Silk fibroin (SF), a protein-based fiber extracted from Bombyx mori cocoons, has recently emerged with great potential for the biomedical field to be used as a biomaterial processable in a variety of formats and applications, due to its natural characteristics. The aims of the present study were to characterize the structural properties of the SF scaffolds, in the format of porous sponges, and to investigate their feasibility to support the adhesion of mesenchymal stromal/stem cells isolated from human Wharton’s jelly of the umbilical cord (WJ-MSC). Adhesion is a prerequisite for using the SF scaffold as biomaterial for supporting three-dimensional (3D) WJ-MSC cultures for several applications. The integration among micro-computed tomography, confocal analysis, and field emission scanning electron microscopy allowed carrying out a deep investigation based on quantitative morphological parameters and qualitative observations at high resolution. High levels of porosity, interconnection, and contact surface–volume ratio confirmed the appropriateness of the designed SF porous scaffolds as supports for cell cultures. WJ-MSC was demonstrated to be capable of adhering to and colonizing the SF scaffold applicable as a 3D cell culture system, of conducting in vitro experiments in a more controlled environment, and possibly of being used in tissue engineering, regenerative medicine, and applications in oncology.

Background A shift from a standard to a personalized prophylaxis has been increasingly adopted in patients with severe haemophilia A (SHA). This approach has raised the likelihood of a significant variability in the prophylactic approaches and the relative Factor VIII (FVIII) consumptions. The aim of our study was to assess the treatment variability of SHA patients without inhibitors and on prophylaxis regimen in Italy. Material and methods Data reported in the National Registry of Congenital Coagulopathies (NRCC) were analysed to assess treatment distribution within SHA patients without inhibitors, focussing on FVIII consumption in 2017, associated with prophylaxis regimen. The analysis was stratified based on age groups and Italian regions to describe the variability of FVIII consumption in Italy. Results In 2017, the Registry reported the therapeutic plans of 1068 SHA patients without inhibitors on prophylaxis. The mean (95% CI) individual consumption ranges from 123 127 IU (99 736-146 518) in the age group 0-6 years to 345 000 IU (336 000-354 000) in the age group >20 years. A significant FVIII consumption variability was identified within the adult population. Regions with less than 50 patients reported the higher variability in mean FVIII consumption per patient-year within the different age groups. Similar difference in FVIII consumption variability was reported also in the age groups comparing "low," "middle" and "high" patient volume regions. Discussion A reliable estimation of FVIII consumption for patients' treatment is necessary to manage and plan the appropriate budget and keep treatment's costs affordable. However, without the implementation of a methodology aiming to assess the overall value produced by these FVIII consumptions, the scenario will keep driven by FVIII consumptions, its costs and the budget available. An effort by haemophilic community, haemophilia treatment centres and institutions is required to develop and share this cultural shift in improving haemophilia management and assessment.

OBJECTIVES:In a period of important therapeutic changes in the field of haemophilia care, we provide updated statistics on children with severe haemophilia (0-12 years of age) in Italy.METHODS:Data presented are from the Italian National Registry of Congenital Coagulopathies (NRCC) - survey 2017.RESULTS:Children with severe Haemophilia A (HA) were 242, those with severe haemophilia B (HB) 48. Prophylaxis was adopted in 92.1% of individuals with severe HA and 88.6% with severe HB. Thirty-nine children (14.8%) were on treatment for inhibitors. FVIII prescribed to children with severe HA represented 11.1% of the total consumption, of which 4.6% was extended half-life (EHL). FIX given to children with HB accounted for 7.2% of the total FIX, of which 19.1% was EHL-FIX.CONCLUSION:The paediatric population analysed is characterized by a great adherence to therapy, so this data may constitute a benchmark for use of new, alternative therapies in the coming years.

BACKGROUNDIn Italy, the National Register of Congenital Coagulopathies (NRCC) collects epidemiological and therapeutic data from patients affected by haemophilia A (HA), haemophilia B (HB), von Willebrand's disease (vWD) and other rare coagulation disorders. Here we present data from the 2016 annual survey.MATERIALS AND METHODSData are provided by the Italian Haemophilia Centres, on a voluntary basis. Information flows from every Centre to a web-based platform of the Italian Association of Haemophilia Centres, shared with the Italian National Institute of Health, in accordance with current privacy laws. Patients are classified by diagnosis, disease severity, age, gender and treatment-related complications.RESULTSIn 2016, the total number of patients with congenital coagulopathies in the NRCC was 10,360: 39.8% of these patients had HA, 31.5% had vWD, 8.5% had HB, and 20.2% had less common factor deficiencies. The overall prevalence of HA and HB was 13.9/100,000 males and 3.0/100,000 males, respectively. The overall prevalence of vWD was 5.4/100,000 inhabitants. During 2016, 126 patients had current alloantibodies to factor VIII (FVIII) or factor IX (FIX) and were under treatment with bypassing agents and/or immune tolerance induction. Overall, 388 patients with a history of alloantibodies were recorded in the NRCC of whom 337 with severe HA and 12 with severe HB. Coagulation factor use, evaluated from treatment plans, was approximately 451,000,000 IU of FVIII for HA patients (7.5 IU/inhabitant), and approximately 53,000,000 IU of FIX for HB patients (0.9 IU/inhabitant).DISCUSSIONThe prevalences of HA and HB fall within the ranges reported in more developed countries; the consumption of FVIII and FIX was in line with that of other European countries (France, United Kingdom) and Canada. The NRCC, with its bleeding disorder dataset, is a helpful tool for shaping public health policies, as well as planning clinical and epidemiological research projects.

Hemophilia is associated with a high financial burden on individuals, healthcare systems, and society. The development of inhibitors significantly increases the socioeconomic burden of the diseases. This study aimed to review and describe the burden of hemophilia with inhibitors, providing a reference scenario to assess the impact of new products in the real word. Two systematic literature reviews were performed to collect data on (i) health economics and (ii) health-related quality of life evidences in hemophilic patients with inhibitors. The costs associated with patients with hemophilia and inhibitors are more than 3 times greater than the costs incurred in those without inhibitors, with an annual cost per patient that can be higher than (sic) 1000000. The costs of bypassing agents account for the large majority of the total healthcare direct costs for hemophilia treatment. The quality of life is more compromised in patients with hemophilia and inhibitors compared to those without inhibitors, in particular the physical domains, whereas mental domains were comparable to that of the general population. The development of an inhibitor has a high impact on costs and quality of life. New treatments have the potential to change positively the management and socioeconomic burden of hemophilia with inhibitors.

In Italy, the surveillance of people with bleeding disorders is based on the National Registry of Congenital Coagulopathies (NRCC) managed by the Italian National Institute of Health (Istituto Superiore di Sanità). The NRCC collects epidemiological and therapeutic data from the 54 Hemophilia Treatment Centers, members of the Italian Association of Hemophilia Centres (AICE). The number of people identified with bleeding disorders has increased over the years, with the number rising from approx. 7000 in 2000 to over 11,000 in 2015. The NRCC includes 4020 patients with hemophilia A and 859 patients with hemophilia B. The prevalence of the rare type 3 vWD is 0.20/100,000 inhabitants. Less common congenital bleeding disorders include the following deficiencies: Factor I (fibrinogen), Factor II (prothrombin), Factor V, Factor VII, Factor X, Factor XI and Factor XIII, which affect 1953 patients. Hepatitis C Virus (HCV) infection affects 1561 patients, more than 200 of whom have two infections (HCV + HIV). Estimated hemophilia-related drug consumption in 2015 was approx. 550 million IU of FVIII for hemophilia A patients and approx. 70 million IU of FIX for hemophilia B patients. The NRCC, with its bleeding disorder data set, is a tool that can provide answers to fundamental questions in public health, monitoring care provision and drug treatment, as well as facilitating clinical and epidemiological research.

Clinical Pharmacology & TherapeuticsVolume 99, Issue 3 p. 271-272 Letter to the Editor Italian Registries on Bleeding Disorders HJ Hassan, Corresponding Author HJ Hassan Istituto Superiore di Sanità, Rome, ItalyCorrespondence: HJ Hassan (jane.hassan@iss.it)Search for more papers by this authorM Morfini, M Morfini Italian Association of Hemophilia Centers, ItalySearch for more papers by this authorD Taruscio, D Taruscio Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorF Abbonizio, F Abbonizio Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorA Giampaolo, A Giampaolo Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorY Kodra, Y Kodra Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorE Oliovecchio, E Oliovecchio Italian Association of Hemophilia Centers, ItalySearch for more papers by this authorL Vittozzi, L Vittozzi Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this author HJ Hassan, Corresponding Author HJ Hassan Istituto Superiore di Sanità, Rome, ItalyCorrespondence: HJ Hassan (jane.hassan@iss.it)Search for more papers by this authorM Morfini, M Morfini Italian Association of Hemophilia Centers, ItalySearch for more papers by this authorD Taruscio, D Taruscio Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorF Abbonizio, F Abbonizio Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorA Giampaolo, A Giampaolo Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorY Kodra, Y Kodra Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this authorE Oliovecchio, E Oliovecchio Italian Association of Hemophilia Centers, ItalySearch for more papers by this authorL Vittozzi, L Vittozzi Istituto Superiore di Sanità, Rome, ItalySearch for more papers by this author First published: 13 November 2015 https://doi.org/10.1002/cpt.301Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume99, Issue3Big DataMarch 2016Pages 271-272 RelatedInformation

