemokine networks governing T cell migration to oesophageal tissue during disease progression. Here, we have identified that circulating T cells in OAC patients, exhibit impaired migratory capacity with decreased frequencies of Th1-associated CXCR3+ and Th17-associated CCR6+ cells. Despite the abundance of Th1 chemokines RANTES (CCL5) and MIP-1α (CCL3) in OAC tumour, enrichments of intratumoural T cells expressing corresponding receptors were not observed. These data suggest that T cell infiltration of oesophageal tissue is compromised in OAC and suggest that future therapies targeting T cell trafficking should occur at the pre-neoplastic stage. This is supported by the finding that antagonism of Th2-biased CCR4 significantly reduces T cell migration in BO but not OAC patients. Since we have previously reported a predominant Th2 immune profile in BO, we suggest that chemokine receptor antagonism may be a viable treatment option to alleviate Th2-predominance in BO and interrupt progression to OAC.

Role of Rho-kinase in guinea-pig gallbladder smooth muscle contraction.

Guinea-pig gallbladder smooth muscle contractions can be elicited pharmacologically by a range of mechanisms. The involvement of Rho-kinase in contractions mediated by receptor-dependent and receptor-independent mechanisms was investigated using the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632). In a separate series of experiments, the role of Rho-kinase in the contractile response to Ca2+ entry through store-operated Ca2+ channels and to electrical field stimulation was also examined. Y-27632 (10 microM), which caused a significant decrease (P<0.0005) in basal resting tone, significantly inhibited gallbladder contractions evoked by cumulative additions of the G-protein-coupled agonists, carbachol (1 nM-100 microM; P<0.05) and cholecystokinin (10 nM-1 microM; P<0.005). Y-27632 also inhibited the contractions evoked by a single addition of the sarcoplasmic reticulum ATPase inhibitor, thapsigargin (1 microM; P<0.0005) and cumulative additions of KCl (10-85 mM; P<0.0005). The contractile response to Ca2+ entry through store-operated Ca2+ channels was significantly inhibited by Y-27632 (P<0.05) as were the contractile responses evoked by electrical field stimulation (2-25 Hz; P<0.0005). In contrast, Y-27632 had no significant effect on contractions evoked by phorbol 12,13-dibutyrate (0.1 nM-1 microM; a protein kinase C activator) or by the phosphatase inhibitor, cantharidin (100 microM). In conclusion, Rho-kinase contributes to the contractile response in guinea-pig gallbladder smooth muscle evoked by both G-protein-coupled and non-G-protein-coupled mechanisms in addition to contributing to the maintenance of basal tone. It also contributes to the contractile responses resulting from electrical field stimulation and store-operated Ca2+ channel entry.

Impact of the inflammatory microenvironment on T-cell phenotype in the progression from reflux oesophagitis to Barrett oesophagus and oesophageal adenocarcinoma.

The incidence of oesophageal adenocarcinoma (OAC), arising from reflux-induced Barrett oesophagus (BO), is increasing dramatically. T-cells have recently been implicated in the initiation of oesophagitis; however, their role in the progression from oesophagitis to BO and OAC has not been fully elucidated. Previous studies have examined the secreted cytokines from oesophageal tissue during disease progression but this study is the first to examine the activation phenotype and the inflammatory profile of CD4(+) and CD8(+) T-cells in human oesophagitis, BO and OAC tissue. Results demonstrated significantly higher levels of IL-4 producing CD4(+) T-cells and secreted levels of IL-6, confirming a Th2 phenotype in BO. In OAC tissue, both pro- and anti-inflammatory cytokines were secreted, with significantly higher levels of IL-6, IL-1ß, TNF-α, IFN-γ, IL-2 and IL-10 compared with normal oesophageal tissue. In addition, CD4(+) T-cells infiltrating OAC tissue displayed a decreased activation profile, with significantly lower CD45RO and CD69 expression compared with normal tissue. Data from this study suggest that factors in the tissue microenvironment may alter T-cell phenotype and function early during oesophageal disease progression and may represent targets for immune intervention.

A proteomic approach to immune-mediated epithelial-mesenchymal transition.

Increasing evidence suggests epithelial-mesenchymal transition (EMT) plays an important role in renal fibrosis. Initial renal injury enables the infiltration of mononuclear cells into the interstitium, and the resulting generation of inflammatory mediators that favour EMT may have a direct role in the development of renal fibrosis. The aim of this study was to investigate the proteome of renal tubular epithelial cells undergoing EMT in vitro. The human proximal tubular cell line (HK-2), exposed to conditioned medium from activated peripheral blood mononuclear cells (PBMC-CM), undergo phenotypic change, from an epithelial towards a fibroblastic phenotype, as evidenced by decreased E-cadherin and increased fibronectin protein expression. Further proteomic analysis, using 2-DE and Progenesis software, revealed the down-regulation of 4 proteins and up-regulation of 23 proteins. MS analysis allowed the positive identification of 15 differentially expressed proteins, including annexin A2, adipocyte plasma membrane-associated protein, T-complex protein 1, reticulocalbin-1 precursor and moesin among others. Western blotting and quantitative real-time PCR confirmed the increase in annexin A2 at the protein and gene level, respectively. Since annexin A2 and S100A6 exist as complexes in B-1 cells, we investigated the S100A6 gene expression further and show an increased expression in HK-2 cells following exposure to activated PBMC-CM. Therefore, we have identified several potential proteins that could play key roles in immune-mediated EMT.