R Lopa

Fondazione IRCCS Ca' Granda - Ospedale Maggiore Policlinico, Milano, Lombardy, Italy

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Publications (17)44.48 Total impact

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    ABSTRACT: The antigens of the Colton blood group system, Co(a) and Co(b), are encoded by a single gene that produces the aquaporin-1 (AQP1) protein, a water channel-forming protein, and are characterized by a single nucleotide polymorphism (SNP). A healthy Caucasoid blood donor originally typed as Co(a-b-) with commercial anti-Co(b) typed Co(a-b+) when retested with another anti-Co(b). Retyped with two different molecular biology methods, the sample came out Co(a)/Co(b). With the aim of understanding these discrepancies, serological, cytometric and molecular biology tests were carried out. Absorption/elution studies with propositus red cells and controls were performed. The region spanning exon 1 to exon 4 of the Colton gene was sequenced, and flow cytometry analyses were carried out. Absorption/elution studies showed the absence of Co(a) and a weak expression of Co(b). DNA sequencing confirmed a CT heterozygosity at nucleotide position 134 (i.e. Co(a)/Co(b)), and an additional heterozygous CT was found at position 112. The presence of the Co(b) allele that encodes for the Co(b) antigen was confirmed. The new allele has the base cytosine at nucleotide 134 (Co(a)), in cis with the new nucleotide 112T. The nucleotide substitution 112C>T causes a missense mutation leading to an amino acid change from proline (CCG) to serine (TCG) at codon 38. The substitution found at codon 38 results in a modified AQP1 protein which explains the Co(a-b+) phenotype and possibly the weak expression of Co(b).
    Vox Sanguinis 03/2010; 99(2):158-62. · 2.85 Impact Factor
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    ABSTRACT: The human liver is a complex tissue consisting of epithelial, endothelial, hematopoietic, and mesenchymal elements that probably derive from multiple lineage-committed progenitors, but no comprehensive study aimed at identifying and characterizing intrahepatic precursors has yet been published. Cell suspensions for this study were obtained by enzymatic digestion of liver specimens taken from 20 patients with chronic liver disease and 13 multiorgan donors. Stem and progenitor cells were first isolated, amplified, and characterized ex vivo according to previously validated methods, and then optimized flow cytometry was used to assess their relative frequencies and characterize their immunophenotypes in the clinical specimens. Stem and progenitor cells committed to hematopoietic, endothelial, epithelial, and mesenchymal lineages were clearly identifiable in livers from both healthy and diseased subjects. Within the mononuclear liver cell compartment, epithelial progenitors [epithelial cell adhesion molecule (EpCAM)(+)/CD49f(+)/CD29(+)/CD45(-)] accounted for 2.7-3.5% whereas hematopoietic (CD34(+)/CD45(+)), endothelial [vascular endothelial growth factor-2 (KDR)(+)/CD146(+)/CD45(-)], and mesenchymal [CD73(+)/CD105(+)/CD90 (Thy-1)(+)/CD45 (-)] stem cells and progenitors accounted for smaller fractions (0.02-0.6%). The patients' livers had higher percentages of hematopoietic and endothelial precursors than those of the donors. In conclusion, we identified and characterized precursors committed to four different lineages in adult human liver. We also optimized a flow cytometry approach that will be useful in exploring the contribution of these cells to the pathogenesis of liver disease.
    Cytometry Part A 12/2009; 77(1):31-40. · 3.71 Impact Factor
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    ABSTRACT: Among the heterogeneous population of circulating hematopoietic and endothelial progenitors, we identified a subpopulation of CD133+ cells displaying myogenic properties. Unexpectedly, we observed the expression of the B-cell marker CD20 in blood-derived CD133+ stem cells. The CD20 antigen plays a role in the modulation of intracellular calcium homeostasis through signaling pathways activation. Several observations suggest that an increase in intracellular calcium concentration ([Ca2+]i) could be involved in the etiology of the Duchenne muscular dystrophy (DMD). Here, we show that a CD20-related signaling pathway able to induce an increase in [Ca2+]i is differently activated after brain derived neurotrophic factor (BDNF) stimulation of normal and dystrophic blood-derived CD133+ stem cells, supporting the assumption of a “CD20-related calcium impairment-affecting dystrophic cells. Presented findings represent the starting point toward the expansion of knowledge on pathways involved in the pathology of DMD and in the behavior of dystrophic blood-derived CD133+ stem cells.
