A miRNA Signature in Human Cord Blood Stem and Progenitor Cells as Potential Biomarker of Specific Acute Myeloid Leukemia Subtypes.

MicroRNAs (miRNAs) are important regulators of several cellular processes. During hematopoiesis, specific expression signatures have been reported in different blood cell lineages and stages of hematopoietic stem cell (HSC) differentiation. Here we explored the expression of miRNAs in umbilical cord blood stem (HSC) and progenitor cells (HPC) and compared it to unilineage granulocyte and granulo-monocyte differentiation as well as to primary blasts from patients with acute myeloid leukemia (AML). CD34 + CD38- ad CD34 + CD38 + cells were profiled using a global array consisting of about 2000 miRNAs. An approach combining bioinformatic prediction of miRNA targets with mRNA expression profiling was used to search for putative biologically enriched functions and networks. At least 15 miRNAs to be differentially expressed between HSC and HPC cell population, a cluster of 7 miRNAs are located in the q32 region of human chromosome 14 (miR-377-3p, -136-5p, 376a-3p, 495-3p, 654-3p, 376c-3p and 381-3p) whose expression decreased during the early stages of normal myelopoiesis but were markedly increased in a small set of AML. Interestingly, miR-4739 and -4516, two novel microRNA whose function and targets are presently unknown, showed specific and peculiar expression profile during the hematopoietic stem cells differentiation into unilineages and resulted strongly upregulated in almost all AML subsets. miR-181, -126-5p, -29b-3p and -22-3p resulted dis-regulated in specific leukemias phenotypes. This study provides the first evidence of a miRNA signature in human cord blood stem and progenitor cells with a potential role in hematopoietic stemness properties and possibly in leukemogenesis of specific AML subtypes.

Ribavirin suppresses erythroid differentiation and proliferation in chronic hepatitis C patients.

Combination therapy with pegylated interferon (pegIFN) plus ribavirin (RBV) is the standard of care for chronic hepatitis C. One of the major treatment-related side effects is anaemia, attributed to RBV-induced haemolysis. However, haemolysis biomarkers are not present in all patients supporting the existence of other pathogenetic mechanisms. We studied the role of RBV in inducing haemolysis and its effects on erythropoiesis. In 18 hepatitis C virus (HCV) genotype 2 patients treated with pegIFN-alpha-2a (180 mcg/week) plus RBV (800 mg/day) for 24 weeks and in 10 hepatitis B virus (HBV) patients treated with pegIFN-alpha-2a (180 mcg/week) for 48 weeks, haemolysis was assessed by serum LDH, haptoglobin and reticulocyte count. Erythropoiesis was evaluated both ex vivo, analysing the clonogenic activity of patients' erythroid progenitors, as well as in vitro adding pegIFN and RBV to liquid cultures obtained from CD34+ cells of healthy volunteers. The majority of patients developed anaemia; the week 4 mean haemoglobin decrease was greater in HCV than in HBV patients (1.7 vs 0.47 g/dL, P = 0.01). Only three HCV patients (17%) and no HBV patients showed signs of haemolysis. The 15 nonhaemolytic HCV patients and all HBV patients showed a delay in erythroid differentiation, with a reduction in colony number and a relative increase in undifferentiated colony percentage. Haemolytic HCV patients had an increase in colony number at week 4 of therapy. In vitro, erythroid cell proliferation and differentiation were inhibited by both pegIFN and RBV. Both pegIFN and RBV have an inhibitory effect on erythroid proliferation and differentiation.

CD20-related signaling pathway is differently activated in normal and dystrophic circulating CD133(+) stem cells.

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 ([Ca(2+)](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 [Ca(2+)](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.

Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients.

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.

Animal model for liver cell banking from non-heart beating donors after prolonged ischaemia time.

BACKGROUND AND AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Evaluation of the effect of cryopreservation on ex vivo expansion of hematopoietic progenitors from cord blood.

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.

High frequency of anti-endomysial reactivity in candidates to heart transplant.

