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Background: In the last decades the replacement of fetal bovine serum (FBS) with human Platelet Lysate (hPL) for ATMPs expansion has been for a long time investigated to overcome FBS-related issues. Despite several studies confirming hPL safety and efficacy in Mesenchymal Stromal Cell (MSC) expansion, there are still gaps in the knowledge of hPL as a supplement, like the composition and release criteria. As growth factors are released after thrombocytes lysis during hPL production, starting platelet concentration may affect hPL quality. This study aimed to investigate hPL starting platelet concentration effects on bone marrow-derived MSC (BM-MSC) ex-vivo expansion. Methods: MSC were isolated from the bone marrow (BM) of 7 donors and cultured from passage 1 to 5 in 4 different conditions: DMEM 10% FBS and DMEM 5% hPL varying starting platelet concentration. Particularly hPL was produced by in-hospital Transfusion Service, in three different starting platelet concentrations (sPLTC): high (4x109 PLTS/ml), medium (2x109 PLTS/ml) and low (1x109 PLTS/ml). The study focused on the analysis of parameters that are mostly affected by hPL such as cell proliferation, immunophenotype, telomeric length, differentiation and senescence. Results: Evaluation of proliferation indexes (PDT and PD) underlined dose-dependent effects of sPLTC, also confirmed by flow cytometry cell cycle analysis. Immunophenotype seems not to be affected by sPLTC. Differences were instead detected by adhesion molecules markers CD10, CD106, CD166, and CD146 expression, as their expression showed a dose-dependent downregulation, based on the sPLTC. Differentiation potential seems to be unaffected by different sPLTC as all cell batches differentiated into osteoblasts, adipocytes and chondrocytes. On the contrary senescence and relative telomeric length RTL (detected by SA-β-GAL activity and PNA-FITC flow cytometry) are strongly affected by sPLTC, in a dose-dependent manner. Particularly high sPLTC results in cell senescence associated with decreased RTL. Conclusions: our data showed that sPLTC affects some BM MSCs properties, underlying its importance during hPL preparation. According to this study, we suggest a medium sPLTC for hPL preparation, as the best compromise between the increase in proliferation index and effects on senescence.

Introduction: Telomere length (TL) is a potential indicator of cancer predisposition; however, the multitude of techniques used to measure it causes the results to be heterogeneous and, in some cases, controversial. In the last years, several studies adopted a strategy based on TL-associated genetic variants to generate a polygenic score, often referred as teloscore, used in lieu of direct TL measurement. For pancreatic neuroendocrine neoplasms (PanNEN), this strategy has not been attempted yet. Methods: A teloscore was generated using 11 SNPs (NAF1-rs7675998, ZNF676-rs409627, TERC-rs10936599, CTC1-rs3027234, PXK-rs6772228, DHX35-rs6028466, OBFC1-rs9420907, ZNF208-rs8105767, ACYP2-rs11125529, TERT-rs2736100, and ZBTB46-rs755017), and 291 PanNEN cases and 1,686 controls collected by the PANcreatic Disease ReseArch (PANDoRA) consortium were genotyped to analyse the association of the teloscore and its individual SNPs with the risk of developing PanNEN. Results: An association between genetically determined long telomeres and the risk of developing PanNEN (OR = 1.99, CI: 1.33–2.98, p = 0.0008) for highest versus median (third) quintile was observed. In addition, two novel SNPs associated with PanNEN risk were identified: ZNF676-rs409627 (ORC/C_vs_G/G = 2.27, CI: 1.58–3.27, p = 8.80 × 10−6) and TERT-rs2736100 (ORC/A_vs_C/C = 2.03, CI: 1.42–2.91, p = 1.06 × 10−4). Conclusion: In conclusion, this study provides for the first time a clear indication of the association between long genetically determined telomeres and increased risk of developing PanNEN.

