Italian Registry of Congenital Bleeding Disorders.

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.

Wharton's jelly mesenchymal stromal cells have contrasting effects on proliferation and phenotype of cancer stem cells from different subtypes of lung cancer.

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.

Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population.

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.

Induction of dopaminergic neurons from human Wharton's jelly mesenchymal stem cell by forskolin.

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.

Valproic acid affects the engraftment of TPO-expanded cord blood cells in NOD/SCID mice.

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.

Italian national survey of blood donors: external quality assessment (EQA) of syphilis testing.

The detection of syphilis among blood donors may reveal high-risk sexual behavior, which can go unreported at the time of donor selection and compromise the safety of the donated blood. In Italy, blood is collected, tested, and distributed by transfusion services (TSs), which also perform outpatient transfusions. Although the TSs must screen for syphilis by law, there are no indications of the specific type of method to be used, generating discrepancies in the results obtained by the different TSs. To determine the proficiency of the TSs in screening for syphilis, we performed an external quality assessment (EQA). The EQA was based on two shipments of serum panels; 133 and 118 of the 326 existing TSs participated in the first and second shipments, respectively. Each panel consisted of both positive and negative serum samples. The results confirmed that the use of a single nontreponemal test (the Venereal Disease Research Laboratory [VDRL] and the rapid plasma reagin [RPR] tests) is the least sensitive means of identifying samples that are positive for syphilis antibodies. We also found that the interpretation of the results of manual techniques, such as the RPR test, the VDRL test, the Treponema pallidum hemagglutination (TPHA) assay, and the T. pallidum particle agglutination (TPPA) assay, can vary greatly among different TSs and operators. Total Ig enzyme immunoassays (EIAs) are the most sensitive. However, the determination of syphilis on the basis of the results of a single test is not sufficient for an accurate screening; and all blood units should thus be assessed by two distinct treponemal tests, that is, a total Ig EIA and the TPHA or the TPPA assay.

Long-term platelet production assessed in NOD/SCID mice injected with cord blood CD34+ cells, thrombopoietin-amplified in clinical grade serum-free culture.

OBJECTIVE: Delayed platelet recovery post-cord blood (CB) transplantation might be due to CB characteristics: low maturity of stem cell compartment, poor production of CD34+/CD41+ cells when induced to differentiate along the megakaryocytic (MK) lineage, retention of a low ploidy in the expanded MKs. Ex vivo expansion of CB hematopoietic progenitor cells for reconstitution of different human hematopoietic lineages has already been developed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. However, optimal conditions for MK-progenitor engraftment to reduce hemorrhaging risk still to be developed. This study assesses the hypothesis that CB-CD34+ amplification with thrombopoietin (TPO) can be applied to a portion of a CB transplant unit to stimulate recovery along MK differentiation program. MATERIALS AND METHODS: Human CB-CD34+ cells were amplified in a serum-free, clinical grade medium with 100 ng/mL TPO alone and in addition to other cytokines (Kit ligand, interleukin-6, and Flt-3 ligand). Seven-day cultured cells were transplanted into irradiated NOD/SCID mice and engraftment, megakaryocytopoiesis, and platelet production were assessed. RESULTS: Platelet release was successful and continuously present for at least 8 weeks in NOD/SCID mice transplanted with CB cells stimulated by TPO. Thrombocytopoiesis was more effective with transplanted TPO-amplified cells than with the cytokine cocktails. CONCLUSION: Platelet number obtained is within the minimum level considered sufficient for hemostasis. Furthermore, amplified cells maintain their self-renewal capacity and multilineage potential differentiation. Thus, transplantation of TPO-expanded CB cells has the potential favoring both platelet recovery and human engraftment.

The first data from the haemovigilance system in Italy.

BACKGROUND: Haemovigilance is defined as the surveillance of adverse reactions occurring in donors and in recipients of blood components and as epidemiological surveillance of donors. The ultimate purpose of haemovigilance is to prevent the repetition of adverse events and reactions. Since the 2002/98/EC Directive came into force, the introduction of haemovigilance systems has become a priority for all countries in the European Community. The Italian haemovigilance system is essentially in line with the Directive, although it does not include surveillance of adverse events in donors and does not have a national level of registration of severe incidents connected with the collection, processing and storage of blood and blood components. Epidemiological surveillance of donors has been performed nationally since 1989 for HIV and since 1999 for HBV, HCV and Treponema pallidum. Surveillance of adverse events in recipients was started at the end of 2004. MATERIALS AND METHODS: The national form proposed for notifying adverse reactions (PETRA) was prepared by the National Institute of Health and distributed to all Transfusion Structures. RESULTS: The data collected (adverse reactions, errors, and near miss errors) came from 21.0% of the Transfusion Structures in 2004 and 38.4% in 2005. The system monitored 49.6 % of all the units distributed in Italy. Overall 1,495 adverse reactions were reported, which is equivalent to 0.8 reactions/1,000 units of blood components distributed. There were 16 reports of errors involving transfusions to the wrong patient. Not all the Transfusion Structures sent their data using the PETRA form. From the 986 PETRA forms received, it was possible to analyse the relevance of the transfusion, the outcome of the patient, the type of blood component involved, the type of error and the type of near miss error. CONCLUSIONS: This study is the first Italian report on transfusion errors and adverse reactions.

Residual risk of transfusion-transmitted human immunodeficiency virus, hepatitis C virus, and hepatitis B virus infections in Italy.

BACKGROUND: Estimating the risk of transfusion-transmitted infections (TTIs) is essential for monitoring blood safety. The residual risk of TTI was estimated for nearly 90 percent of the blood supply in Italy. STUDY DESIGN AND METHODS: Data were analyzed from 1,079,281 repeat donors, corresponding to 5,361,000 donations made in blood transfusion centers throughout Italy in the period 1999 through 2001. The residual risk of transfusion-transmitted human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections was estimated with the incidence rate-window period model. The denominator for the incidence rate (i.e., the number of person-years at risk) was estimated on a sample of 5850 donors. RESULTS: The risk of an infectious donation entering the blood supply, per 1 million donations, was 1.91 (probable range, 0.52-3.32) for HIV, 16.74 (9.57-24.01) for HCV, and 69.16 (43.12-102.70) for total HBV (adjusted for vaccination and hepatitis B surface antigen transience). CONCLUSION: In Italy, the estimated residual risk of TTI is apparently low, particularly for HIV infection. Although the estimated risks are higher for HCV and HBV, the introduction of mandatory viral detection tests for HCV in 2002 should account for an 80 percent reduction in the HCV risk. Moreover, the ongoing HBV vaccination program will contribute to reducing the risk of transfusion-transmitted HBV.