BACKGROUND: A possible link between coeliac disease and dilated cardiomyopathy has recently been suggested. AIMS: . To assess the frequency of anti-endomysial antibodies, the marker for coeliac disease, in patients with different forms of heart failure, and to establish the clinical features of those endomysial antibody positive. SUBJECTS AND METHODS: . A total of 642 consecutive patients entering the waiting list for heart transplantation from 1995 through 1997 were studied. The prevalence of endomysial IgA antibodies, determined by indirect immunofluorescence, was compared to that observed in three surveys conducted in the Italian general population. RESULTS: Of the 642 patients, 12 (1.9%; 95% confidence interval 0.97-3.2) resulted endomysial antibody positive, versus 34/9,720 healthy controls (0.35%; 95% confidence interval, 0.23-0.47), accounting for a relative risk of 5.3 (95% confidence interval, 2.8-10.3). Anti-endomysial antibodies were found in 6/275 patients with dilated cardiomyopathy and 6/367 with other forms of heart failure (2.2% versus 1.6%; 95% confidence interval 0.8-4.7 and 0.6-3.5), with no statistical difference. The 12 endomysial antibody positive patients were leaner (body mass index, 22.0 +/- 1.9 vs 24.2 +/- 3. 1, p<0. 05) than 36 seronegative patients matched for baseline demographics and aetiology of cardiomyopathy No differences were observed as regards clinical, biochemical and echocardiographic features, mortality in waiting list and 2-year post-transplant survival. CONCLUSIONS: Patients with end-stage heart failure are at increased risk for coeliac disease as compared to the general population.

Evaluation of a multicolor, single-tube technique to enumerate lymphocyte subpopulations.

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.

Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection.

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin-derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP-labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L-lactide and trimethylene carbonate (PLA-TMC) copolymer and a collagen-based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S-100 and glial fibrillary acidic protein. RT-PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.

Frozen platelet plates for platelet antibody detection and cross-match.

We performed 2 studies aimed at developing a frozen platelet panel suitable for platelet cross-matching. The stability of the most important platelet membrane glycoproteins and the reactivity of antigens of the human platelet antigen (HPA) and of the human leukocyte antigen (HLA) systems were evaluated with the platelet suspension immunofluorescence test (PSIFT) in a panel of platelets frozen in microplates with 6% dimethylsulfoxide. In study No. 1 we evaluated platelet reaction with a broad-spectrum weak anti-HLA and a potent anti-HPA-1a antiserum and the expression of glycoproteins Ib and IIb/IIIa complex on platelet membrane before freezing and after 0.5, 1, 2, 3, 4, 5, 6 and 12 months of storage at -80 degrees C. In study No. 2 we examined platelet reactivity with anti-HPA-1b, -HPA-2a, -HPA-3a, -HLA-A2, -HLA-A3 of platelets stored frozen for 12 months in parallel with fresh platelets from the same donors. Study No. 1 showed that glycoprotein expression was stable and that the weak anti-HLA and the potent anti-HPA-1a antibodies were clearly detected during 12 months at -80 degrees C. Of the 35 paired PSIFT performed in study No. 2 with fresh and frozen/thawed platelets incubated with anti-HPA-1b, -HPA-2a, -HPA-3a, -HLA-A2, -HLA-A3 antisera and AB serum, concordant reactions were obtained in all cases with the exception of 1 case of HLA-A3-positive platelets incubated with anti-HLA-A3 antiserum, that was reactive with frozen/thawed platelets but nonreactive with fresh platelets from the same donor.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet quality after 15-day storage of platelet concentrates prepared from buffy coats and stored in a glucose-free crystalloid medium.

It has been reported previously that platelet concentrates (PCs) prepared from buffy coat pools diluted in a simple, glucose-free medium (BC-PCs) are effective in thrombocytopenic patients after 4 to 12 days of storage. Such preparations produce platelet increments similar to those of traditional PCs prepared from platelet-rich plasma (PRP-PCs) stored for 1 to 3 days. The purpose of this study was to obtain a series of in vitro measurements during storage to allow a more detailed characterization of BC-PCs and a more detailed comparison of BC-PCs with PRP-PCs. At the beginning of storage, the level of (alpha granule membrane protein-140 (GMP-140), a marker of platelet activation, was significantly higher on PRP-PC platelets, and BC-PCs were superior in measurements reflecting platelet function, such as osmotic reversal, ATP release, and aggregation with collagen. Compared to PRP-PCs, BC-PCs were superior in the percentage of discs, total ATP, and glycoprotein lb expression by Day 7. This superiority became more striking on Day 11. Overall, 15-day-old BC-PCs compared favorably to 7-day old PRP-PCs: BC-PCs were superior in ATP release and aggregation with collagen, but they were not significantly different for all other measurements reflecting platelet quality and function. Thus, the quality of platelets in BC-PCs was superior on Day 1, and this superiority progressed as storage continued. In addition, the metabolism of BC-PCs was favorable.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of platelet activation and membrane glycoprotein Ib and IIb-IIIa expression after filtration through three different leukocyte removal filters.

