Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients.

PURPOSE: We undertook the current study with untreated breast cancer to (1) role the variations in the plasma levels of cfDNA and the size distribution in early stage, (2) determine the frequency in plasma of methylation of three candidate genes, RASSF1A, MAL, and SFRP1, and (3) to determine whether detection of cfDNA variations and methylation changes in plasma might have specific clinical utility. METHODS AND MATERIALS: Thirty-nine patients woman patients (median age 64 years; range, 36-90 years) who underwent surgery for primary BR and 49 healthy females' subjects (control group without any breast lesion) were evaluated. The cfDNA levels were analyzed using quantitative real-time polymerase chain reaction of ß-globin. Based on the ALU repeats, the cfDNA was considered as either total (fragments of 115 bp, ALU115) or tumoral (fragments of 247 bp, ALU247). The association between the levels of the ALU247, ALU115 repeat, and ALU 247/115and the pathologic tumor characteristics was analyzed. Used methylight qPCR method, cfDNA from plasma samples of healthy donors and patients with breast cancer were evaluated for the diagnotic value of the methylation status of three genes (RASSF1A, MAL, SFRP1) frequently methylated in breast cancer. RESULTS: The baseline levels of cfDNA were significantly higher in the patients with cancer, and the level of ALU247 was the most accurate circulating cfDNA marker in discriminating the cancer from non-cancer subjects. A high statistical significance was found by considering the T stage and patients with regional LN metastasis positive cancers showed significantly higher cfDNA level of ALU247. Moreover, patients with methylation of at least one of the gene under investigate showed a higher quantity of cfDNA ALU115 (p< 0.0001) and ALU247 level (p< 0.0001). CONCLUSIONS: We observed that necrosis could be a potential source of circulating tumour-specific cfDNA ALU247; and that cfDNA ALU247 and methylated cfDNA (RASSF1A, MAL and SFRP1) are both a phenotypic feature of tumour biology.

Differentiation of mesenchymal stem cells derived from pancreatic islets and bone marrow into islet-like cell phenotype.

BACKGROUND: Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. CONCLUSIONS/SIGNIFICANCE: Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.

Anti-endomysium antibodies detection in the celiac disease screening: Indirect immunofluorescence pattern using umbilical cord sections as substrate.

Celiac disease screening test with anti-endomysial antibodies detection is limited by the use of endangered species tissue (monkey distal oesophagus) as well as by the high costs of commercial kits. Due to such limitations new tests are developed to avoid the use of monkey tissue: an IFA test utilising umbilical cord sections (a discarded tissue) and an ELISA test detecting anti-transglutaminase antibodies. In most cases umbilical cord anti-endomysium test, is not routinely used due to the different pattern of EmA IFA test with monkey oesophagus: in the present article we show some pattern obtained with this new method in order to clarify possible reading misinterpretation.

Endomysium Antibodies Detection with Umbilical Cord Sections.

Anti-Endomysium antibodies (EmA) detection is the most reliable test for the diagnosis of coeliac disease. Usually monkey distal oesophagus sections are used. Tests with ELISA anti-gliadin (AGA) antibodies show that umbilical cord EmA tests can effectively substitute monkey oesophagus EmA tests.