Effect of dietary status on plasma-fractionated metanephrines in healthy individuals measured by Elisa.

Pheochromocytoma is a rare tumor that typically originates in the adrenal glands, often causing the over-production of catecholamines. The aim of this study was to determine whether dietary status could affect the concentration of plasma-fractionated metanephrines. This study was conducted at the Chemical Pathology Laboratory, Royal Hospital, Oman. Three plasma samples were collected from each participant (16 male and 16 female) over three consecutive days (day one: dietary restriction,day two: excess intake of restricted foods, day three: random sample following the typical diet for each participant). Samples were collected and centrifuged to obtain the plasma, which was then stored at -20°C prior to analysis. Metanephrine and normetanephrine concentrations were measured by comparative ELISA. Plasma metanephrine and normetanephrine measured under the three different dietary conditions for each individual were not significantly different and within normal range. Pearson correlation coefficient analysis of plasma concentration of metanephrines within individual patients under the three dietary conditions revealed positive correlation. We found no significant effect of dietary status on plasma metanephrine or normetanephrine concentration. Therefore, samples taken under any dietary condition may be used to determine plasma MN and NMN concentration. However, dietary restrictions in the diagnosis of Pheochromocytoma need further investigations.

TET-2 up-regulation is associated with the anti-inflammatory action of Vicenin-2.

Vicenin-2, a C-glycoside flavone that is present in many plant sources, exerts potent anti-inflammatory effects in a number of cell and animal models of inflammation. Ten-eleven translocation (TET)-2 has recently gained considerable attention due to the role it plays in regulating the inflammasome. We studied the ability of Vicenin-2 (V-2) to regulate a range of lipopolysaccharide (LPS) stimulated inflammatory activities in PMA-differentiated THP-1 cells and human primary mononuclear cells. We also investigated the action of V-2 on the secretion of NLRP3 inflammasome regulated cytokines (IL-1ß and IL-18) by ELISA, and determined if V-2 can regulate the expression of NLRP3, IL-10, IL-1Ra and TET-2. The effect of V-2 on NF-κB signalling was investigated by fluorescence microscopy and gene reporter assay. Additionally, the effect of V-2 on LPS-induced phosphorylation of IKB-α was also investigated by Western blot analysis. V-2 down-regulated LPS-induced secretion of proinflammatory cytokines (TNF-α and IL-1ß), in both THP-1 and primary mononuclear cells. V-2 also decreased the LPS-stimulated secretion of IL-18 in THP-1 cells. V-2 significantly down-regulated TNF-α induced NF-κB reporter activity in HEK293T transfected cells and attenuated IKB-α phosphorylation in THP-1 cells. V-2 treatment also induced enhanced nuclear staining of the p50 subunit and reduced p65 subunit of NF-κB. V-2 treatment alone increased the expression of anti-inflammatory cytokine, IL-10, and the regulator of the inflammasome; IL-1Ra, in the presence of LPS. V-2 also significantly decreased LPS-induced NLRP3 expression while concomitantly increasing TET-2 expression. This study demonstrates that the anti-inflammatory actions of V-2 are associated not only with increased IL-10 and IL-1Ra expression, but also with TET-2 up-regulation. Further work is required to establish if the effects of V-2 can be definitively linked to TET-2 activity and that these actions are mirrored in a range of relevant cell types.

Secreted HMGB1 from Wnt activated intestinal cells is required to maintain a crypt progenitor phenotype.

BACKGROUND AND AIMS: Colorectal cancer (CRC) arises via multiple genetic changes. Mutation of the tumour suppressor gene APC, a key regulator of Wnt signalling, is recognised as a frequent early driving mutation in CRC. We have previously shown that conditional loss of Apc within the murine small intestine (Apcfloxmice) results in acute Wnt signalling activation, altered crypt-villus architecture and many hallmarks of neoplasia. Our transctipomic profiling (Affymetrix Microarrays) and proteomic profiling (iTRAQ-QSTAR) of Apc-deficient intestine inferred the involvement of High Mobility Group Box 1 (Hmgb1) in CRC pathogenesis. Here we assess the contribution of HMGB1 to the crypt progenitor phenotype seen following Apc loss. RESULTS: Elevated HMGB1 was confirmed in intestinal epithelia and serum following conditional loss of Apc. Treatment of Apcflox mice with anti-HMGB1 neutralising antibody significantly reduced many of the crypt progenitor phenotypes associated with Apc loss; proliferation and apoptosis levels were reduced, cell differentiation was restored and the expansion of stem cell marker expression was eradicated. METHODS: Hmgb1 levels in intestinal epithelia and serum in Apcflox and ApcMin mice were assessed using qRT-PCR, Western blot and ELISA assays. The functional importance of elevated extracellular Hmgb1 was assessed using an anti-HMGB1 neutralising antibody in Apcflox mice. CONCLUSIONS: HMGB1 is expressed and secreted from intestinal epithelial cells in response to Wnt signalling activation. This secreted HMGB1 is required to maintain nearly all aspects of the crypt progenitor phenotype observed following Apc loss and add to the body of accumulating evidence indicating that targeting HMGB1 may be a viable novel therapeutic approach.

Direct formation of thienopyridine-derived nitrosothiols--just add nitrite!

Thienopyridines (ticlopidine, clopidogrel and prasugrel) are pro-drugs that require metabolism to exhibit a critical thiol group in the active form that binds to the P2Y12 receptor to inhibit platelet activation and prevent thrombus formation in vivo. We investigated whether these thienopyridines participate in S-nitrosation (SNO) reactions that might exhibit direct anti-platelet behaviour. Optimum conditions for in vitro formation of thienopyridine-SNO formation were studied by crushing ticlopidine, clopidogrel or prasugrel into aqueous solution and adding sodium nitrite, or albumin-SNO. Ozone-based chemiluminescence techniques were utilised to specifically detect NO release from the SNO produced. Effect on agonist-induced platelet aggregation was monitored using light transmittance in a 96 well microplate assay. Pharmaceutical grade preparations of ticlopidine, clopidogrel and prasugrel were found to exhibit significant free thiol and formed SNO derivatives directly from anionic nitrite in water under laboratory conditions without the need for prior metabolism. Thienopyridine-SNO formation was dependent on pH, duration of mixing and nitrite concentration, with prasugrel-SNO being more favourably formed. The SNO moiety readily participated in trans-nitrosation reactions with albumin and plasma. Prasugrel-SNO showed significantly better inhibition of platelet aggregation compared with clopidogrel-SNO, however when compared on the basis of SNO concentration these were equally effective (IC50=7.91 ± 1.03 v/s 10.56 ± 1.43 µM, ns). Thienopyridine-derived SNO is formed directly from the respective base drug without the need for prior in vivo metabolism and therefore may be an important additional contributor to the pharmacological effectiveness of thienopyridines not previously considered.