We evaluated three filters used for leukocyte removal from platelet concentrates: Imugard IG 500, Pall PL100 and Sepacell PL-10A. Filter performance, platelet activation and expression of membrane glycoproteins Ib and IIb-IIIa were evaluated. Imugard, Pall and Sepacell showed median postfiltration in vitro platelet recoveries of 88, 84 and 80%, and total residual leukocyte counts of 16.1, 7.5 and 0.6 x 10(6)/pool of 8 platelet concentrates, respectively. Mean platelet volume was reduced after filtration with all filters. Postfiltration values of glycoproteins Ib and IIb-IIIa, and of activation markers GMP 140 and gp 53 were not significantly different from prefiltration values. Filtration through Imugard, Pall and Sepacell did not induce significant platelet activation or modifications of platelet membrane glycoproteins Ib and IIb-IIIa.

Long-term expansion and maintenance of cord blood haematopoietic stem cells using thrombopoietin, Flt3-ligand, interleukin (IL)-6 and IL-11 in a serum-free and stroma-free culture system.

Although cord blood (CB) compares favourably with other haematopoietic stem cell (HSCs) sources, its use in large patients is limited by the low number of cells available. Ex vivo expansion of CB HSCs has been used to overcome this limitation. In this study, we investigated the effect of different cytokine cocktails, including interleukin (IL)-6, IL-11, Flt3-ligand (FL) and thrombopoietin (TPO) combined with serum or serum-free medium on the ex vivo expansion of CD34+ cells from CB. Initial experiments showed that expansion could be slightly improved using serum, but we chose to use serum-free medium in the subsequent investigations to apply good medical practice (GMP) conditions suitable for clinical use. The highest expansion of CD34+ cells was obtained with a cocktail containing FL + TPO + IL-6 + IL-11. The median (range) fold expansions of CD34+ cells at 5 and 10 weeks with serum-free medium were 235.6 (131.3-340) and 5205.6 (4736.6-5674.7) respectively. The absence of IL-11 was associated with a similar fold expansion after 5 weeks (median 215.6, range 149.8-281.5), but after 10 weeks expansion was slightly lower (median 1314.7, range 645-1984.4). Our data support the possibility of maintaining long-term expansion of CB HSCs in a simple stroma- and serum-free system.

Role of lactate in platelet storage lesion.

It is known that lactate accumulation may cause a pH fall in platelet concentrates (PC) during storage, and this phenomenon causes platelet morphological lesions and loss of platelet in vivo viability. In this study, we added increasing amounts of lactate to identical PC in order to evaluate the role of hydrogen ion accumulation in determining platelet activation and lesion during storage. Six hours after PC preparation, lactate was added to PC1 and PC2 at 20 and 12 mM final concentrations, respectively, while PC3 served as control. In PC1, pH was lower than 6.3, and platelet function and discoid morphology were lost. PC2 were stored for 7 days at pH values ranging from 6.4 to 6.6, and most results of in vitro measurements reflecting platelet function such as osmotic reversal, ATP release and aggregation in response to different stimuli were not significantly inferior when compared to controls. The addition of lactate had no apparent effect on the rise of platelet activation markers P-Selectin, lysosome-like protein gp 53, platelet-bound fibrinogen and granulophysin, while a reduction of borderline significance was observed in glycoprotein Ib expression after pH reduction to values lower than 6.6. It is concluded that the rise of platelet activation markers during storage reflects platelet lesions different from those determined by lactate per se.