Background Manufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. Materials and methods The study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. Results MycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. Conclusions MycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

IntroductionOnly a small number of risk factors for pancreatic ductal adenocarcinoma (PDAC) has been established. Several studies identified a role of epigenetics and of deregulation of DNA methylation. DNA methylation is variable across a lifetime and in different tissues; nevertheless, its levels can be regulated by genetic variants like methylation quantitative trait loci (mQTLs), which can be used as a surrogate.Materials and methodsWe scanned the whole genome for mQTLs and performed an association study in 14 705 PDAC cases and 246 921 controls. The methylation data were obtained from whole blood and pancreatic cancer tissue through online databases. We used the Pancreatic Cancer Cohort Consortium and the Pancreatic Cancer Case–Control Consortium genome-wide association study (GWAS) data as discovery phase and the Pancreatic Disease Research consortium, the FinnGen project and the Japan Pancreatic Cancer Research consortium GWAS as replication phase.ResultsThe C allele of 15q26.1-rs12905855 showed an association with a decreased risk of PDAC (OR=0.90, 95% CI 0.87 to 0.94, p=4.93×10−8in the overall meta-analysis), reaching genome-level statistical significance. 15q26.1-rs12905855 decreases the methylation of a 'C-phosphate-G' (CpG) site located in the promoter region of theRCCD1antisense (RCCD1-AS1) gene which, when expressed, decreases the expression of the RCC1 domain-containing (RCCD1) gene (part of a histone demethylase complex). Thus, it is possible that the rs12905855 C-allele has a protective role in PDAC development through an increase ofRCCD1gene expression, made possible by the inactivity ofRCCD1-AS1.ConclusionWe identified a novel PDAC risk locus which modulates cancer risk by controlling gene expression through DNA methylation.

Book chapter revisiting perforators with hemodynamic view of it.&nbsp

Book chapter discussing reflux patterns and types of reflux to prepare for saphenous preserving procedures.&nbsp

Background: Hematopoietic progenitor cells (HPCs) cryopreservation have applications, especially in the autologous setting, allowing therapeutic use several years after collection. Cryopreservation...

Background PBSC collection using a blood cell separator in very low weight patients can be frequently complicated by severe adverse effects and technical difficulties. Material and methods From March 2013 to January 2017, 14 PBSC collections were performed in 12 children weighing less than 10 kg, affected by different solid tumours. PBSC collection was performed with a "homemade" aseptically assembled circuit. The circuit is composed by a 150 mL collection bag connected with a 4 stopcock ramp, perfused with ACD. This circuit allows collection of a specific total blood amount from CVC, depending on CD34+ /kg target. Results Mean CD34+ cell performance per collection was 9.3 × 106 /kg. Tolerance to the procedure was very good as none of the patients experienced complications, with the exception of a patient who showed mild cyanosis and pallor after collection. Moreover, no bleeding or thrombotic complications have been observed. To date, 16 PBSC reinfusions have been performed in 7 children with a mean CD34+ cells viability of 98.1% ± 2.7 and mean WBC viability of 57% ± 10. Cell recovery after thawing was 87% ± 10.8. A rapid graft intake for both neutrophils and platelets, between day 7 and 20 after reinfusion was observed. Discussion The procedure of total blood collection without the use of a cell separator is feasible and allows a good PBSC collection without significant side effects in very low-weight children. Moreover, this method could represent a valid and safe alternative to leukapheresis in patients where classic procedure could be difficult to apply.

