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 vector's 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.

SummaryMyelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective haematopoiesis and blood cytopenias. The present study investigated the potential of bone marrow CD34 + progenitors in MDS patients to proliferate and differentiate into dendritic cells (DCs) in a cytokine-supplemented liquid culture system and analysed the status of blood DC subsets in these patients. CD34 + progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34 + cell was significantly lower compared with controls (median value 0AE2 vs. 4, P ¼ 0AE003). In patients, the survival and proliferation of CD34 + cells in culture was not correlated to the degree of apoptosis. Phenotypically and functionally CD34 + -derived DCs were similar in MDS patients and normal subjects. The percentage of both circulating DC subsets in patients was extremely diminished compared with controls (myeloid DC: 0AE10 ± 0AE10% vs. 0AE35 ± 0AE13%, P < 0AE001; plasmacytoid DC: 0AE11 ± 0AE10% vs. 0AE37 ± 0AE14%, P < 0AE001). In cases with the 5q deletion both CD34-derived DCs and blood DCs harboured the cytogenetic abnormality. Our results indicate that, in MDS, the production of DCs is affected by the neoplastic process resulting in ineffective 'dendritopoiesis' with low blood DC precursor numbers. This quantitative DC defect probably contributes to the poor immune response against infectious agents and to the escape of the malignant clone from immune recognition with disease progression towards acute leukaemia.

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Focal adhesion kinase (FAK) and its downstream signaling targets are implicated in the process of apoptosis induced by external stimuli, in several mammalian systems. In this report, we demonstrate, that medfly (Ceratitis capitata) hemocytes do undergo apoptosis during larval development. In particular, we show using Western blot, ELISA and flow cytometry analysis, that FAK expression silencing in transfected by FAK double-stranded RNA (dsRNA) hemocytes, enhances twofold hemocyte apoptosis, by signaling through Src, MEK/ERK, and PI-3K/Akt signaling pathways. FAK expression silencing, in response to FAK dsRNA treatment, blocks partially the phosphorylation of its downstream targets. Pre-incubation of hemocytes, with specific inhibitors of FAK downstream signaling molecules, demonstrated that all these inhibitors reduced hemocyte viability and enhanced the magnitude of apoptosis about threefold. This data suggest that these pathways contribute to hemocyte survival and/or death during development. The expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK signaling molecules appear to be dependent upon developmental stages. The expression and phosphorylation of the above signaling molecules, in annexin-positive and annexin-negative hemocytes is also distinct. The maximum expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK appeared in annexin-positive hemocytes, in both early and late apoptotic hemocytes. The novel aspect of this report is based on the fact that hemocytes attempt to suppress apoptosis, by increasing the expression/phosphorylation of FAK and, hence its downstream targets signaling molecules Src, ERK, PI-3K p85a, and Akt. Evidently, the basic survival pathways among insects and mammals appear to remain unchanged, during evolution.

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INTRODUCTION: Hypomethylating agents have become a standard therapy for high-risk myelodysplastic syndromes (MDS) and elderly patients with acute myeloid leukemia (AML). AIM:The aim of the study was to assess the efficacy of azacitidine treatment in patients with MDS and AML followed for 18 months. MATERIALS AND METHODS: Twenty-seven patients with MDS and AML treated in theClinic of Hematology at St. Marina University Hospital, Varna were included in the study. Azacitidine was administered subcutaneously at a dose of 75 mg/m 2 for 7 days. Disease assessment was performed on the 3 rd month, 6 th month, and at progression. RESULTS: Twenty-seven patients were analyzed. Their median age was 71.5 years. Nine had refractory anemia with excess of blasts II (RAEB II), 5 had chronic myelomonocytic leukemia II (CMML II), 1 was with unclassifiable MDS (MDS-U), and 12 with AML. The median number of administered cycles was 6 (1-19). Eleven patients completed 6 cycles of azacitidine. Partial response was achieved in 9 patients (33%) (7 MDS and 2 AML), stable disease in 8 (29%) (5 MDS and 3 AML). Progressive disease was observed in 10 patients (37%). The response correlated with the type of the disease (p=0.03), cytogenetic risk (p=0.01), and survival (p=0.000). At 18 months, 60% of MDS patients were alive compared to 41.7% in the AML group. The median time to death in the AML patient group was 2.5 months. The mean overall survival was 10.4 months (12.6 months for MDS patients and 5.4 months for AML patients). CONCLUSION:The therapy with azacitidine is an option for elderly patients with high-risk MDS. In patients with AML a rapid progression is observed during the first two cycles with mortality rate of 58.3%. Scr Sci Med. 2018;50(1):31-35

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