Glucose >200 mg/dL during Continuous Glucose Monitoring Identifies Adult Patients at Risk for Development of Cystic Fibrosis Related Diabetes.

Rationale. Cystic fibrosis related diabetes (CFRD) is the most common comorbidity in patients with CF. In spite of increased screening, diagnosis, and treatment of CFRD, the mortality rate in patients with CFRD still far exceeds the mortality rate in those without CFRD. Guidelines suggest that screening for CFRD be performed annually using the 2-hour 75-gram oral glucose tolerance test (OGTT). Adherence to recommended screening has been poor, with only approximately one-quarter of adults with CF undergoing OGTT in 2014. Use of continuous glucose monitoring (CGM) for diagnosis may become an alternative. Objectives. Our objective was to determine whether abnormal CGM predicts subsequent development of CFRD, lung function, and body mass index (BMI) decline and increased rate of CF pulmonary exacerbations in adults with CF. Methods. In a prospective single center pilot trial from September 2009 to September 2010, 21 adult patients due for routine OGTT were recruited to complete simultaneous 3-day CGM and 2-hour 75 gram OGTT. Subsequently, clinical information was reviewed from 2008 to 2015. Conclusions. There was a moderate correlation between interpreted results of 2-hour OGTT and CGM (p = 0.03); CGM indicated a greater level of glucose impairment than OGTT. Glucose >200 mg/dL by CGM predicted development of CFRD (p = 0.0002).

miR-19-Mediated Inhibition of Transglutaminase-2 Leads to Enhanced Invasion and Metastasis in Colorectal Cancer.

UNLABELLED: Transglutaminase-2 (TG2) is a critical cross-linking enzyme in the extracellular matrix (ECM) and tumor microenvironment (TME). Although its expression has been linked to colorectal cancer, its functional role in the processes that drive disease appears to be context dependent. There is now considerable evidence of a role for microRNAs (miRNA) in the development and progression of cancer, including metastasis. A cell model of metastatic colon adenocarcinoma was used to investigate the contribution of miRNAs to the differential expression of TG2, and functional effects on inflammatory and invasive behavior. The impact of TG2 in colorectal cancer was analyzed in human colorectal tumor specimens and by manipulations in SW480 and SW620 cells. Effects on invasive behavior were measured using Transwell invasion assays, and cytokine production was assessed by ELISA. TG2 was identified as a target for miR-19 by in silico analysis, which was confirmed experimentally. Functional effects were evaluated by overexpression of pre-miR-19a in SW480 cells. Expression of TG2 correlated inversely with invasive behavior, with knockdown in SW480 cells leading to enhanced invasion, and overexpression in SW620 cells the opposite. TG2 expression was observed in colorectal cancer primary tumors but lost in liver metastases. Finally, miR-19 overexpression and subsequent decreased TG2 expression was linked to chromosome-13 amplification events, leading to altered invasive behavior in colorectal cancer cells. IMPLICATIONS: Chromosome-13 amplification in advanced colorectal cancer contributes to invasion and metastasis by upregulating miR-19, which targets TG2.

Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.

The oncogene microRNA-21 (miRNA; miR-21) is overexpressed in most solid organ tumours; however, a recent examination of stage II colorectal cancer (CRC) specimens suggests this may be a stromal phenomenon and not only a feature of cancer cells. In vitro and in vivo studies show that miR-21 has potent pro-metastatic effects in various malignant carcinoma cell lines. The tumour microenvironment has also been identified as a key actor during the metastatic cascade; however to date the significance of deregulated miR-21 expression within the cancer-associated stroma has not been examined. In the present study, a quantitative RT-PCR-based analysis of laser microdissected tissue confirmed that miR-21 expression is associated with a four-fold mean increase in CRC stroma compared with normal tissue. In situ hybridisation using locked nucleic acid probes localised miR-21 expression predominantly to fibroblasts within tumour-associated stroma. To study the molecular and biological impact of deregulated stromal miR-21 in CRC, stable ectopic expression was induced in immortalised fibroblasts. This resulted in upregulated α-smooth muscle actin expression implying miR-21 overexpression is driving the fibroblast-to-myofibroblast transdifferentiation. Conditioned medium from miR-21-overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. 3D organotypic co-cultures containing fibroblasts and CRC cells revealed that ectopic stromal miR-21 expression was associated with increased epithelial invasiveness. Reversion-inducing cysteine-rich protein with kazal motifs, an inhibitor of matrix-remodelling enzyme MMP2, was significantly downregulated by ectopic miR-21 in established and primary colorectal fibroblasts with a reciprocal rise in MMP2 activity. Inhibition of MMP2 abrogated the invasion-promoting effects of ectopic miR-21. This data, which characterises a novel pro-metastatic mechanism mediated by miR-21 in the CRC stroma, highlights the importance of miRNA deregulation within the tumour microenvironment and identifies a potential application for stromal miRNAs as biomarkers in cancer.