    Cellular and Molecular Life Sciences CMLS 02/2009; 66(4):697-710. · 5.62 Impact Factor
  • Journal of Hepatology - J HEPATOL. 01/2009; 50.
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    ABSTRACT: Volume reduction of cord blood units decreases the cost of cryogenic storage. This study reports the analysis of a 10-year quality control program of a semiautomated cord blood volume reduction procedure. Cord blood was collected in a plastic bag containing 29 mL citrate-phosphate-dextrose, centrifuged at 2124 x g for 12 minutes, and processed with a semiautomated device. The procedure was aimed at removing most red blood cells and plasma and concentrating hematopoietic progenitors in the buffy coat (BC), thus reducing the unit volume and saving cryogenic space. Finally, the BC was cryopreserved with an equal volume of 20 percent dimethyl sulfoxide. Total nucleated cells (TNCs) were counted before and after processing in the 4311 units banked from 1998 through 2007, whereas CD34+ cells and colony-forming units-granulocyte-macrophage (CFU-GM) were counted in 420 random units from 2001 through 2007. Mean postvolume reduction annual recoveries of TNCs, CD34+ cells, and CFU-GM ranged from 82.8 +/- 12.3 (standard deviation) to 91.4 +/- 6.4 percent, from 87.8 +/- 14.1 to 95.2 +/- 23.8 percent, and from 101.5 +/- 51.4 to 117.8 +/- 59.5 percent, respectively. Very strong correlations were found (r > 0.87) between postprocessing versus preprocessing TNCs, CD34+ cells, and CFU-GM; a moderate correlation between initial TNC count and unit's volume (r = 0.51); and no correlation between TNC percentage of recovery in the BC and initial unit's volume. The latter data indicate that most TNCs concentrate in the BC. The semiautomated procedure of cord blood unit volume reduction used in this study provides high and stable cellular recoveries during several years of routine cord blood banking.
    Transfusion 12/2008; 49(3):563-9. · 3.53 Impact Factor
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    ABSTRACT: To evaluate the fully automated FACSCanto software, we compared lymphocyte subpopulation counts obtained using three-color FACSCalibur-CELLQuest and six-color FACSCanto-FACSCanto software techniques. High correlations were observed between data obtained with these techniques. Our study indicated that FACSCanto clinical software is accurate and sensitive in single-platform lymphocyte immunophenotyping.
    Clinical and vaccine Immunology: CVI 08/2008; 15(7):1124-7. · 2.60 Impact Factor
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    ABSTRACT: Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.
    Cell Transplantation 02/2007; 16(6):563-77. · 4.42 Impact Factor
  • Neuromuscular Disorders - NEUROMUSCULAR DISORD. 01/2007; 17(9):876-876.
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    ABSTRACT: Although there is a growing interest on the use of non-heart beating donors to enlarge the liver donor pool, livers with prolonged warm ischaemia time are not currently considered for organ transplantation. We hypothesised that these organs may represent a source of hepatocytes for cell transplantation and/or use in bioartificial liver devices. Thus, we investigated if prolonged ischaemia could influence the recovery and viability of functional hepatocytes dissociated from rat livers. Hepatocytes were isolated from the liver within 15 min after death (t=15 min) and after 4, 8 and 12h of ischaemia. Cells were either maintained in culture or cryopreserved. In all products, we evaluated cell recovery and viability, hepatocyte markers and cellular functions, including albumin and urea production. The number of cells per gram of tissue was similar at 15 min, 4 and 8h, while it was significantly decreased at 12h. About 0.2 x 10(6) viable cells expressing hepatocyte markers and producing albumin and urea were isolated up to 8h of ischaemia per gram of tissue. Recovery of viable and functional hepatocytes seems possible after prolonged ischaemia time. These data warrant the evaluation of hepatocyte isolation from human livers of non-heart beating donors.
