We report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34+ cells, with transfection efficiencies of 46.3 ± 5.2%, 23.0 ± 2.1% and 24.2 ± 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of γ-globin mRNA by 3.3 ± 0.13, of γ-globin protein by 6.75 ± 3.25 and HbF protein by 2 ± 0.2 fold, while the vector remained episomal and non integrated. In murine β-YAC BMCs the vector mediated the activation of the silent human γ-globin gene and in CD34+ cells, increased γ-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of β-globin Replicator, transferred into CD34+ cells, produced CD34+ eGFP+ cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 ± 0.13 fold increased γ-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors.

The newer implantable loop recorders recently introduced (Reveal Plus. Medtronic Inc, Minneapolis, MN, U.S.A.). equipped with auto activation capabilities, are expected to expand our diagnostic abilities in the investigation of syncopal episodes. The case presented here is that of a man with pre-syncopal and syncopal episodes, without organic heart disease, in whom the device was auto-activated due to undersensing and the recorded strip imitated complete heart block. Increased attention should be given by the interpreter of the device's recordings to avoid false positive diagnoses that might lead to serious therapeutic actions.

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Introduction: Multiple myeloma (MM) is a plasma cell dyscrasia that is heavily dependent on a permissive BM micro-environment. Components of the WNT pathway have been implicated in this process and specifically, WNT-inhibitors such as Dkk1, have been blamed for the osteolytic lesions of MM. WNT signaling is required for proper osteoblast development while the pathway is also involved in enhancing the self renewal potential of various stem cells. A source of WNT ligands in the BM are the T-cells; when stimulated by parathormone, T-cells secrete wnt10b that affects hematopoietic stem cell numbers. In the present study, we evaluated WNT signaling in the T-cells of patients with MM in order to test whether these bystander T-cells have an altered phenotype that could influence disease pathology. Methods: A total of 18 MM patients was analyzed after informed consent was obtained; the patients underwent BM biopsy for diagnostic and staging purposes. T-cells were isolated from BM mononuclear cells using magnetic beads with a purity of around 90%. Normal controls were T-cells isolated from healthy subjects. RNA was isolated and analyzed by RT-PCR for expression of PTH receptor (PTH1R), wnt10b and the canonical WNT signal transducer, b-catenin (bCAT). Results and discussion: BM concentration of T-cells was 15%, close to the normal values, indicating no major deviation from normal conditions. Activity of the WNT pathway was estimated by bCAT mRNA levels; relative to the control, bCAT levels in MM T-cells were uniformily suppressed. In a total of 8 evaluable samples, there was a 3x decrease in bCAT levels. Although WNT activity could be influenced by different factors, there is a possibility that it may be related to PTH activity and PTH receptor expression on patient T-cells. In all our cases, calcium was recorded within normal range and PTH values were also normal. However, PTH1R mRNA levels in MM T-cells were 14.2x lower compared to normal T-cells, a value that argues for a biological role in disease pathogenesis. A direct consequence of low PTH1R mRNA levels, according to our hypothesis, would lead to a decrease in WNT10b. Indeed, WNT10b levels were on average 90% of the normal with relative wide variation; in 3 samples, there was an average 3.2x increase while in 8, the levels were on average 21% of normal. On the clinical level, lower wnt10b activity, was more often associated with bone pathology while higher wnt10b activity was more often associated with extramedullary plasmacytomas. Our data show that T-cells may not be bystanders in the MM disease progression and certain manifestations sush as bone disease, could be associated with, aberrant T cell function. Ongoing studies are under way to link clinical and laboratory data. Disclosures No relevant conflicts of interest to declare.

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