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

BACKGROUND AND AIMS: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD). METHODS: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. RESULTS: TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. CONCLUSIONS: Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

CC-10004 but not thalidomide or lenalidomide inhibits lamina propria mononuclear cell TNF-α and MMP-3 production in patients with inflammatory bowel disease.

BACKGROUND: Thalidomide, one of whose activities is to inhibit Tumour Necrosis Factor (TNF)-α production, has been reported to be an effective treatment for refractory inflammatory bowel disease (IBD). TNF-α driven production of matrix metalloproteinase (MMP)-3 by gut lamina propria mononuclear cells (LPMCs) is a major pathway of tissue injury in IBD; however the effect of thalidomide and newer more potent immunomodulatory derivatives on this pathway has not been studied. AIM: To investigate the effect of thalidomide, CC-4047 (pomalidomide), CC-5013 (lenalidomide), and CC-10004 (apremilast) on gut LPMC TNFα and MMP-3 production in patients with IBD. METHODS: Gut LPMCs and myofibroblasts were isolated from patients with IBD, and cultured with thalidomide, CC-4047, CC-5013, and CC-10004. MMP-3 and TIMP-1 levels were determined by western blotting and real-time PCR, and TNF-α levels by ELISA. RESULTS: CC-10004 significantly reduced both TNF-α production and MMP-3 production by cultured LPMCs. Thalidomide and CC-4047 and CC-5013 had no significant effect on the production of TNF-α or MMP-3 by LPMCs. CONCLUSION: These results provides a mechanistic rationale for both the failure of lenalidomide (CC-5013) in a recent randomised controlled trial in Crohn's disease, and for the evaluation of CC-10004 as a novel oral therapy in the treatment of CD and UC.

MicroRNAs: key players in carcinogenesis and novel therapeutic targets.

MicroRNAs (miRNAs) represent a recently uncovered class of small and endogenous non-coding RNAs. MiRNA function is critical to normal cellular processes such as differentiation and apoptosis, and recent studies have demonstrated that deregulated miRNA expression contributes to the malignant phenotype. The purpose of this review is to summarise these findings in relation to the most common human malignancies, and to analyse the clinical and therapeutic opportunities they provide.

Blockade of transforming growth factor beta upregulates T-box transcription factor T-bet, and increases T helper cell type 1 cytokine and matrix metalloproteinase-3 production in the human gut mucosa.

BACKGROUND AND AIMS: The role of transforming growth factor beta (TGFbeta) in inhibiting T cell function in the normal gut has been studied in animal models. However, the impact of TGFbeta inhibition on T cells in the normal human gut remains poorly understood. The effect of TGFbeta blockade in normal intestinal biopsies grown ex vivo and lamina propria mononuclear cells (LPMCs) on T-bet, a T-box transcription factor required for T helper cell type (Th)1 differentiation, interferon gamma (IFN gamma) production, T cell apoptosis and matrix metalloproteinase (MMP)-3 production has therefore been tested. METHODS: TGFbeta transcripts were determined by quantitative reverse transcription-PCR in laser-captured gut epithelium and lamina propria. Biopsies and LPMCs were cultured with anti-TGFbeta neutralising antibody. After 24 h culture, T-bet was determined by immunoblotting, and T cell apoptosis was assessed by flow cytometry. IFN gamma, tumour necrosis factor alpha (TNFalpha), interleukin (IL) 2, IL6, IL8, IL10, IL12p70 and IL17 were measured by ELISA. MMP-3 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 were assessed by immunoblotting. RESULTS: A higher number of TGFbeta transcripts was found in the lamina propria than in the epithelium in normal gut. T-bet expression was significantly higher in biopsies and LPMCs cultured with anti-TGFbeta antibody than in those cultured with control antibody. TGFbeta blockade downregulated T cell apoptosis, and induced a significant increase in IFN gamma, TNFalpha, IL2, IL6, IL8 and IL17 production. A higher expression of MMP-3, but not TIMP-1, was observed in the tissue and supernatant of biopsies treated with anti-TGFbeta antibody. CONCLUSIONS: The findings support a crucial role for TGFbeta in dampening T cell-mediated tissue-damaging responses in the human gut.