    Digestive and Liver Disease 01/2007; 38(12):905-11. · 3.16 Impact Factor
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    ABSTRACT: Transfusion-related acute lung injury (TRALI) is a rare but serious complication which can occur after transfusion of blood components. In this report we describe our flow-cytometry approach to the laboratory diagnosis of a case of TRALI in a recipient of fresh frozen plasma containing human leukocyte antigen (HLA) class II antibodies. The post-transfusion reaction work-up included the direct and indirect Granulocyte Immunofluorescence Test (GIFT) on the recipient's neutrophils collected before and after the reaction and on the serum from the recipient and from all implicated donors; flow-cytometry bead-based screening and identification assay for HLA class I and II antibodies in donor sera and flow cytometry cross-matching on T and B patient's lymphocytes. Finally, we investigated the reactivity of one donor serum, containing HLA class II antibodies, with the patient's neutrophils activated in vitro to induce expression of HLA class II. We found an increased level of IgG bound on patient's granulocytes collected after TRALI, in the absence of detectable granulocyte and HLA class I antibodies in the five implicated donors. One of them showed HLA-DR 1 and -DR 51 antibodies, which determined a positive cross-match with patient's B lymphocytes and in vitro activated granulocytes. Both HLA class II antigens were present in the recipient and absent in the donor. In some pathological conditions, HLA class II antibodies can react with activated granulocytes expressing HLA-DR antigens, and activate TRALI reaction. HLA class II antibodies screening and flow cytometry cross-matching techniques should be added to the current diagnostic algorithm of TRALI.
    European Journal Of Haematology 11/2004; 73(4):295-9. · 2.55 Impact Factor
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    ABSTRACT: Recent evidence indicates that neural stem cell properties can be found among a mammalian skin-derived multipotent population. A major barrier in the further characterization of the human skin-derived neural progenitors is the inability to isolate this population based on expression of cell surface markers. Our work has been devoted to purified human skin-derived stem cells that are capable of neural differentiation, based on the presence or absence of the AC133 cell surface marker. The enriched skin-derived AC133(+) cells express the CD34 and Thy-1 antigens. These cells cultured in a growth medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) proliferate, forming spheres, and differentiate in vitro into neurons, astrocytes, and rarely into oligodendrocytes. Single cells from sphere cultures initiated from human purified AC133(+) cells were replated as single cells and were able to generate new spheres, demonstrating the self-renewing ability of these stem cell populations. Brain engraftment of cells obtained from human purified AC133(+)-derived spheres generated different neural phenotypes: immature neurons and a most abundant population of well differentiated astrocytes. The AC133-derived astrocytes assumed perivascular locations in the frontal cortex. No donor-derived oligodendrocytes were found in the transplanted mouse brains. Several donor small, rounded cells that expressed endothelial markers were found close to the host vessel and near the subventricular zone. Thus, mammalian skin AC133-derived cells behave as a multipotent population with the capacity to differentiate into neural lineages in vitro and, prevalently, endothelium and astrocytes in vivo, demonstrating the great plasticity of these cells and suggesting potential clinical application.
    Journal of Neuroscience Research 09/2004; 77(4):475-86. · 2.97 Impact Factor
  • Digestive and Liver Disease 06/2003; 35(5):370-1. · 3.16 Impact Factor
  • Digestive and Liver Disease. 05/2003; 35(5):370–371.
