Adele Giampaolo

The costimulatory molecule B7 is expressed on human microglia in culture and in multiple sclerosis acute lesions

B7 is a constimulatory molecule which is expressed on antigen-presenting cells and which plays a pivotal role in T cell activation and proliferation. To elucidate mechanisms regulating intracerebral immune responses, expression of B7 was examined in cultured microglial cells and in brain tissue from control and multiple sclerosis patients. Using immunocytochemical and polymerase chain reaction techniques, we show that B7 was expressed in cultured microglial cells from the human embryonic brain. Microglia also bound the soluble form of the B7 receptor CTLA-4 (CTLA-4-Ig). B7 gene expression and binding of anti-B7 antibodies and CTLA-4-Ig increased after treatment with interferom B7 was not inducible in human astrocytes. Human microglia expressed other costimulatory molecules, such as intercellular adhesion molecule-I, LFA-I and LFA-3. In sections of multiple sclerosis brains, B7 immunoreactivity was detected on activated microglia and infiltrating macrophages within active lesions. In chronic lesions, only perivascular cells were stained. B7 immunoreactivity was undetectable in sections from Alzheimers disease or normal brain tissue. These data suggest that B7 may be involved in T cell activation and lesion development in multiple sclerosis and that the regulated expression of B7 on microglia may contribute to the local stimulation of T cell proliferation and effector functions.

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.

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

BackgroundThe 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).MethodsRhabdospheres 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.ResultsMEK/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.ConclusionsOur 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.

Factor-V expression in platelets from human megakaryocytic culture.

The origin of platelet‐factor‐V has long been discussed. To elucidate whether and when human platelet‐factor‐V is synthesized by megakaryocytes, we utilized in vitro‐generated megakaryocytes capable of producing platelets. Factor‐V gene was silent in purified progenitors and megakaryocytic precursors but was expressed in late culture phase and maintained also in platelets. Similarly, factor‐V protein was expressed in mature proplatelet‐bearing megakaryocytes (immunofluorescence analysis); it was also detectable in cultured megakaryocytes and platelets (Western blotting) and within permeabilized cultured platelets (flow cytometry). The absence of other cells in our culture system indicates conclusively that human megakaryocytes synthesize factor‐V.

Induction of Dopaminergic Neurons From Human Wharton's Jelly Mesenchymal Stem Cell by Forskolin

The purpose of this study was to investigate the Whartons jelly mesenchymal stem cells differentiation ability toward neuronal fate. Human Whartons 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.

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.

Therapeutic management and costs of severe haemophilia A patients with inhibitors in Italy

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 45 000 000 IU of FVIII (median consumption/patient of 1 200 000 IU year−1). Patients receiving bypassing agents utilized 21 000 000 IU of aPCC (median consumption/patient of 360 000 IU year−1), and 38 000 mg of rFVIIa (median consumption/patient of 440 mg year−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.

Current status of Italian Registries on inherited bleeding disorders.

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.

Cord blood CD34+ cells expanded on Wharton's jelly multipotent mesenchymal stromal cells improve the hematopoietic engraftment in NOD/SCID mice

This study aims to investigate the capability of Whartons 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.

Molecular heterogeneity of beta thalassaemia in the Italian population

Summary Fifty‐one subjects originating from Southern Italy and affected by Cooleys anaemia have been studied in order to define the degree of heterogeneity of β thalassaemia mutations in this high incidence area. Restriction endonuclease mapping has been carried out on genomic DNA by the Southern blot technique both to exclude the existence of gross deletions or rearrangements and to establish the relative frequency of four polymorphic restriction sites (i.e. Gγ and Aγ Hind III, β Ava II and β Bam HI) within the γδβ gene region. In 28 subjects unequivocal linkage of the four polymorphic sites has been determined leading to the identification of seven different chromosome haplotypes, six of which had previously been reported associated with specific β0 and β+ thalassaemia mutations. Globin chain synthesis studies on peripheral blood reticulocytes indicated that subjects carrying the same genotype may behave differently as far as the β chain production is concerned relative to both the a and the non‐α chains. Thus, β thalassaemia turns out to be quite heterogeneous even in this limited geographical area. β+ mutations appear to be predominant, particularly those affecting nuclear precursor RNA splicing to mature β globin mRNA.