Effetti clinici e biologici della terapia epigenetica nelle sindromi mielodisplastiche a basso rischio

Background: Nucleoside 5-Azacitidine (5-Aza) in high risk MDS patients (pts) at a dose of 75mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates (hematologic improvement (HI) 50-60%, complete remission (CR) 10-30%) and prolongation of survival (at 2 years 50,8%). Aim: The role of 5-Aza in low-risk MDS patients is not well defined but its use in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial a low-dose schedule of 5-Aza (75 mg/mq daily for 5 consecutive days every 28 days) was given to low-risk MDS pts in order to evaluate its efficacy and tolerability and to identify biological markers to predict the response. Methods: From September 2008 to February 2010, 34 patients were enrolled into the study. Fifteen patients had refractory anemia (RA), 5 patients refractory anemia with ringed sideroblasts (RARS), 7 patients refractory cytopenia with multilineage dysplasia (RCMD) and 7 patients refractory anemia with excess blasts-1 (RAEB-1). All patients failed previously EPO therapy and were in chronic red blood cell (RBC) supportive care with a median transfusions requirement of 4 units/monthly. The response treatment criteria was according to IWG 2006. Results: At present time 31 out of 34 pts are evaluable: 12/31 pts (39%) completed the treatment plan (8 courses), 7/31 pts (22%) performed the first 4 courses, 8/31 (26%) made 1 to 3 courses and 4/31 (13%) died during the treatment period. Out of 12 pts who completed the 8 courses of therapy 10 (83%) obtained an HI, 2/12 (17%) maintained a stable disease. Out of 10 pts who obtained HI, 4 pts (40%) achieved a CR. Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%) but they were transitory and usually no delay of treatment was necessary. 2/4 pts died early after the 1th cycle for septic shock and gastrointestinal hemorrage respectively whereas 2/4 pts died in a condition of stable disease after the 4th cycle for pneumonia and respiratory distress. Samples for biologic studies have been collected from the pts before starting the therapy and at the end of 4th and 8th course. Preliminary data on the lipid signalling pathways suggested a direct correlation between PI-PLC-β1 gene expression and 5-Aza responsiveness. Conclusion: Interim analysis of our study based on the small number of cases who completed the treatment program, shows that 83% of pts obtain an HI and 40% obtain a CR. 4 patients died during the treatment and even if the causes were reported as no related to the therapy it has been considered that caution has to be reserved in given 5-Aza in these pts who are elderly and frail. Preliminary data of PI-PLC-β1 gene expression suggest that this and probably other biological markers could help us to know a priori who are the patients who have more chances to respond.

Stenodactilina e lanceolina: due nuove potenti “ribosome-inactivating proteins” bicatenarie. Purificazione, caratterizzazione chimico-fisica e studio dei meccanismi citotossici

Two lectins, called lanceolin and stenodactylin, were purified by affinity chromatography on CL Sepharose 6B from the caudices of the Passifloraceae Adenia lanceolata and Adenia stenodactyla, respectively. They are glycoproteins with Mw of 61,243 (lanceolin) and 63,131 daltons (stenodactylin), consisting of an enzymatic A chain linked to a larger B chain with lectin properties, with N-terminal amino acid sequences similar to that of volkensin, the toxic lectin from Adenia volkensii. These two lectins agglutinate red blood cells, inhibit protein synthesis in a cell-free system as well as in whole cells, and depurinate ribosomes and DNA, but not tRNA or poly(A). They are highly toxic to cells, in which they induce apoptosis and strongly inhibit protein synthesis, and to mice, with LD50s 8.16 mg/kg (lanceolin) and 2.76 mg/kg (stenodactylin) at 48 hours after administration. Thus, lanceolin and stenodactylin have all the properties of the toxic type 2 ribosomeinactivating proteins (RIPs). Further experiments were conducted in order to clarify the effects of these RIPs in cells. We investigated the cronological relationship between cytotoxic activity, indirectly evaluated as inhibition of protein synthesis, and loss of cell viability in NB100 cell line. The induction of apoptosis was assessed by determining caspases 3 and 7 levels, which increase 8-16 hours earlier than the beginning of protein synthesis inhibition. This suggest that the arrest of protein synthesis is not a central event in the pathway of cell poisoning by RIPs. The high toxicity and the induction of cell death only by apoptosis and not by necrosis in two muscular cell lines (TE671 and RD/18) suggest that lanceolin and stenodactylin may be potential candidates for experimental chemoablation in strabism and blepharospasm. These results show that lanceolin and stenodactylin are amongst the most potent toxins of plant origin.

The biological basis of autism spectrum disorders: evaluation of oxidative stress and erytrocyte membrane alterations

This case-control study involved a total of 29 autistic children (Au) aged 6 to 12 years, and 28 gender and age-matched typically developing children (TD). We evaluated a high number of peripheral oxidative stress parameters, erythrocyte and lymphocyte membrane functional features and membrane lipid composition of erythrocyte. Erythrocyte TBARS, Peroxiredoxin II, Protein Carbonyl Groups and urinary HEL and isoprostane levels were elevated in AU (confirming an imbalance of the redox status of Au); other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma Total antioxidant capacity and plasma carbonyl groups, erythrocyte SOD and catalase activities) were unchanged, whilst peroxiredoxin I showed a trend of elevated levels in red blood cells of Au children. A very significant reduction of both erythrocyte and lymphocyte Na+, K+-ATPase activity (NKA), a reduction of erythrocyte membrane fluidity, a reduction of phospatydyl serine exposition on erythrocyte membranes, an alteration in erythrocyte fatty acid membrane profile (increase in MUFA and in ω6/ω3 ratio due to decrease in EPA and DHA) and a reduction of cholesterol content of erythrocyte membrane were found in Au compared to TD, without change in erythrocyte membrane sialic acid content and in lymphocyte membrane fluidity. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity, and ADOS and CARS score are inversely related to peroxiredoxin II levels. Oxidative stress and erythrocyte structural and functional alterations may play a role in the pathogenesis of Autism Spectrum Disorders and could be potentially utilized as peripheral biomarkers.

Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery

Critical lower limb ischemia is a severe disease. A common approach is infrainguinal bypass. Synthetic vascular prosthesis, are good conduits in high-flow low-resistance conditions but have difficulty in their performance as small diameter vessel grafts. A new approach is the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to synthetic and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods. Umbilical cord-derived mesenchymal cells isolated from human umbilical cord tissue were expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize vessel segments. Cells were seeded cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, mimicking the cardiovascular pulsatile flow. After dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Discussion. Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedures