Aglaia Athanassiadou

Prasugrel overcomes high on-clopidogrel platelet reactivity post-stenting more effectively than high-dose (150-mg) clopidogrel: the importance of CYP2C19*2 genotyping.

OBJECTIVES The primary aim of the study was to determine the antiplatelet effects of prasugrel versus high-dose clopidogrel in patients with high on-treatment platelet reactivity (HTPR) after percutaneous coronary intervention (PCI) and, secondarily, their relation to cytochrome (CYP) 2C19*2 carriage. BACKGROUND High on-treatment platelet reactivity after clopidogrel administration after PCI is linked to the loss-of-function CYP2C19*2 allele and accompanied by an increased risk of adverse events. METHODS We performed a prospective, randomized, single-blind, crossover study of platelet inhibition by prasugrel 10 mg/day versus high-dose 150 mg/day clopidogrel in 71 (of 210 screened; 33.8%) post-PCI patients with HTPR. Platelet function was assessed by the VerifyNow assay (Accumetrics, San Diego, California), and real-time polymerase chain reaction genotyping was performed for CYP2C19*2 carriage. RESULTS The primary endpoint of platelet reactivity (measured in platelet reactivity units) at the end of the 2 treatment periods was lower after prasugrel compared with clopidogrel (least-squares estimates 129.4, 95% confidence interval [CI]: 111.1 to 147.7 versus 201.7, 95% CI: 183.2 to 220.2; p < 0.001). The least-squares mean difference between the 2 treatments was -122.9 (95% CI: -166.7 to -79.2, p < 0.001), and -47.5 (95% CI: -79.5 to -15.4, p = 0.004), in carriers and noncarriers of at least 1 mutant allele, respectively. The HTPR rates were lower for prasugrel than for clopidogrel, in all patients (7.5% vs. 35.8%, p < 0.001), in carriers (5.3% vs. 47.4%, p = 0.007), and in noncarriers (8.8% vs. 29.4%, p = 0.005), respectively. CONCLUSIONS In patients with HTPR after PCI, prasugrel is more effective compared with high clopidogrel in reducing platelet reactivity, particularly in CYP2C19*2 carriers. Genotyping guidance might be helpful only in case an increased clopidogrel maintenance dose is considered. (Prasugrel Versus High Dose Clopidogrel in Clopidogrel Resistant Patients Post Percutaneous Coronary Intervention (PCI); NCT01109784).

Genetic Analysis of Families with Parkinson Disease that Carry the Ala53Thr Mutation in the Gene Encoding α-Synuclein

We wish to thank Drs. C. Bissas, N. Georgopoulos, P. Ghikas, C. Kremmydas, P. Leonardos, S. Papapetropoulos, and, last but not least, A. Protonotariou, for their great help in sample collection. This work was supported by European Framework Program EPET II grant 236/234/603 to G.M.M.

Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element

Episomally maintained self-replicating systems present attractive alternative vehicles for gene therapy applications. Recent insights into the ability of chromosomal scaffold/matrix attachment regions (S/MARs) to mediate episomal maintenance of genetic elements allowed the development of a small circular episomal vector that functions independently of virally encoded proteins. In this study, we investigated the potential of this vector, pEPI-eGFP, to mediate gene transfer in hematopoietic progenitor cell lines and primary human cells. pEPI-eGFP was episomally maintained and conferred sustained eGFP expression even in nonselective conditions in the human cell line, K562, as well as in primary human fibroblast-like cells. In contrast, in the murine erythroleukemia cell line, MEL, transgene expression was silenced through histone deacetylation, despite the vectors episomal persistence. Hematopoietic semisolid cell colonies derived from transfected human cord blood CD34+ cells expressed eGFP, albeit at low levels. After 4 weeks, the vector is retained in approximately 1% of progeny cells. Our results provide the first evidence that S/MAR-based plasmids can function as stable episomes in primary human cells, supporting long-term transgene expression. However, they do not display universal behavior in all cell types.

