COLD-PCR Amplification of Bisulfite-Converted DNA Allows the Enrichment and Sequencing of Rare Un-Methylated Genomic Regions

Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes

Portal Vein Stent Placement in Anastomotic Stenosis After Deceased Donor Liver Transplantation: A Case Report

Vascular complications (VCs) after liver transplantation (LT) frequently result in graft and patient loss. The smaller vessels and the insufficient length for reconstruction in living donor LT and pediatric transplantation predispose patients to a higher incidence of VCs. Herein we present a case of portal vein stenosis (PVS) in an adult deceased donor LT recipient with portal vein thrombosis requiring extended thrombectomy at the time of LT. He presented with ascites 4 months after LT, was diagnosed with PVS, and was successfully treated with percutaneous transhepatic venoplasty and placement of a portal stent. This case highlights the importance of Doppler ultrasound as a screening modality for detection of VCs after LT and the pivotal role of endovascular repair as a first-line treatment for PVS

Detection of un-methylated DNA within different abundances of methylated DNA background by conventional or <i>fast</i>-COLD-MS-PCR.

<p><b>Panel A.</b> Post-PCR melting profile of the 255 bp bisulfite-converted-specific amplicon after conventional PCR or COLD-PCR. Serial dilutions of un-methylated (U) to methylated (M) genomic DNA are depicted (top half). Higher abundances of un-methylated DNA can be discriminated from methylated DNA by the melt peak, whereas lower abundances are only detectable if <i>fast</i>-COLD-MS-PCR replaces conventional PCR. <b>Panel B.</b> Sanger sequencing results of the 0.05% un-methylated (U): methylated (M) DNA sample as amplified by conventional and <i>fast</i>-COLD-MS-PCR are shown (bottom half). Chromatograms are aligned and compared using SeqDoc, and the CpG methylation positions are revealed in the middle panel.</p

Melting profiles of bisulfite-converted DNA from clinical samples following conventional PCR.

<p>Post-PCR melting profiles of the 255 bp bisulfite-converted <i>MGMT</i> gene amplicon after conventional PCR. Examples of fully un-methylated DNA samples isolated from infant blood (<b>Panel A</b>) and glioma samples (<b>Panel B</b>) are depicted. 100% methylated (M) and 100% un-methylated (U) DNA controls are used as reference standards, demonstrating a ∼5°C melting temperature difference among the two. <b>Panel C.</b> A glioma sample with mixed methylation/unmethylation pattern is shown.</p

Melting profiles of bisulfite-converted DNA from clinical samples following <i>fast</i>-COLD-MS-PCR.

<p>The effect of lowering the denaturation temperature in PCR is depicted. <b>Panel A.</b> Glioma sample no. 3 was subjected to different critical denaturation temperature-T_c during <i>fast</i>-COLD-MS-PCR. The modulation of the preferential amplification of the un-methylated DNA fraction is shown. <b>Panel B. </b><i>fast</i>-COLD-MS-PCR performed at a T_c of 84°C demonstrates that the amplification of the methylated DNA fraction is completely inhibited.</p

Intestinal mucosal proliferation, apoptosis and oxidative stress in patients with liver cirrhosis

Background. Intestinal mucosal barrier dysfunction in liver cirrhosis and its implicated mechanisms is of great clinical importance because it is associated with the development of serious complications from diverse organs through promotion of systemic endotoxemia.Aim. The present study was designed to investigate whether enterocytes’ proliferation, apoptosis and intestinal oxidative stress are altered in the intestinal mucosa of patients with compensated and decompensated liver cirrhosis.Material and methods. Twelve healthy controls (group A) and twenty four cirrhotic patients at a compensated (n = 12, group B) or decompensated condition (n = 12, group C) were subjected to duodenal biopsy. In intestinal specimens mucosal apoptotic and mitotic activity and their ratio were recorded by means of morphological assessment and mucosal lipid hydroperoxides were measured. Plasma endotoxin concentration, an index of gut barrier function, was also determined.Results. Cirrhotic patients presented significantly higher serum endotoxin concentrations as compared to healthy controls (P < 0.001), whilst endotoxemia was higher in decompensated disease (P < 0.05 vs. compensated cirrhosis). Intestinal mucosal mitotic count was significantly lower in patients with compensated and decompensated cirrhosis compared to controls (P < 0.01, respectively), whilst a trend towards increased apoptosis was recorded. The mitotic/apoptotic ratio was significantly reduced in groups B (P < 0.05) and C (P < 0.01) as compared to controls. Intestinal lipid peroxidation was significantly increased in decompensated cirrhotics (P < 0.001 vs. groups A and B).Conclusions. The present study demonstrates for the first time that human liver cirrhosis is associated with decreased intestinal mucosal proliferation and proliferation/apoptosis ratio even at early stages of cirrhosis and increased intestinal oxidative stress in advanced liver disease

Neutrophil extracellular traps in giant cell arteritis biopsies: presentation, localization and co-expression with inflammatory cytokines

Objectives To explore the presence of neutrophil extracellular traps (NETs) in inflamed temporal artery biopsies (TABs) of patients with GCA. Methods Ten patients with GCA [five with limited and five with associated generalized vascular involvement, as defined by F-18-fluorodeoxyglucose PET with CT (PET/CT)] and eight with PMR were studied. The presence, location, quantitation and decoration of NETs with IL-6, IL-1 beta and IL-17A were assessed in TABs at the time of disease diagnosis by tissue immunofluorescence and confocal microscopy. Paired serum levels of IL-6 and IL-17A were also evaluated in all patients. Results All temporal artery biopsies from GCA, but not PMR, patients had NETs located mainly in the adventitia, adjacent to the vasa vasorum. NETs decorated with IL-6 were present in 8/10 TABs of GCA patients, of whom 5 were PET/CT(+) and 3 PET/CT(-) patients. IL-17A(+) NETs were observed in all GCA patients. IL-1 beta(+) NETs were not detected in any GCA patient. No relation was found between serum IL-6 and IL-17A levels and NETs containing IL-6 and/or IL-17A. Conclusions NETs bearing pro-inflammatory cytokines are present in inflamed GCA-TABs. Future studies with a larger number of patients from different centres will show whether the findings regarding neutrophils/NETs in the TAB are consistent and disclose their clinical impact