An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC

Transitional mucosa of the colon and tumor growth factors

Transitional mucosa of the colon is the tissue immediately adjacent to a cancer, which has histochemical and ultrastructural features similar to those seen in neoplasia. Therefore, it has been hypothesized in the past that this tissue represents the premalignant antecedent to colonic adenocarcinoma. Other investigators have disputed this explanation because similar changes are found adjacent to colonic lesions other than adenocarcinoma.The hypothesis offered here suggests that transitional mucosa may result from the paracrine influence of tumor growth factors released by the tumor. Candidate peptide hormones known to be produced by tumors are proposed, and a framework is outlined to explain the occurrence of transitional mucosa adjacent to non-neoplastic lesions. Transitional mucosa may be both a genuine reflection of the transformed phenotype and yet a "reactive" phenomenon secondary to the presence of the adjacent tumor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26787/1/0000343.pd

Prohibitin 1 Modulates Mitochondrial Stress-Related Autophagy in Human Colonic Epithelial Cells

Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A(1) or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease

The clinical significance of MiR-148a as a predictive biomarker in patients with advanced colorectal cancer

Aim: Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies. Methods: We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival. Results: Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93). Discussion: MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy

Post-translational processing of gastrin in neoplastic human colonic tissues

Gastrin has been postulated to stimulate proliferation in colorectal neoplasms. Although gastrin mRNA has been demonstrated to be present in colon cancer cell lines, the intact peptide had not been recovered from human colorectal neoplasms.We demonstrate that gastrin and its precursors are present in both colorectal neoplasia and adjacent normal-appearing colonic mucosa. In colonic tissue, the glycine-extended precursor form of the peptide is over 10-fold more abundant than the amidated gastrin, and progastrin is more than 700-fold more abundant. In contrast, amidated gastrin in the human antrum is the predominant form of gastrin by a factor of 10. Furthermore, the ratio of gastrin precursors to gastrin is significantly increased in neoplastic colonic mucosa when compared with normal colonic tissue. These data suggest that the processing of gastrin is unique in the human colon and that further differences in processing occur in neoplastic colonic tissue.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29682/1/0000009.pd

JC Virus Mediates Invasion and Migration in Colorectal Metastasis

INTRODUCTION:JC Virus (JCV), a human polyomavirus, is frequently present in colorectal cancers (CRCs). JCV large T-Ag (T-Ag) expressed in approximately half of all CRC's, however, its functional role in CRC is poorly understood. We hypothesized that JCV T-Ag may mediate metastasis in CRC cells through increased migration and invasion. MATERIAL AND METHODS:CRC cell lines (HCT116 and SW837) were stably transfected with JCV early transcript sequences cloned into pCR3 or empty vectors. Migration and invasion assays were performed using Boyden chambers. Global gene expression analysis was performed to identify genetic targets and pathways altered by T-Ag expression. Microarray results were validated by qRT-PCR, protein expression analyses and immunohistochemistry. Matching primary CRCs and liver metastases from 33 patients were analyzed for T-Ag expression by immunohistochemistry. RESULTS:T-Ag expressing cell lines showed 2 to 3-fold increase in migration and invasion compared to controls. JCV T-Ag expression resulted in differential expression of several genetic targets, including genes that mediate cell migration and invasion. Pathway analysis suggested a significant involvement of these genes with AKT and MAPK signaling. Treatment with selective PI3K/AKT and MAPK pathway inhibitors resulted in reduced migration and invasion. In support of our in-vitro results, immunohistochemical staining of the advanced stage tumors revealed frequent JCV T-Ag expression in metastatic primary tumors (92%) as well as in their matching liver metastasis (73%). CONCLUSION:These data suggest that JCV T-Ag expression in CRC associates with a metastatic phenotype, which may partly be mediated through the AKT/MAPK signaling pathway. Frequent expression of JCV T-Ag in CRC liver metastasis provides further clues supporting a mechanistic role for JCV as a possible mediator of cellular motility and invasion in CRC

The effectiveness, safety and cost-effectiveness of cytisine versus varenicline for smoking cessation in an Australian population: a study protocol for a randomized controlled non-inferiority trial

Smoking cessation medications are effective but often underutilised because of costs and side effects. Cytisine is a plant-based smoking cessation medication with over 50 years of use in Central and Eastern Europe. While cytisine has been found to be well-tolerated and more effective than nicotine replacement therapy, direct comparison with varenicline have not been conducted. This study evaluates the effectiveness, safety and cost-effectiveness of cytisine compared with varenicline.Two arm, parallel group, randomised, non-inferiority trial, with allocation concealment and blinded outcome assessment.Australian population-based study.Adult daily smokers (N=1266) interested in quitting will be recruited through advertisements and Quitline telephone-based cessation support services.Eligible participants will be randomised (1:1 ratio) to receive either cytisine capsules (25-day supply) or varenicline tablets (12-week supply), prescribed in accordance with the manufacturer's recommended dosing regimen. The medication will be mailed to each participant's nominated residential address. All participants will also be offered standard Quitline behavioural support (up to six 10-12 minute sessions).Assessments will be undertaken by telephone at baseline, 4- and 7-months post-randomisation. Participants will also be contacted twice (two and four weeks post-randomisation) to ascertain adverse events, treatment adherence and smoking status. The primary outcome will be self-reported 6-month continuous abstinence from smoking, verified by carbon monoxide at 7-month follow-up. We will also evaluate the relative safety and cost-effectiveness of cytisine compared with varenicline. Secondary outcomes will include self-reported continuous and 7-day point prevalence abstinence and cigarette consumption at each follow-up interview.If cytisine is as effective as varenicline, its lower cost and natural plant-based composition may make it an acceptable and affordable smoking cessation medication that could save millions of lives worldwide