Genetic modification of hematopoietic stem cells with nonviral systems: past progress and future prospects

Serious unwanted complications provoked by retroviral gene transfer into hematopoietic stem cells (HSCs) have recently raised the need for the development and assessment of alternative gene transfer vectors. Within this context, nonviral gene transfer systems are attracting increasing interest. Their main advantages include low cost, ease of handling and large-scale production, large packaging capacity and, most importantly, biosafety. While nonviral gene transfer into HSCs has been restricted in the past by poor transfection efficiency and transient maintenance, in recent years, biotechnological developments are converting nonviral transfer into a realistic approach for genetic modification of cells of hematopoietic origin. Herein we provide an overview of past accomplishments in the field of nonviral gene transfer into hematopoietic progenitor/stem cells and we point at future challenges. We argue that episomally maintained self-replicating vectors combined with physical methods of delivery show the greatest promise among nonviral gene transfer strategies for the treatment of disorders of the hematopoietic system.

Nitric oxide synthase expression, enzyme activity and NO production during angiogenesis in the chick chorioallantoic membrane

In order to elucidate further the role of nitric oxide (NO) as an endogenous antiangiogenic mediator, mRNA expression of inducible nitric oxide synthase (iNOS), enzyme activity and production of NO were determined in the chick chorioallantoic membrane (CAM), an in vivo model of angiogenesis. In this model, maximum angiogenesis is reached between days 9–12 of chick embryo development. After that period, vascular density remains constant. Inducible NO synthase (iNOS) mRNA expression, determined by reverse transcriptase polymerase chain reaction (RT–PCR), increased from the 8th day reaching a maximum (70% increase) at days 10–11. NO synthase activity, determined as citrulline formation in the presence of calcium, also increased from day 8 reaching a maximum around day 10 (100% increase). Similar results were obtained in the absence of calcium suggesting that the NOS determined was the inducible form. Nitric oxide production, determined as nitrites, increased from day 8 reaching a maximum around day 10 (64% increase) and remaining stable at day 13. Finally, the bacterial lipopolysaccharide LPS (which activates transcriptionally iNOS), inhibited dose dependently angiogenesis in the CAM. These results in connection with previous findings from this laboratory, showing that NO inhibits angiogenesis in the CAM, suggest that increases in iNOS expression, enzyme activity and NO production closely parallel the progression of angiogenesis in the CAM, thus providing an endogenous brake to control this process.

Hematopoietic Stem Cell Mobilization for Gene Therapy of Adult Patients With Severe β-Thalassemia: Results of Clinical Trials Using G-CSF or Plerixafor in Splenectomized and Nonsplenectomized Subjects

The safety and efficacy of hematopoietic stem cell (HSC) mobilization was investigated in adult splenectomized (SPL) and non-SPL patients with thalassemia major, in two clinical trials, using different mobilization modes: granulocyte-colony-stimulating factor (G-CSF)-alone, G-CSF following pretreatment with hydroxyurea (HU), plerixafor-alone. G-CSF-mobilization was both safe and effective in non-SPL patients. However, in SPL patients the procedure resulted in excessive response to G-CSF, expressed as early hyperleukocytosis necessitating significant dose reduction, and suboptimal CD34(+) cells yields. One-month HU-pretreatment prevented hyperleukocytosis and allowed successful CD34(+) cell collections when an optimal washout period was maintained, but it significantly prolonged the mobilization procedure. Plerixafor resulted in rapid and effective mobilization in both SPL and non-SPL patients and was well-tolerated. For gene therapy of thalassemia, G-CSF or Plerixafor could be used as mobilization agents in non-SPL patients whereas Plerixafor appears to be the mobilization agent of choice in SPL adult thalassemics in terms of safety and efficacy.

