A silenced allele in the Colton blood group system.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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).

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.

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.

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.

Oligohydrosis and fever in pediatric patients treated with zonisamide.

Zonisamide is an antiepileptic drug developed and first marketed in Japan in 1989. Cases of oligohydrosis, characterized by deficient production and secretion of sweat, were reported in children treated with zonisamide in Japan during development and in the postmarketing period. Zonisamide was approved in the United States in March 2000 for adjunctive treatment of partial seizures in adults. Searching the Food and Drug Administration's Adverse Events Reporting System, we identified six domestic cases of zonisamide-associated oligohydrosis and/or fever, all in patients

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.

The 'cherry buffy-coat syndrome', a cause of decreased platelet yield in platelet concentrates obtained from buffy-coats.

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Isolation and characterization of the human A-myb promoter: regulation by NF-Y and Sp1.

The A-myb transcription factor shows a restricted tissue distribution and is cell cycle regulated. Furthermore its deregulation has profound effects on the growth and/or differentiation of the cells in which it is normally expressed. We have therefore characterized its promoter. A 12 kb genomic clone was isolated that comprises the first exon, part of the first intron as well as upstream regulatory sequences. Multiple transcription start sites have been identified which operate in both B lymphocytes and epithelial cells and the upsteam region was shown to have promoter, activity. The boundaries of the minimal promoter region (-183-14), of a positive upstream (-538-183) and a negative downstream regulatory region (NRE) (+83+374) have been defined. The NRE is promoter- and orientation-independent but position specific. The A-myb minimal promoter is GC-rich, does not contain any TATA box but has a functional CCAAT box. The CCAAT box and minimal promoter is highly conserved in the corresponding murine sequence. The CCAAT box efficiently binds the NF-Y complex and its mutation decreases basal promoter activity by 50%. Two Sp1 binding sites are present upstream from the CCAAT box which can bind Spl and contribute to A-myb promoter activity by 70 and 30%, respectively. The two Sp1 sites and CCAAT box together contribute to over 80% of A-myb basal promoter activity and are therefore the major regulatory elements. Finally, we show that the promoter is cell cycle regulated and that the SP1 and CCAAT elements are required for S phase induction.