Selenoprotein expression in endothelial cells from different human vasculature and species.

Selenium (Se) can protect endothelial cells (EC) from oxidative damage by altering the expression of selenoproteins with antioxidant function such as cytoplasmic glutathione peroxidase (cyGPX), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and thioredoxin reductase (TR). If the role of Se on EC function is to be studied, it is essential that a model system be chosen which reflects selenoprotein expression in human EC derived from vessels prone to developing atheroma. We have used [75Se]-selenite labelling and selenoenzyme measurements to compare the selenoproteins expressed by cultures of EC isolated from different human vasculature with EC bovine and porcine aorta. Only small differences were observed in selenoprotein expression and activity in EC originating from human coronary artery, human umbilical vein (HUVEC), human umbilical artery and the human EC line EAhy926. The selenoprotein profile in HUVEC was consistent over eight passages and HUVEC isolated from four cords also showed little variability. In contrast, EC isolated from pig and bovine aorta showed marked differences in selenoprotein expression when compared to human cells. This study firmly establishes the suitability and consistency of using HUVEC (and possibly the human cell line EAhy926) as a model to study the effects of Se on EC function in relation to atheroma development in the coronary artery. Bovine or porcine EC appear to be an inappropriate model.

Selenite protects human endothelial cells from oxidative damage and induces thioredoxin reductase.

The ability of selenium to protect cultured human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) from oxidative damage induced by 100 microM t-butyl hydroperoxide (t-BuOOH) was compared. Preincubation of human endothelial cells for 24 h with sodium selenite at concentrations as low as 5 nM provided significant protection against the harmful effects of 100 microM t-BuOOH, with complete protection being achieved with 40 nM selenite. The preincubation period was required for selenite to exert this protective effect on endothelial cells. When compared with selenium-deficient cells, the activities of cytoplasmic glutathione peroxidase (GPX-1), phospholipid hydroperoxide glutathione peroxidase (GPX-4) and thioredoxin reductase (TR) were each induced approx. 3--4-fold by 40 nM selenite. HCAEC and HUVEC showed great similarity in their relative abilities to resist oxidative damage in the presence and absence of selenite, and the activities of TR and the GPXs were also similar in these cell types. BAEC were more susceptible to damage by 100 microM t-BuOOH than were human endothelial cells, and could not be protected completely by incubation with selenite at concentrations up to 160 nM. The activity of TR in human endothelial cells was approx. 25-fold greater than that in BAEC of a similar selenium status, but GPX-1 and GPX-4 activities were not significantly different between the human and bovine cells. These studies, although performed with a small number of cultures, show for the first time that selenium at low doses can provide significant protection of the human coronary artery endothelium against damage by oxidative stress. TR may be an important antioxidant selenoprotein in this regard, in addition to the GPXs. The data also suggest that HUVEC, but not BAEC, represent a suitable model system in which to study the effects of selenium on the endothelium of human coronary arteries.

Effects of organic and inorganic selenium supplementation on selenoenzyme activity in blood lymphocytes, granulocytes, platelets and erythrocytes.