Allelic imbalance of expression and epigenetic regulation within the alpha-synuclein wild-type and p.Ala53Thr alleles in Parkinson disease†

Genetic alterations in the alpha‐synuclein (SNCA) gene have been implicated in Parkinson Disease (PD), including point mutations, gene multiplications, and sequence variations within the promoter. Such alterations may be involved in pathology through structural changes or overexpression of the protein leading to protein aggregation, as well as through impaired gene expression. It is, therefore, of importance to specify the parameters that regulate SNCA expression in its normal and mutated state. We studied the expression of SNCA alleles in a lymphoblastoid cell line and in the blood cells of a patient heterozygous for p.Ala53Thr, the first mutation to be implicated in PD pathogenesis. Here, we provide evidence that: (1) SNCA shows monoallelic expression in this patient, (2) epigenetic silencing of the mutated allele involves histone modifications but not DNA methylation, and (3) steady‐state mRNA levels deriving from the normal SNCA allele in this patient exceed those of the two normal SNCA alleles combined, in matching, control individuals. An imbalanced SNCA expression in this patient is thus documented, with silencing of the p.Ala53Thr allele and upregulation of the wild‐type‐allele. This phenomenon is demonstrated for a first time in the SNCA gene, and may have important implications for PD pathogenesis. Hum Mutat 31:1–7, 2010.

Genetic assessment of familial and early-onset Parkinson's disease in a Greek population

Although the first mutation associated with Parkinsons disease (PD) was identified several years ago in the alpha‐synuclein (SNCA) gene in families of Greek and Italian ancestry, a more systematic study of this and other known PD mutations has not been performed in the Greek population.

Genotypic heterogeneity and correlation to intergenic haplotype within high HbF β-thalassemia intermedia

Abstract:  Objectives: A molecular study was carried out of β‐thalassemia intermedia patients, compound heterozygotes for mutations usually found in β‐thalassemia major, with high levels of HbF in the absence of hereditary persistence of fetal hemoglobin (HPFH) syndrome. Our objective was to locate cis‐DNA structures, DNA haplotypes, motifs, or polymorphisms that may correlate with the presence of high HbF. Methods: Allele‐specific oligonucleotide (ASO) hybridization was used for the detection of mutations and restriction fragment length polymorphism (RFLP) analysis and automated sequencing for motifs, haplotypes, and polymorphisms. Southern blot was used for investigating α‐thalassemia and/or α‐ or γ‐globin genes triplications. RNA extracted from burst forming unit‐erythroid (BFU‐e) colonies of peripheral blood mononuclear cell cultures was used in reverse transcriptase‐polymerase chain reaction (RT‐PCR) to investigate intergenic transcription. Results: We established that (i) the combination: T haplotype of the Aγ‐δ‐globin intergenic region, the motif (TA)9N10(TA)10 in the HS2 site of locus control region (LCR), and TAG pre‐Gγ haplotype is sufficient but not necessary for high HbF, (ii) the genetic determinant(s) for high HbF involves an element associated with this combination and must be present in the specific R haplotype occurring in β‐thalassemia intermedia and (iii) the genetic determinant(s) for high HbF does not involve the abolition of intergenic transcription in the Aγ‐δ‐globin intergenic region. Conclusions: The genetic determinant(s) of high HbF in the absence of HPFH is linked to intergenic haplotype T and does not disrupt intergenic transcription.

Environmental Mycobacteria in Drinking Water Before and After Replacement of the Water Distribution Network

Environmental mycobacteria (M. chelonae (8), M.flavescens (6), M. gordonae (6), M. fortuitum (5), M. kansasii (4), M. phlei (2) and M. terrae (2)) were isolated from21.3% of drinking water samples before replacement of thedistribution network of Patras. After replacement of the networkonly 1.8% of the samples contained environmental mycobacteria(M. chelonae (2)). The identification of environmentalmycobacteria was performed by Restriction EnzymeAnalysis-Polymerase Chain Reaction. Our results showed no statistically significant correlationbetween the presence of mycobacteria and the bacteriologicalfecal indicators (p >0.05). Moreover, we determined thatconcentrations of free residual chlorine equal to or greaterthan 0.5 mg L-1 in the water supply network were needed toeliminate environmental mycobacteria.We conclude that the presence of a biofilm in the old Patrasdrinking water network significantly increased the number ofenvironmental mycobacteria in the drinking water. This problemwas resorted after the replacement of the network pipes.