ALBERT

Description: Saliva is an interesting, non-conventional, valuable diagnostic fluid. It can be collected using standardized sampling device; thus, its sampling is easy and non-invasive, it contains a variety of organic metabolites that reflect blood composition. The aim of this study was to validate a user-friendly method for the simultaneous determination of low molecular weight metabolites in saliva. We have optimized and validated a high throughput, direct, low-cost reversed phase liquid chromatographic method with diode array detection method without any pre- or post-column derivatization. We indexed salivary biomolecules in 35 whole non-stimulated saliva samples collected in 8 individuals in different days, including organic acids and amino acids and other carbonyl compounds. Among these, 16 whole saliva samples were collected by a single individual over three weeks before, during and after treatment with antibiotic in order to investigate the dynamics of metabolites. The concentrations of the metabolites were compared with the literature data. The multianalyte method here proposed requires a minimal sample handling and it is cost-effectiveness as it makes possible to analyze a high number of samples with basic instrumentation. The identification and quantitation of salivary metabolites may allow the definition of potential biomarkers for non-invasive “personal monitoring” during drug treatments, work out, or life habits over time.

Description: Allogeneic stem-cell transplantation is a potential curative option in multiple myeloma (MM). Reduced-intensity conditioning regimens (allo-RIC) result in a lower transplant related mortality (TRM) compared to conventional conditioning, despite of a higher relapse rate. Several prospective studies compared single or tandem autologous stem cell transplantation (SCT) with planned tandem autologous-reduced intensity allogeneic SCT, with discordant results in overall and progression-free survival (OS and PFS). Many studies were conducted using a “mini-allo-SCT”, a regimen containing a low-dose total body irradiation (TBI) and Fludarabine (Flu). Moreover, introduction of new drugs (bortezomib, thalidomide or lenalidomide) in the first decade of 2000 changed the biological history of MM. We analyzed the results of ten-year experience with mini-allo-SCT in patients with MM in our institution. Patients, materials and methods: Between June 2000 and December 2010, 21 patients (9 M, 12 F, median age 54 – range 36-66) received a mini-allo-SCT, 17 from an HLA identical sibling donor, 4 from a MUD full-matched. The source of stem cell was the peripheral blood in all patients. All grafts were not manipulated. At the time of diagnosis, Durie-Salmon (DS) stage was I in 5 patients (23.8%), II in 3 patients and III in 13 patients (61.9%). Disease status at the time of transplant was partial response (PR) in 17 patients (81%), 13 of them in first PR, 4 in second or more PR; 4 patients received allo-SCT as salvage therapy in active ore refractory disease. Eleven patients (52.4%) underwent to transplant after one line of treatment, 5 patients after 2 lines, 5 patients after 3 or more lines. Five patients (23.8%) were treated with new drugs. Auto-SCT is included in previous lines. Nine patients received one auto-SCT before the mini-allo-SCT; ten patients (47.6%) underwent transplantation after two auto-SCT. Two patients were allo-grafted frontline. Conditioning regimen was Flu-TBI in all patients. Graft versus Host Disease (GvHD) prophylaxis consisted on cyclosporine and MMF in all. Results: Overall response rate was 76%, 5 PR and 11 complete remission (CR). One patient developed progression next allo-SCT. Four patients died in the first 100 days after allo-SCT, and they are censored for OS and PFS analyses. Of 17 pre-transplant PR, 11 achieved CR (64%), 4 maintained PR, 2 died before response evaluation. Of 4 patients who underwent allo-SCT in active disease, only 1 obtained a PR, whereas the other 3 patients developed progression or were not-valuable. Six patients (28.6%) developed acute GvHD, but no one died for complicated acute GvHD. Eleven patients (52.4%) had chronic GvHD. Follow up range was from 4 to 96 months. The median time was 19 months. The relapse/progression rate in course of follow up was 29%. Two patients progressed after PR (40%), 3 after CR (5.9%). At the time of the last follow-up 8 patients died (47%), 3 of them for progression of MM. Survival analyses: TRM at 1 and 3 years was respectively 24% and 31%. OS at five years was 51%, with a plateau trend after 3 years (Fig. 1). In univariate statistical analysis, early DS stage at diagnosis (I-II), double auto-transplant, development of chronic GvHD have a significant impact (p value .05 ) Fig. 3a:. OS in patients previously treated or not with new drugs ( p value 〉 .05 ); Fig. 3b: PFS in patients previously treated or not with new drugs ( p value 〉 .05 ) Disclosures No relevant conflicts of interest to declare.

Description: Metabolomic profiling of cell lines has shown many potential applications and advantages compared to animal models and human subjects, and an accurate cellular metabolite analysis is critical to understanding both the intracellular and extracellular environments in cell culture. This study provides a fast protocol to investigate in vitro metabolites of immortalized hippocampal neurons HN9.10e with minimal perturbation of the cell system using a targeted approach. HN9.10e neurons represent a reliable model of one of the most vulnerable regions of the central nervous system. Here, the assessment of their extracellular metabolic profile was performed by studying the cell culture medium before and after cell growth under standard conditions. The targeted analysis was performed by a direct, easy, high-throughput reversed-phase liquid chromatography with diode array detector (RP-HPLC-DAD) method and by headspace solid-phase microextraction–gas chromatography–mass spectrometry (HS-SPME-GC-MS) for the study of volatile organic compounds (VOCs). The analysis of six different batches of cells has allowed to investigate the metabolic reproducibility of neuronal cells and to describe the metabolic “starting” conditions that are mandatory for a well-grounded interpretation of the results of any following cellular treatment. An accurate study of the metabolic profile of the HN9.10e cell line has never been performed before, and it could represent a quality parameter before any other targeting assay or further exploration.