Detection of internal tandem duplications in the FLT3 gene by different electrophoretic methods

In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of theA complex evaluation of the analytical properties of the three most frequently used detection methods – PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE) – was performed on 95 DNA samples obtained from 73 AML patients.All the three methods verified the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1%–2% mutant allele, while the detection limit of CE was 0.28%. However, acceptable reproducibility (inter-assay CV <25%) of the mutant allele rate determination was only achievable above 1.5% mutant/total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7% lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes.This study suggests the use of PCR+CE in the diagnostics and the follow-up of AML patients. The data further supports the importance of proper analytical evaluation of home-made molecular biological diagnostic tests.

Relationship Between Galectin-10 Expression and Severity of Celiac Disease Abolished in the Presence of Gammadelta-Positive T-Cell Clonal Expansion

Celiac disease (CD) patients are prone to develop T-cell lymphoma throughout a progressive accumulation of aberrant, clonal gd+ T-cells. It is supposed that high production of pro-inflammatory cytokines, and chronic antigenic stimulation, due to gluten ingestion, plays a key role in inducing inflammation, resistance to apoptosis and the emergence of these T-clones. Regulatory T-cells maintain immunological self-tolerance by active suppression of auto aggressive T-cells. Among them, Tr1 subset plays a role in the suppression of naïve and memory T-cells. The role of Tr1 in human diseases is not well understood, even because there are no specific markers able to identify these cells, however recently, Galectin-10 has been proposed as a marker for functional Tr1. To better understand pathogenetic mechanisms associated with CD and clonal T-cell proliferations we investigated galectin-10 expression from gut epithelium by 2D-DIGE approaches. Patients were selected and grouped for histological inflammatory degree and for gd+T pattern defined by g-TCR genescan analysis. Groups consisted of 7 individuals with Marsh-0 (4/7 oligoclonal), 3 with a Marsh-1 or -2 (3/3 polyclonal) and 5 with Marsh-3 (2/5 clonal gd+T). Control consisted of 4 individuals with excluded CD. We found, a parallel increase in galectin-10 levels and Marsh index in individuals with polyclonal gd+T cells (p=0.0092), while reduced levels were evidenced from patients with clonal gd+T cells and Marsh-3 (p=0.017). We assume that galectin-10 up-expression is induce to an attempt to extinguish inflammation. If these clones are those more susceptible to malignant progression reserve further exploration.

Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for the Molecular Subtyping of Enterobacter sakazakii

ABSTRACT The genomic content of Enterobacter sakazakii strain ATCC BAA-894 was analyzed for variable-number tandem repeats (VNTRs). In this study we report the development of a multiple-locus VNTR analysis (MLVA) strategy for the subtyping of E. sakazakii. The method is based on a GeneScan analysis of four VNTR loci labeled with multiple fluorescent dyes. This approach was applied to a collection of 112 isolates representing all 16 of the currently defined E. sakazakii biogroups. MLVA successfully discriminated among these isolates and compared favorably with pulsed-field gel electrophoresis. The method was relatively fast and easy to perform. The potential value of MLVA as an epidemiological tool is discussed.