Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes

Chronic cerebrospinal venous insufficiency in multiple sclerosis: a highly prevalent age-dependent phenomenon

BACKGROUND: This study aimed to investigate the prevalence and clinical relevance of chronic cerebrospinal venous insufficiency (CCSVI) in multiple sclerosis (MS) patients and healthy controls using extra- and intracranial colour Doppler sonography. METHODS: We examined 146 MS patients, presenting with a clinically isolated syndrome, relapsing-remitting, secondary progressive, or primary progressive MS, and 38 healthy controls. Sonographic examination was performed according to Zamboni’s protocol and was performed by three independent sonographers. The results of sonographic examination were compared with clinical and demographic characteristics of the patients. RESULTS: CCSVI, defined as the presence of at least two positive Zamboni’s criteria, was found in 76% of MS patients and 16% of control subjects. B-mode anomalies of internal jugular veins, such as stenosis, malformed valves, annuli, and septa were the most common lesions detected in MS patients (80.8%) and controls (47.4%). We observed a positive correlation between sonographic diagnosis of CCSVI and the patients’ age (p = 0.003). However, such a correlation was not found in controls (p = 0.635). Notably, no significant correlations were found between sonographic signs of CCSVI and clinical characteristics of MS, except for absent flow in the jugular veins, which was found more often in primary (p<0.005) and secondary (p<0.05) progressive patients compared with non-progressive patients. Absent flow in jugular veins was significantly correlated with patients’ age (p < 0.0001). CONCLUSIONS: Sonographically defined CCSVI is common in MS patients. However, CCSVI appears to be primarily associated with the patient’s age, and poorly correlated with the clinical course of the disease

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

BACKGROUND: Celiac Disease (CD) is both a frequent disease (1:100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosine Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies

Sistemi polimerici multicomponenti bioriassorbibili per applicazioni biomediche e farmaceutiche

Sono statti preparati copolimeri anfifilici a blocchi ed a innesto, basati su policaprolattone e poliossietilene, con diverse architetture: lineare, ramificata, a pettine. Sono state studiate le proprietà di autorganizzazione, sia in massa che in soluzione acquosa, di questi sistemi e le loro potenzialità come nanovettori per il rilascio mirato di farmaci e come agenti compatibilizzanti di miscele polimeriche utilizzabili in ingegneria tissutale

Duodenal carcinoma at ligament of Treitz. Case report and review of the literature

We report a case of adenocarcinoma of the duodenojejunal angle and remark the rarity of this pathology, the difficulty of diagnosis and treatment peculiar to tumours of the duodenum. Thise rare tumour ischaracterized by polymorphic and non specific symptomatology. The possible therapy is surgery. Radio and chemotherapy don't significantly improve surviva

Immiscible Poly(L-lactide)/Poly(e-caprolactone) Blends: Influence of the Addition of a Poly(L-lactide)/poly(oxyethylene) Block Copolymer on Thermal Behaviour an Morphology

A binary PLLA/PCL (70/30 by wt) and ternary (70/30/2) blends, containing a diblock PLLA-PEO or a PLLA-PCL-PLLA triblock copolymer as third components, were obtained by melt extrusion and studied by DSC, POM and SEM techniques. The size of the dispersed PCL domains were strongly reduced in the presence of the copolymer and the emulsifying effect was enhanced with the PLLA-PEO copolymer. The results were analyzed on the basis of the solubility parameters of the blend components