The blood selenium (Se) concentration in the U.K. population has declined by approx. 50% between 1974 and 1991, reflecting a large decrease in dietary Se supply, with intakes only half the reference nutrient intake of 1 microg/kg body weight. Tissue levels of Se are readily influenced by dietary intake. Therefore selenoprotein activity may be sub-optimal due to low Se status, and thus compromise normal cell function. To examine the effects of changing Se intake on selenoproteins, we have determined the relative effectiveness of organic selenomethionine and inorganic sodium selenite (50 microg of Se daily for 28 days) in modulating glutathione peroxidase activities in blood cells from 45 healthy men and women, from a U.K. population. Transient and acute changes in lymphocyte, granulocyte and platelet phospholipid-hydroperoxide glutathione peroxidase (GPx4) activity occurred by day 7 or 14 of sodium selenite treatment and by day 7 in lymphocytes from selenomethionine-treated subjects compared with controls taking a placebo. In contrast, GPx4 activity in granulocytes and platelets in the selenomethionine group increased gradually over the 28 days. Cytosolic glutathione peroxidase (GPx1) activity in these blood cells from both treatment groups increased gradually over the 28 days. For each cellular selenoenzyme activity a significant inter-individual difference (P<0.001) in the extent of the response to Se supplementation was observed, but this was not related to blood Se concentrations either before or after treatments. Significant inverse correlations were evident between baseline enzyme activities and percentage change in activity after 28 days of supplementation [e.g. lymphocyte GPx4, r=-0.695 (P<0.001)], indicating that pre-treatment activity may be sub-optimal as a result of poor Se status. The different and contrasting effects that Se supplementation had on blood selenoenzyme activities may be indicative of a difference in metabolic need for Se regulated at the level of Se-dependent cell function.

The regulation of transaminative flux of methionine in rat liver mitochondria.

We have demonstrated methanethiol production from methionine in isolated rat liver mitochondria and shown how it is affected by other metabolites. The enzymes involved include several transaminases, branched chain 2-oxoacid dehydrogenase, acyl-CoA dehydrogenase, and crotonase. Methanethiol production from methionine in mitochondria isolated from rat liver was increased by 50% after the rats had been given a single injection of glucagon, but was reduced by 25% when the rats had been starved for 24 h. These results indicate the physiological importance of the transaminative pathway of methionine metabolism.

Methionine transamination--metabolic function and subcellular compartmentation.

Enzymatic activities catalysing the inter-conversion of L-methionine and its oxy analogue 4-methylthio-2-oxobutyric acid (2,4-KMB) were detected in the liver, skeletal muscle and heart of the laboratory rat and of sheep. In both species the highest activity of methionine transamination was found in the liver and was located in the cytoplasm and mitochondria. We propose that physiological and nutritional role of the cytoplasmic methionine transamination is amination of 2,4 KMB and formation of L-methionine while in mitochondria the activity is responsible for disposal of excess methionine is oxidised through oxidative decarboxylation of 2,4 KMB.

Determination of L-methionine-dl-sulphoxide in tissue extracts.

The amino acid fraction from rat liver, heart and skeletal muscle was prepared by the separation of sulphosalicylic acid extract on Dowex 50 H+ form. The presence of L-methionine-dl-sulphoxide in these extracts was identified and compared by three independent chromatographic methods: ion-exchange, Pico-Tag and reversed-phase high-performance liquid chromatography after precolumn derivatisation with diethylethoxymethylenemalonate. Quantitative data indicate that L-methionine-dl-sulphoxide is present in the intracellular pool at the levels of free methionine.

Urea synthesis in rats fed diet containing kidney beans.

When rats were fed a diet containing kidney bean (Phaesolus vulgaris) urea excretion was increased 3-5 fold. Isolated liver mitochondria from rats fed the kidney bean diet produced 40% more citrulline in the presence of arginine than mitochondria isolated from control rats. Mitochondrial activities of urea cycle enzymes and N-acetylglutamate synthetase were similar in animals fed diets containing kidney bean or lactalbumin. The possible mechanisms causing acute urea production in rats fed with kidney bean are discussed.

Differential visual field-interhemispheric transfer: can it explain sex and handedness differences in lateralization?

104 men and women were tested for visual field-hemispheric transfer of spatial information on a dot-localization task. Right-handed subjects showed significant improvement when stimuli were presented to the left visual field of the right hemisphere (LVF-RH) after practice on the same task presented to the right visual field of the left hemisphere (RVF-LH) first. No improvement was found when the task was presented in the reverse order (LVF-RH first followed by RVF-LH). It was concluded that, for right-handers, transfer of spatial information to the right hemisphere is facilitated while transfer to the left hemisphere is inhibited. Left-handed subjects demonstrated no significant improvement in either condition, suggesting inhibition or lack of transfer of spatial information in either direction. No sex differences were found in either right-handed or left-handed subjects. The findings suggest that there may be different mechanisms underlying the similarities in functional lateralization of women and left-handers.