BACKGROUND:The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD).METHODS:Rhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium.RESULTS:MEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells.CONCLUSIONS:Our results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance. Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.

Studies on the role of multipotent mesenchymal stromal cells (MSC) on tumor growth have reported both a tumor promoting and a suppressive effect. The aim of the present study was to determine the effect of MSC isolated from Wharton's jelly of umbilical cord (WJMSC) on lung cancer stem cells (LCSC) derived from human lung tumors: two adenocarcinomas (AC) and two squamous cell carcinomas (SCC). LCSC derived from SCC and AC expressed, to varying extents, the more relevant stem cell markers. The effect of WJMSC on LCSC was investigated in vitro using conditioned medium (WJ-CM): a proliferation increase in AC-LCSC was observed, with an increase in the ALDH+ and in the CD133+ cell population. By contrast, WJ-CM hampered the growth of SCC-LCSC, with an increase in the pre-G1 phase indicating the induction of apoptosis. Furthermore, the ALDH+ and CD133+ population was also reduced. In vivo, subcutaneous co-transplantation of AC-LCSC/WJMSC generated larger tumors than AC-LCSC alone, characterized by an increased percentage of CD133+ and CD166+ cells. By contrast, co-transplantation of WJMSC and SCC-LCSC did not affect the tumor size. Our results strongly suggest that WJMSC exert, both in vitro and in vivo, contrasting effects on LCSC derived from different lung tumor subtypes.

The purpose of this study was to investigate the Wharton's jelly mesenchymal stem cells differentiation ability toward neuronal fate. Human Wharton's jelly mesenchymal stem cells (hWJMSC) have been isolated from human umbilical cord of full‐term births and characterized by flow cytometry analysis for their stem mesenchymal properties through specific surface markers expression (CD73, CD90, and CD105). hWJMSC mesodermal lineage differentiation ability and karyotype analysis were assessed. The trans‐differentiation of hWJMSC into neural lineage was investigated in presence of forskolin, an agent known to increase the intracellular levels of cAMP. A molecular profile of differentiated hWJMSC was performed by microarray technology which revealed 1,532 statistically significant modulated genes respect to control cells. Most of these genes are mainly involved in functional neuronal signaling pathways and part of them are specifically required for the neuronal dopaminergic induction. The acquisition of the dopaminergic phenotype was evaluated via immunocytochemistry and Western blot analysis revealed the significant induction of Nurr1, NeuroD1, and TH proteins expression in forskolin‐induced hWJMSC. Moreover, the treatment with forskolin promoted, in hWJMSC, a strong upregulation of the neurotrophin Trk receptors related to the high release of brain‐derived neurotrophic factor. Taken together these findings show that hWJMSC may be represent an optimal therapeutic strategy for neurological diseases. J. Cell. Physiol. 229: 232–244, 2014. © 2013 Wiley Periodicals, Inc.

ObjectiveThis study aims to investigate the capability of Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSC) to support the in vitro expansion of hematopoietic stem/progenitor cells (HSPC) derived from cord blood (CB) in the absence of exogenous cytokines, and the effect on engraftment of the expanded cells in a mouse model.MethodsCB-CD34+ cells were seeded on WJ-MSC layer and cultured in HP01 serum-free medium. Day-7 and day-13 expanded cells were transplanted in NOD/SCID mice. After 8wk, engraftment was evaluated in mouse bone marrow as percentage of human CD45+ cells.ResultsCD34+ population was expanded without increasing the differentiation rate. Co-culture increased the expansion of the CD34+ cells by 2.0 and 7.3 times after 7 and 13d, respectively, and maintained the CD34+ cells up to day 20. In particular, earlier CD34+/CD90+ and CD34+/CD33- subtypes were increased. An advantage of the day-7 co-cultured HSPC in respect of HSPC at day 0 in the engraftment of NOD/SCID mice was obtained both as percentage of mice engrafted (100% vs. 75%) and as percentage of chimerism.ConclusionsAlthough the increase in hematopoietic progenitors is not dramatic as in the presence of added cytokines, this study demonstrates the effectiveness of the WJ-MSC not only to preserve the CD34+ population but also to improve the repopulating efficacy of the amplified HSPC, also in the absence of added cytokines and growth factors.