Role of prostaglandin E 2 in cholinergic-mediated glycoprotein synthesis in canine antrum

We studied the mechanism of cholinergic stimulation of mucin synthesis in canine antral explants, including the role of PGE 2 as an intermediate messenger. Isolated antral mucosa was incubated with 10 −5 M carbachol (Cb), 10 −5 M indomethacin (IND), 10 −5 M pirenzepine (PZ), 10 −5 M Cb+10 −5 M PZ, 10 −5 M Cb+10 −5 M IND, and 10 −5 M IND +PGE 2 (10 −8 , 10 −7 and 10 −6 M) in the presence or absence of [ 3 H]glucosamine. After 24 hr, total glycoprotein synthesis was quantitated by Sepharose-4B chromatography and by 10% TCA/1%PTA precipitation with lipid extraction. PGE 2 released into the media was measured by radioimmunoassay (RIA). Cb significantly increased total glycoprotein synthesis and produced a significant increase in PGE 2 release. The increase in glycoprotein synthesis and the release of PGE 2 was blocked by the addition of muscarinic antagonist PZ. The addition of IND significantly inhibited glycoprotein synthesis and almost entirely suppressed PGE 2 secretion. IND also inhibited the effect of Cb on glycoprotein synthesis and PGE 2 release. Moreover, PGE 2 (10 −6 and 10 −7 M) significantly increased the glycoprotein synthesis in the canine stomach. This suggests the coordinate participation of PGE 2 -releasing cell population in modulation of glycoprotein synthesis in gastric mucosa.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44416/1/10620_2005_Article_BF01300285.pd

Beef production from feedstuffs conserved using new technologies to reduce negative environmental impacts

End of project reportMost (ca. 86%) Irish farms make some silage. Besides directly providing feed for livestock, the provision of grass silage within integrated grassland systems makes an important positive contribution to effective grazing management and improved forage utilisation by grazing animals, and to effective feed budgeting by farmers. It can also contribute to maintaining the content of desirable species in pastures, and to livestock not succumbing to parasites at sensitive times of the year. Furthermore, the optimal recycling of nutrients collected from housed livestock can often be best achieved by spreading the manures on the land used for producing the conserved feed. On most Irish farms, grass silage will remain the main conserved forage for feeding to livestock during winter for the foreseeable future. However, on some farms high yields of whole-crop (i.e. grain + straw) cereals such as wheat, barley and triticale, and of forage maize, will be an alternative option provided that losses during harvesting, storage and feedout are minimised and that input costs are restrained. These alternative forages have the potential to reliably support high levels of animal performance while avoiding the production of effluent. Their production and use however will need to advantageously integrate into ruminant production systems. A range of technologies can be employed for crop production and conservation, and for beef production, and the optimal options need to be identified. Beef cattle being finished indoors are offered concentrate feedstuffs at rates that range from modest inputs through to ad libitum access. Such concentrates frequently contain high levels of cereals such as barley or wheat. These cereals are generally between 14% to 18% moisture content and tend to be rolled shortly before being included in coarse rations or are more finely processed prior to pelleting. Farmers thinking of using ‘high-moisture grain’ techniques for preserving and processing cereal grains destined for feeding to beef cattle need to know how the yield, conservation efficiency and feeding value of such grains compares with grains conserved using more conventional techniques. European Union policy strongly encourages a sustainable and multifunctional agriculture. Therefore, in addition to providing European consumers with quality food produced within approved systems, agriculture must also contribute positively to the conservation of natural resources and the upkeep of the rural landscape. Plastics are widely used in agriculture and their post-use fate on farms must not harm the environment - they must be managed to support the enduring sustainability of farming systems. There is an absence of information on the efficacy of some new options for covering and sealing silage with plastic sheeting and tyres, and an absence of an inventory of the use, re-use and post-use fate of plastic film on farms. Irish cattle farmers operate a large number of beef production systems, half of which use dairy bred calves. In the current, continuously changing production and market conditions, new beef systems must be considered. A computer package is required that will allow the rapid, repeatable simulation and assessment of alternate beef production systems using appropriate, standardised procedures. There is thus a need to construct, evaluate and utilise computer models of components of beef production systems and to develop mathematical relationships to link system components into a network that would support their integration into an optimal system model. This will provide a framework to integrate physical and financial on-farm conditions with models for estimating feed supply and animal growth patterns. Cash flow and profit/loss results will be developed. This will help identify optimal systems, indicate the cause of failure of imperfect systems and identify areas where applied research data are currently lacking, or more basic research is required