Anna Vaiopoulou

OCT4 spliced variant OCT4B1 is expressed in human colorectal cancer

OCT4, a POU‐domain transcription factor is considered to be a key factor in maintaining the pluripotency of stem cells. Several OCT4 isoforms are differentially expressed in human pluripotent and non‐pluripotent cells. Reactivation of OCT4 expression is postulated to occur in differentiated cells that have undergone tumorigenesis. To examine OCT4 expression in colorectal cancer (CRC) tissues, and to assess the efficacy of OCT4 as a potential biomarker for CRC, in this study, we investigated its expression in CRC tissues, evaluated its relationship to various clinicopathological parameters and defined the isoform of OCT4 that was found to be expressed in CRC cases. Primary tumor tissues and matching adjacent non‐cancerous tissues were obtained from 84 CRC patients. OCT4 expression and isoform determination were documented by reverse transcription‐PCR and real‐time PCR. OCT4, Sox‐2, and NANOG localization were performed using immunohistochemistry. The isoforms expressed in the studied cases were confirmed by sequencing. Twenty biopsy specimens representing healthy tissues, retrieved from colonoscopy were studied in parallel as controls. OCT4 expression levels were higher in CRC tissues compared to matching, adjacent non‐cancerous tissues, and healthy controls. Additionally, the levels of OCT4 expression in CRC tissues correlated with tumor stage. OCT4 and Sox‐2 were localized in the nuclei and the cytoplasm of CRC cells. In all CRC cases, we found that the OCT4B1 isoform is expressed. Over‐expression of OCT4B1 was found in poorly and moderately differentiated CRC tissues. In conclusion, the data imply that OCT4B1 isoform may represent a potential biomarker for the initiation, progression, and differentiation of CRC. Mol. Carcinog.

Polymorphisms of caspase 8 and caspase 9 gene and colorectal cancer susceptibility and prognosis.

PurposeCaspase-8 (CASP8) and caspase-9 (CASP9) play crucial roles in regulating apoptosis, and their functional polymorphisms may alter cancer risk. Our aim was to investigate the association between CASP8 and CASP9 gene polymorphisms and colorectal cancer (CRC) susceptibility.MethodsA case–control study at 402 CRC patients and 480 healthy controls was undertaken in order to investigate the association between the genotype and allelic frequencies of CASP8 −652 6N ins/del and CASP9 −1263 A>G polymorphisms and the CRC susceptibility. The polymerase chain reaction (PCR) restriction fragment length polymorphism method was used and the incidence of polymorphisms on messenger RNA (mRNA) expression levels was detected by quantitative reverse-transcriptase PCR in CRC tissues.ResultsNo statistical significant association was observed between CASP8 −652 6N ins/del polymorphism frequencies and CRC susceptibility. CASP9 −1263 G allele was observed to be significant associated with reduced risk of CRC. Homozygotes for the −1263 GG CASP9 genotype, and hetrozygotes for the −1263 AG genotype expressed 6.64- and 3.69-fold higher mRNA levels of Caspase-9, respectively compared to the −1263 AA genotype cases. No significant association was observed between CASP9 −1263 A>G polymorphism and tumor characteristics. The CASP9 −1263 GG genotype was associated with increased overall survival in CRC patients.ConclusionThe CASP9 −1263 A>G polymorphism was observed to play a protective role in CRC predisposition, while the CASP9 −1263 GG genotype may confer a better prognosis at CRC patients.

Association of the clock genes polymorphisms with colorectal cancer susceptibility.

The circadian rhythm regulates the cell cycle progression and DNA damage response. The aim of our study was to investigate the association between polymorphisms in the CLOCK1, PER2, and PER3 genes with the colorectal cancer (CRC) susceptibility and clinicopathological variables.

Evaluation of the performance of selected in-house and commercially available PCR and real-time PCR assays for the detection of Leishmania DNA in canine clinical samples.

Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.

Molecular basis of the irritable bowel syndrome

Irritable bowel syndrome (IBS) is a functional disorder characterized by abdominal pain, discomfort and bloating. The pathophysiology of IBS is poorly understood, but the presence of psychosocial basis is now known. There is an increasing number of publications supporting the role of genetics in IBS. Most of the variations are found in genes associated with the brain-gut axis, revealing the strong correlation of brain-gut axis and IBS. miRNAs, which play critical roles in physiological processes, are not well studied in IBS. However, so far there is found an involvement of alterations in miRNA expression or sequence, in IBS symptoms. IBS phenotype is affected by epigenetic alteration and environment. Changes in DNA and histone methylation are observed in patients who suffered childhood trauma or abuse, resulting in altered gene expression, such as the glucocorticoid receptor gene. Finally, diet is another factor associated with IBS, which may contribute to symptom onset. Certain foods may affect on bacterial metabolism and epigenetic modifications, predisposing to IBS.

Current Advantages in the Application of Proteomics in Inflammatory Bowel Disease

Since the formulation of the concept of proteomics, a plethora of proteomic technologies have been developed in order to study proteomes. In inflammatory bowel disease (IBD), several studies use proteomics to try to better understand the disease and discover molecules which can be used as biomarkers. Biomarkers should be able to be used for diagnosis, therapy and prognosis. Although several biomarkers have been discovered, few biomarkers have clinical value. In this review, we analyze and report the current use of proteomic techniques to highlight biomarkers characterizing IBD, and different stages of disease activity. We also report the biomarkers and their potential clinical value.

Serum protein profiling of adults and children with Crohn disease.