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    ABSTRACT: In previous studies, we identified a cytokine cocktail including thrombopoietin, Flt-3 ligand, interleukin (IL)-6 and IL-11 in serum-free medium, suitable to induce significant and sustained ex vivo expansion of primitive hematopoietic stem cells (HSCs) from cord blood (CB) for up to 10 weeks. The aim of the present study was to evaluate the effects of cryopreservation on ex vivo expansion of HSCs and their committed progenitors. CD34+ cells were purified from CB units, each of which was processed in part as such and in part as cryopreserved and thawed, then expanded for 5 weeks in serum-free medium with the cytokine cocktail described above. We determined the number of nucleated cells (NC), CD34+, CD34+/38(-)/33(-), CD34+/61+, CD61+ cells and the clonogenic potential. After 2 weeks the median fold expansion of NC, CD34+ and CD34+/38(-)/33(-) cells was around two log both with fresh and cryopreserved CB and the expansion continued similarly until week 5. Our data suggest that this serum free protocol induces similar ex vivo expansion of HSCs and their committed progenitors from both fresh and cryopreserved CB. Our findings can be useful in view of clinical applications, since CB used for transplantation is stored in the cryopreserved state.
    Bone Marrow Transplantation 11/2001; 28(7):693-8. · 3.54 Impact Factor
  • Transfusion 06/2001; 41(5):718-9. · 3.53 Impact Factor
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    ABSTRACT: A large number of European blood centres, including our own, use the buffy-coat method for platelet production. In this article we describe a previously unnoticed phenomenon shown by a proportion of buffy-coats, which display an unusually bright cherry colour and low platelet counts. We performed bacterial cultures, platelet counts, pO2, pCO2 and pH, and evaluated platelet activation by flow cytometry in cherry versus normal-colour (control) buffy-coats. In addition, we compared donor characteristics in the two groups and platelet counts in the packed red blood cells (RBC) obtained from the original donations. Finally, we monitored the frequency of cherry buffy-coats in the bags of three manufacturers, and determined the concordance rate of two trained technicians in detecting cherry buffy-coats. Bacterial cultures were negative. Cherry buffy-coats contained significantly fewer platelets, more O2, less CO2 and had a significantly higher pH than normal buffy coats. Platelet activation was slightly higher in cherry buffy-coats. RBC from donations yielding cherry buffy-coats contained a significantly higher number of platelets than controls. Donor characteristics were not significantly different. Cherry buffy-coats were significantly more frequent with bags from one manufacturer (24%) than from others (9% and 11.6%). The concordance study showed excellent agreement. Our hypothesis is that the cherry colour is caused by O2 accumulation in buffy-coats with low platelet counts. The latter may be caused by platelet activation and aggregation during blood processing. Further work is needed to determine the cause of this phenomenon, its frequency in different laboratories and means to prevent it.
    Vox Sanguinis 02/2001; 80(1):57-60. · 2.85 Impact Factor
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    ABSTRACT: We evaluated the influence of mode of delivery on the hematopoietic stem cell (HSCs) content (colony-forming cells, CFC, and the number of CD34+ cells) of 120 consecutive umbilical cord blood (UCB) units collected in our bank iin 1999. Ninety-eight units were collected after vaginal delivery (VD) and 22 after cesarean section (CS). Table 1 reports mean and SD of the HSCs content of VD and CS units and the results of the statistical analysis:CS-UCB showed a significant higher number of CD34+ cell counts not associated with a corresponding increment of CFC counts.In order to expand this observation we evaluated in another series of 22 VD-UCB and 12 CS-UCB, the percentages of apoptotic (Annexin-V+/7-aminoactinomicin−) and necrotic (Annexin-V+/7-aminoactinomicin+) CD34+ cells by flow-cytometry. Table 2 reports the results (mean and SD) and statistical analysis of this part of the study.Our data suggest that increased % of necrotic CD34+ cells may be the cause of decreased clonogenic potential of CS-UCB.
    Experimental Hematology - EXP HEMATOL. 01/2000; 28(7):73-74.