Inherited bleeding disorders are a group of congenital coagulopathies resulting when deficiencies of the proteins responsible for coagulation, platelet function or fibrinolysis occur. Haemophilia A (HA, deficiency of factor VIII), Haemophilia B (HB, deficiency of factor IX) and von Willebrand Disease (vWD) are the most frequent, being >90% of all inherited bleeding disorders with a prevalence of 0.5, 0.1 and 1–5/10,000 respectively1–3. Other rare inherited bleeding disorders are represented by deficiencies of factor I, II, V, VII, X, XI, XII, XIII, combined V+VIII, combined vitamin K-dependent factors, with a general population prevalence between 1/500,000 and 1/2,000,0004. Inherited bleeding disorders require wide-ranging care and effective management within a multidisciplinary team setting. The modern treatments of inherited bleeding disorders are now remarkably effective, although expensive. In Italy the epidemiological data on inherited bleeding disorders derive from three registries: The Italian National Registry for Rare Diseases (RNMR); The National Registry of Congenital Coagulopathies (RNCC); The Haemophilia Database of the Italian Association of Haemophilia Centres (AICE). These registries differ in terms of organisation, purpose and data collection process. A general description and results for each registry is reported as follow.

Haemophilia A (HA) patients with high responding inhibitors require therapies with bypassing agents to control bleedings or Immune Tolerance Induction (ITI) to attempt inhibitor eradication and restore FVIII therapy. The aim of this study was to assess the therapeutic management and product consumption of HA inhibitor patients and the relative costs in Italy. A retrospective survey was performed utilizing data from the National Registry of Congenital Coagulopathies and from a specific questionnaire on product consumption of HA inhibitor patients over the year 2011. Among HA patients, 10% had currently detectable inhibitors; 24% of patients were undergoing ITI (mostly children) and 76% utilized bypassing agents. Patients on ITI consumed 45000000IU of FVIII (median consumption/patient of 1200000IUyear-1). Patients receiving bypassing agents utilized 21000000IU of aPCC (median consumption/patient of 360000IUyear-1), and 38000mg of rFVIIa (median consumption/patient of 440mgyear-1). The annual cost/patient on ITI and on bypassing agents therapy was analysed. Recombinant products represen-ted the product of choice for children therapies in >90% of the cases. FVIII prophylaxis of severe HA patients without inhibitor costs about half than therapy with bypassing agents and is three times less expensive than prophylaxis with such agents. Therefore, the possibility to restore FVIII prophylaxis, having eradicated the inhibitor through ITI, can justify the high costs of ITI treatment needed in the short term. Consistent with this notion, over the last years a 50% increase in the number of patients undergoing ITI in Italy was registered.

Hematopoietic stem and progenitor cells (HSPC) can improve the long-term outcome of transplanted individuals and reduce the relapse rate. Valproic acid (VPA), an inhibitor of histone deacetylase, when combined with different cytokine cocktails, induces the expansion of CD34 + cell populations derived from cord blood (CB) and other sources. We evaluated the effect of VPA, in combination with thrombopoietin (TPO), on the viability and expansion of CB-HSPCs and on short- and long-term engraftability in the NOD/SCID mouse model. In vitro, VPA + TPO inhibited HSPC differentiation and preserved the CD34 + cell fraction; the self-renewal of the CD34 + TPO + VPA-treated cells was suggested by the increased replating efficiency. In vivo, short- and long-term engraftment was determined after 6 and 20 weeks. After 6 weeks, the median chimerism percentage was 13.0% in mice transplanted with TPO-treated cells and only 1.4% in those transplanted with TPO + VPA-treated cells. By contrast, after 20 weeks, the engraftment induced by the TPO + VPA-treated cells was three times more effective than that induced by TPO alone, and over ten times more effective compared to the short-term engraftment induced by the TPO + VPA-treated cells. The in vivo results are consistent with the higher secondary plating efficiency of the TPO + VPA-treated cells in vitro.