Background & Aim Background In the last decades the replacement of fetal bovine serum (FBS)with human Platelet Lysate (hPL) for ATMPs clinical expansion has been for a long time investigated to overcome FBS related issues.Despite several studies confirm hPL safety and efficiency in Mesenchymal Stromal Cell(MSC)expansion, there are still some gaps in the knowledge of hPL as cell culture supplement, starting from composition and quality release criteria. Aim of the study As growth factors are released after thrombocytes lysis during hPL production, starting platelet concentration may affect hPL quality.The aim of this study was to investigate hPL starting platelet concentration effects on bone marrow derived MSC(BM-MSC). Methods, Results & Conclusion Material and methods MSC were isolated from the bone marrow of 7 donors and cultured from p1 to p7 in 4 different conditions: DMEM 10% FBS and DMEM 5% hPL varying starting platelet concentration.Particularly hPL was produced according to standardized protocol by in-hospital Transfusion Service, in three different starting platelet concentrations (sPLTC): high sPLTC(4 × 109 PLTS/ml), medium(2 × 109 PLTS/ml) and low(1 × 109 PLTS/ml).The study focused on analysis of parameters that are mostly affected by hPL such as proliferation, immunophenotype, telomeric length, differentiation and cell senescence. Results and Conclusions Evaluation of proliferation indexes(PDT and PD) underlined dose dependent effects of sPLTC (figure 1), also confirmed by flow cytometry cell cycle analysis of BrdU incorporation. Immunophenotype seems not to be affected by sPLTC, as classical MSC markers meet ISCT criteria (table 1). Differences were instead detected by analyzing mean fluorescence intensity (MFI) in a dose dependent manner (figure 2), both for intrinsic and extrinsic parameters. Differentiation potential seems to be unaffected by different sPLTC as all cells batches differentiated into osteoblasts, adipocytes and chondrocytes. On the contrary senescence and relative telomeric length RTL (detected by SA-β-GAL activity and PNA-FITC flow cytometry) are strongly affected by sPLTC, in a dose dependent manner. Particularly high sPLTC results in increased senescent cells associated with decreased RTL (figure 3). In conclusion our data showed that sPLTC affects some BM MSCs properties, underlying its importance during hPL preparation. According to this study we suggest a medium sPLTC for hPL preparation, as best compromise between increase in proliferation index and effects on senescence.

The GvHD is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) represents an alternative therapeutic strategy to immunosuppressive therapy. Although ECP is used since 1990s, the mechanism of action has not yet been completely clarified. We analyzed cells collected from 20 ECP procedures of 4 patients affected by chronic GvHD and, for comparison, Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy donors undergoing from same type of photochemiotherapy, evaluating by flow cytometry, the effects before and after photoactivation with 8-MOP. The analysis showed a significant increase in cell death after ECP in particular in CD4 T lymphocytes as described in literature correlated with haematocrit value. Most interesting data emerge from the analysis of cytotoxic activity of NK cells, using flow cytometry analysis of surface expression of CD107a in the presence of target cells (K562). In all analyzed samples it was possible to document a statistically significant reduction of the cytotoxic activity of NK cells after photoactivation. The decrease of the cytotoxic activity was related to hematocrit value of leukoapheresis: in fact, lower HCT values were associated with a more marked reduction of cytotoxic activity. The study confirms literature data about the increase of cellular mortality induce by ECP. Furthermore, for the first time it is demonstrated that the ECP exerts a marked and significant inhibitory effect on the cytotoxic activity of NK cells. Our study suggests that lower values of hematocrit are associated with better treatment outcome.

Background The use of platelet lysate (PL) for the ex-vivo expansion of mesenchymal stromal/stem cells (MSCs) was initially proposed by Doucet et al. in 2005, as an alternative to animal serum. Moreover, regulatory authorities discourage the use of fetal bovine serum (FBS) or other animal derivatives, to avoid risk of zoonoses and xenogeneic immune reactions. Even if many studies investigated PL composition, there still are some open issues related to its use in ex-vivo MSC expansion, especially according to good manufacturing practice (GMP) grade protocols. Methods As an authorized cell factory, we report our experience using standardized PL produced by Azienda Ospedaliero Universitaria Meyer Transfusion Service for MSC expansion according to a GMP grade clinical protocol. As suggested by other authors, we performed an in-vitro test on MSCs versus MSCs cultured with FBS that still represents the best way to test PL batches. We compared 12 MSC batches cultured with DMEM 5% PL with similar batches cultured with DMEM 10% FBS, focusing on the MSC proliferation rate, MSC surface marker expression, MSC immunomodulatory and differentiation potential, and finally MSC relative telomere length. Results Results confirmed the literature data as PL increases cell proliferation without affecting the MSC immunophenotype, immunomodulatory potential, differentiation potential and relative telomere length. Conclusions PL can be considered a safe alternative to FBS for ex-vivo expansion of MSC according to a GMP grade protocol. Our experience confirms the literature data: a large number of MSCs for clinical applications can be obtained by expansion with PL, without affecting the MSC main features. Our experience underlines the benefits of a close collaboration between the PL producers (transfusion service) and the end users (cell factory) in a synergy of skills and experiences that can lead to standardized PL production. Electronic supplementary material The online version of this article (10.1186/s13287-018-0863-8) contains supplementary material, which is available to authorized users.

