Detection of methylated tumor DNA in serum as a marker for the colorectale carcinoma

Kolorektale Karzinome sind die dritthäufigsten bösartigen Tumore weltweit. Daher ist es wichtig eine sensitive und verlässliche Methode für die Diagnose und die Verlaufskontrolle dieser Erkrankung zu finden. Ein Ansatz für diesen Zweck ist, epigenetisch veränderte DNA im Serum als Marker für das kolorekale Karzinom zu nützen. Daher war das Ziel unseres Projektes eine Methode für die Detektion und Quantifizierung von methyliertem RASSF1A und P16 im Serum zu finden. Nach der Etablierung einer Standardkurve, verglichen wir zwei Ansätze für die Berechnung des Methylierungslevels einer Probe. Die Berechnung des %Methylierung Werts lieferte akkuratere Ergebnisse, während die „Percentage of Methylated Reference“ Methode praktikabler ist. In einem weiteren Schritt untersuchten wir die Effizienz fünf verschiedener, kommerziell erhältlicher Kits für die Aufreinigung von DNA. Wir konnten zeigen, dass der Kit, der auf einer Methode basiert, die magnetic beads nützt, die besten Ergebnisse erzielte. Wir testeten auch eine Bisulfit-unabhängige, auf Restriktionsenzymen basierende Methode für die quantitative Detektion von methylierten DNA Sequenzen im Serum, die aber keine Verbesserung der Sensitivität brachte. Anschließend benutzen wir das geeignetste Protokoll um Serumproben von Patienten mit kleinzelligen Lungenkarzinom und kolorektalem Karzinom zu untersuchen. Wir konnten bei 2 von 8 (25%) Bronchialkarzinom Patienten und 0 von 9 (0%) CRC Patienten methyliertes RASSF1A detektieren. Bei der Untersuchung auf P16 waren alle Proben aus beiden Gruppen negativ.Colorectal carcinomas (CRCs) are the third most common malignant tumors worldwide. Thus, it is important to find sensitive and reliable methods for the diagnosis and the clinical monitoring of this disease. An approach for this purpose is the use of epigentically altered DNA as a marker for the CRC. Therefore, the aim of our project was to establish methods to detect and quantify the methylated forms of the genes P16 and RASSF1A in serum. After the establishment of the standard curve, we compared two approaches for calculating the methylation level of a sample. We obtained more accurate results using the %methylation approach, on the other hand the calculation of the Percentage of Methylation Reference (PMR) is more reasonable and more practicable to perform. Second we compared the efficiency of five different, commercially available DNA isolation kits. We found that the kit applying magnetic beads provided the best extraction rates. Finally, we tested also another, sodium-bisulfite independent, restriction enzyme based method to quantitatively detect methylated DNA sequences in serum. But it did not improve the sensitivity of the method. Using the most efficient protocol, we analyzed serum samples of small cell lung cancer (SCLC) and of CRC patients. We could detect methylation of RASSF1A in 2 of 8 (25%) lung cancer patients and in 0 of 9 (0%) of CRC patients. When using the primer and probes for P16 all samples of both cancer groups were negative

Oral administration of dextran sodium sulphate induces a caecum-localized colitis in rabbits

Trichuris suis ova (TSO) have shown promising results in the treatment of inflammatory bowel disease (IBD) but the mechanisms which underlies this therapeutic effect cannot be studied in mice and rats as T. suis fails to colonize the rodent intestine, whilst hatching in humans and rabbits. As a suitable rabbit IBD model is currently not available, we developed a rabbit colitis model by administration of dextran sodium sulphate (DSS). White Himalayan rabbits (n = 12) received 0.1% DSS in the daily water supply for five days. Clinical symptoms were monitored daily, and rabbits were sacrificed at different time points. A genomewide expression analysis was performed with RNA isolated from caecal lamina propria mononuclear cells (LPMC) and intestinal epithelial cells (IEC). The disease activity index of DSS rabbits increased up to 2.1 ± 0.4 (n = 6) at day 10 (controls <0.5). DSS induced a caecum-localized pathology with crypt architectural distortion, stunted villous surface and inflammatory infiltrate in the lamina propria. The histopathology score reached a peak of 14.2 ± 4.9 (n = 4) at day 10 (controls 7.7 ± 0.9, n = 5). Expression profiling revealed an enrichment of IBD-related genes in both LPMC and IEC. Innate inflammatory response, Th17 signalling and chemotaxis were among the pathways affected significantly. We describe a reproducible and reliable rabbit model of DSS colitis. Localization of the inflammation in the caecum and its similarities to IBD make this model particularly suitable to study TSO therapy in vivo

Downregulation of duodenal SLC transporters and activation of proinflammatory signaling constitute the early response to high altitude in humans