Objectives: Crohn disease (CD) and ulcerative colitis (UC), known collectively as inflammatory bowel diseases (IBDs), are chronic immunoinflammatory pathologies of unknown aetiology. Despite the frequent use of biomarkers in medical practice, there is a relative lack of information regarding validated paediatric biomarkers for IBD. Furthermore, biomarkers proved to be efficacious in adults are frequently extrapolated to the paediatric clinical setting without considering that the pathogenesis of many diseases is distinctly different in children. In the present study, proteomics technology was used to monitor differences in protein expression among adult and young patients with CD, identify a panel of candidate protein biomarkers that may be used to improve prognostic–diagnostic accuracy, and advance paediatric medical care. Methods: Male and female serum samples from 12 adults and 12 children with active CD were subjected to 2-dimensional gel electrophoresis. Following the relative quantitation of protein spots exhibiting a differential expression between the 2 groups by densitometry, the spots were further characterized by matrix-assisted laser desorption tandem time-of-flight mass spectrometer. The results were confirmed by Western blot analysis. Results: Clusterin was found to be significantly overexpressed in adults with CD, whereas ceruloplasmin and apolipoprotein B-100 were found to be significantly overexpressed in children, indicating that the expression of these proteins may be implicated in the onset or progression of CD in these 2 subgroups of patients. Conclusions: Interestingly, we found a differential expression of several proteins in adults versus paediatric patients with CD. Undoubtedly, future experiments using a larger cohort of patients with CD are needed to evaluate the relevance of our preliminary findings.

Pancreatic neuroendocrine tumors: current opinions on a rare, but potentially curable neoplasm.

Pancreatic neuroendocrine tumors (PNETs) share a unique genetic identity, functional behavior, and clinical course. Compared with tumors of the exocrine pancreas, they are rare and show a different biologic behavior and prognosis. On the basis of data from recent studies, all PNETs, outside of small insulinomas, should be considered potentially malignant and treated accordingly. Untreated tumors have a high possibility to grow locally into adjacent structures or spread to distant organs. Although surgical excision irrespective of tumor functioning or nonfunctioning state remains the cornerstone of therapy, providing the best disease-free and survival rates to date, the understanding of the genetic nature of the disease yields new ‘targets’ to consider in drug development. The aim of this review is to summarize all recent advances of genetic research and new drug development in terms of PNETs, especially their genetic identity and subsequent alterations leading to the development of near or total malignant activity, and the new medical treatment strategies of this potentially curable disease on the basis of therapeutical agents acting, where possible, at the genetic level.

Evaluation of the microbial safety of child food of animal origin in Greece.

Foodborne illness is a major cause of morbidity and mortality especially for children, even in the developed world. The aim of this study was to assess the microbial safety of food of animal origin intended for consumption by children in Greece. Sampling involved 8 categories of retail products and was completed with a collection of 850 samples. These were tested by PCR and/or culture for Listeria monocytogenes, Campylobacter spp., Escherichia coli O157, Salmonella spp., Cronobacter sakazakii, Brucella spp., and Mycobacterium avium subsp paratuberculosis (MAP). The number of positive results recorded collectively for the pathogens under investigation over the total number of samples tested was 3.52% and 0.12% by PCR and culture, respectively. The most frequently detected pathogen was enterohemorrhagic E. coli (1.29%) followed by Brucella (0.82%) and Listeria (0.82%). DNA belonging to MAP was detected in 0.35% of samples, which was also the percentage of positivity recorded for Campylobacter. The percentage for Salmonella was 0.12%. It can be concluded from the results that there is no indication of noncompliance for the tested food samples. However, detection of DNA belonging to pathogens that are transmissible to humans through food is indicative that constant vigilance regarding food safety is an absolute necessity.

mTOR downstream effectors, 4EBP1 and eIF4E, are overexpressed and associated with HPV status in precancerous lesions and carcinomas of the uterine cervix

The present study aims to investigate the expression levels of two critical mammalian target of rapamycin (mTOR) downstream effectors, 4E binding protein 1 (4EBP1) and eukaryotic initiation factor 4E (eIF4E) proteins, in precancerous squamous intraepithelial lesions and cancer of the uterine cervix, and their association with human papilloma virus (HPV) infection status. Uterine cervical biopsies from 73 patients were obtained, including 40 fresh-frozen samples and 42 archival formalin-fixed, paraffin-embedded tissue specimens. Whole protein extracts were analyzed for the expression of 4EBP1 and eIF4E proteins using western blotting. In addition, distribution of 4EBP1 and eIF4E protein expression and 4EBP1 phosphorylation (P-4EBP1) were analyzed by immunohistochemistry in archival tissues and correlated with the degree of dysplasia. The presence of high-risk HPV (HR-HPV) types was assessed by polymerase chain reaction. Using western blot analysis, high expression levels of 4EBP1 and eIF4E were observed in all uterine cervical carcinomas, which significantly correlated with the degree of dysplasia. By immunohistochemistry, overexpression of 4EBP1 and eIF4E was detected in 20 of 21 (95%) and 17 of 21 (81%) samples, respectively, in patients with high-grade dysplasia and carcinomas, compared with 1 of 20 (5%) and 2 of 20 (10%) samples, respectively, in patients with low-grade lesions or normal histology. All 4EBP1-positive cases tested were also positive for P-4EBP1. Furthermore, overexpression of 4EBP1 and eIF4E significantly correlated with the presence of HR-HPV oncogenic types. The present study demonstrated that critical effectors of mTOR signaling, which control protein synthesis initiation, are overexpressed in cervical high-grade dysplasia and cancer, and their levels correlate with oncogenic HPV types. These findings may provide novel targets for investigational therapeutic approaches in patients with cancer of the uterine cervix.