The thyrocytes are exposed to high levels of oxidative stress which could induce DNA damages. Base excision repair (BER) is one of the principal mechanisms of defense against oxidative DNA damage, however recent evidences suggest that also nucleotide excision repair (NER) could be involved. The aim of present work was to identify novel differentiated thyroid cancer (DTC) risk variants in BER and NER genes. For this purpose, the most strongly associated SNPs within NER and BER genes found in our previous GWAS on DTC were selected and replicated in an independent series of samples for a new case-control study. Although a positive signal was detected at the nominal level of 0.05 for rs7689099 (encoding for an aminoacid change proline to arginine at codon 117 within NEIL3), none of the considered SNPs (i.e. rs7990340 and rs690860 within RFC3, rs3744767 and rs1131636 within RPA1, rs16962916 and rs3136166 in ERCC4, and rs17739370 and rs7689099 in NEIL3) was associated with the risk of DTC when the correction of multiple testing was applied. In conclusion, a role of NER and BER pathways was evoked in the susceptibility to DTC. However, this seemed to be limited to few polymorphic genes and the overall effect size appeared weak.

Background Leukapheresis for autologous stem cell transplantation represents an efficient technique for the reconstitution of haematopoietic system in patients subjected to a high-dose chemotherapy for the treatment of haematological malignancies. The current regulations emphasise first steps of leukapheresis procedure but do not recommend methods for thawing, only suggesting that it must be performed as soon as possible in a 37 °C thermostatic bath. Aim of the Study We compared the classic method of thawing with an innovative and fully traceable method that uses WSCFD ® Stem Cell Fast Thawer KW. Materials and Methods The first part of the study was focused on the thermodynamic process of the two methods, thawing 6 "simulated" leukapheresis (buffy coats of healthy donors cryopreserved with saline solution, 5% HSA and 5% DMSO) and analysing the thawing curve obtained, by using an inside probe. In the second part, we focused on the recovery of viable CD34+ cells and leukocytes, thawing 20 real leukapheresis from paediatric patients. In this phase we also analyse final core bag temperature, time of procedure, cellular recovery with ISHAGE single platform flow cytometry assay and clonogenic potential performing a CFU assay. Results We found no significant differences between the two methods, both for thermodynamic aspect and cellular recovery. Thawing curves were similar and the paired Student's-t test used for statistical analysis showed a CD34+ cells recovery of 92.2% ± 11.4 using WSCFD ® versus 90% ± 11.1 of thermostatic bath. Data were similar even for leukocytes recovery (80.8% ± 9.5 with WSCFD ® and 79.2% ± 14.4 with thermostatic bath). All thawed products never exceeded the core temperature of 30 °C and no differences were found about the post-thaw clonogenic potential (614 × 10 4 ± 98.3 total CFU using WSCFD ® versus 592 × 10 4 ± 78.5 using thermostatic bath). The only difference observed was about the thawing time: WSCFD method requires a slightly longer time but, on the other hand, it correlates with reduced mean increase in temperature per minute, as a result of a more linear thawing curve. Conclusions WSCFD ® can replace the 37 °C thermostatic bath thawing procedure for leukapheresis, providing more security and fully traceable process data.

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Talvolta il rapporto tra il Medico e la Chimica non e dei piu rosei, e cio forse deriva dal fatto che tale materia spesso e stata insegnata nel corso degli anni dell’Universita senza collegamenti con la realta in cui vive il futuro Medico: la Chimica si muove su materiale “non vivente” e il Medico e portato a pensare che non serva alla sua futura missione.