Solute carrier (SLC) transporters mediate the uptake of biologically active compounds in the intestine. Reduced oxygenation (hypoxia) is an important factor influencing intestinal homeostasis. The aim of this study was to investigate the pathophysiological consequences of hypoxia on the expression and function of SLCs in human intestine. Hypoxia was induced in human intestinal epithelial cells (IECs) in vitro (0.2; 1% O2 or CoCl2). For human in vivo studies, duodenal biopsies and serum samples were obtained from individuals (n = 16) acutely exposed to 4,554 meters above sea levels. Expression of relevant targets was analyzed by quantitative PCR, Western blotting, or immunofluorescence. Serum levels of inflammatory mediators and nucleosides were determined by ELISA and LC/MS-MS, respectively. In the duodenum of volunteers exposed to high altitude we observed decreased mRNA levels of apical sodium-dependent bile acid transporter (ASBT), concentrative nucleoside transporters 1/2 (CNT1/2), organic anion transporting polypeptide 2B1 (OATP2B1), organic cation transporter 2 (OCTN2), peptide transporter 1 (PEPT1), serotonin transporter (SERT), and higher levels of IFN-γ, IL-6, and IL-17A. Serum levels of IL-10, IFN-γ, matrix metalloproteinase-2 (MMP-2), and serotonin were elevated, whereas the levels of uridine decreased upon exposure to hypoxia. Hypoxic IECs showed reduced levels of equilibrative nucleoside transporter 2 (ENT2), OCTN2, and SERT mRNAs in vitro, which was confirmed on the protein level and was accompanied by activation of ERK1/2, increase of hypoxia-inducible factor (HIF) proteins, and production of IL-8 mRNA. Costimulation with IFN-γ and IL-6 during hypoxia further decreased the expression of SERT, ENT2, and CNT2 in vitro. Reduced oxygen supply affects the expression pattern of duodenal SLCs that is accompanied by changes in serum levels of proinflammatory cytokines and biologically active compounds demonstrating that intestinal transport is affected during systemic exposure to hypoxia in humans

Antidiabetic effect of Melicope glabra (Blume) T.G. Hartley (Rutaceae) in high-fat diet/streptozotocin-induced diabetic rats and its bioactive components

Melicope glabra (Blume) T.G. Hartley is a plant of Rutaceae family. The present study aimed to evaluate the in vivo hypoglycemic effect of M. glabra leaves extract on diabetic rats and identify its bioactive components. The induc-tion of diabetes in Sprague-Dawley rats was done by a combination of high-fat diet and low-dose streptozotocin injection. The M. glabra extracts and standard sitagliptin were orally administered once daily to the diabetic rats for 28 days. The diabetic rats showed drastic weight loss, hyperglycemia, poor glucose tolerance, hyperlipidemia, and disturbances in the liver and kidney functions. However, oral administration of M. glabra extracts to the dia-betic rats significantly lowered the DPP-4 level and increased glucagon-like peptide-1 (GLP-1) level. Scopoletin, pachypodol, and stigmasterol were revealed as the active DPP-4 and α-amylase inhibitors. This study revealed novel findings which suggest the potential of M. glabra as alternative antidiabetic treatment and source of anti-diabetic agent

Effects of retinoids in mouse models of colitis: benefit or danger to the gastrointestinal tract?

BACKGROUND: In vitro and in vivo data have shown that retinoid treatment promotes an anti-inflammatory milieu with few adverse effects toward the gastrointestinal tract. The in vivo studies reported here further evaluate retinoid effects in 2 mouse models of inflammatory bowel disease. METHOD: Chronic dextran sulfate sodium colitis was induced in age- and weight-matched C57Bl/6 mice by 4 cycles of dextran sulfate sodium administration (6-8 animals/group). At cycle 4, animals were administered 13-cis-retinoic acid (isotretinoin, 30 mg/kg) or vehicle (oral gavage) or 4-oxo-13-cis-retinoic acid (15 mg/kg, intraperitoneal) daily. T-cell transfer colitis was induced in CB17 SCID mice by transfer of naive CD4CD62L T cells and treated by transfer of regulatory CD4CD25 T cells (4-6 animals/group); isolated from BALB/c mice after treatment with isotretinoin or vehicle, as above, for 2 weeks. Assessments included endoscopic and histological scores, myeloperoxidase activity, serum cytokines, and plasma isotretinoin levels. RESULTS: Retinoid-treated animals with colitis showed comparable changes in myeloperoxidase activity, and endoscopic and histological scores, versus untreated animals with colitis. Modest and comparable changes were seen in body weight and colon length in animals injected with naive T cells from isotretinoin-treated donors versus those injected with T cells from vehicle-treated donors. Retinoid treatment was consistently associated with lower interleukin-12 levels, which, after the transfer of naive T cells from isotretinoin-treated donors, supported isotretinoin-mediated predisposition of naive T cells toward reduced proinflammatory cytokine expression. Colitis had no effect on isotretinoin exposure. CONCLUSIONS: Retinoids attenuate the proinflammatory cytokine response in vivo, with only modest effects on body weight and parameters of gastrointestinal morphology