Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for the Detection of Adenovirus 40 and 41.

Human adenoviruses (hAdVs) of subgroup F (enteric serotypes 40 and 41) display characteristic gut tropism, in vivo, fastidious growth characteristics in cell culture, and are estimated to be associated with 5-20% worldwide of acute gastroenteritis cases among infants and young children. Adequate hAdV gastroenteritis case management requires laboratory-based diagnosis. The present study aimed to the development and evaluation of a simple and cost-effective, one-step, single-tube adenovirus type 40/41 specific loop-mediated isothermal amplification (LAMP) assay for the detection of hAdV40/41 DNA in environmental and/or clinical samples, since no LAMP assay has previously been reported for the detection of these virus types. The assay targeted the hexon gene and had the advantages of being rapid, simple, specific, and sensitive. Results could be obtained within 60 min, under isothermal conditions at 69 °C. The detection limits for hAdV genomes were between 50 and 100 copies/reaction for hAdV40 and hAdV41, and no cross-reactions with other selected viruses, were found. The assay was evaluated with clinical as well as environmental samples. The developed assay is expected to provide a potential molecular tool in obtaining greater knowledge of the hAdV40/41 importance in the epidemiology and clinical manifestations of gastroenteritis.

c-Jun N-terminal kinase activation failure is a new mechanism of anthracycline resistance in acute myeloid leukemia.

Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML). Besides the P-glycoprotein efflux, additional cellular factors may contribute to drug resistance in AML. c-Jun N-terminal kinase (JNK) is activated after exposure of cells to chemotherapeutics. We asked whether chemoresistance in AML is attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative cell lines U937R and URD40 showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. RNA interference-based depletion of JNK1 in drug-sensitive U937 cells made them chemoresistant, whereas selective restoration of the inactive JNK pathway in the resistant U937R cells sensitized them to anthracyclines. Short-term in vitro exposure of primary AML cells (n=29) to daunorubicin showed a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels and the response of patients to standard induction chemotherapy (P=0.012). We conclude that JNK activation failure confers another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.

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 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.