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Real-time technology eliminates many of the pitfalls of diagnostic PCR, but this method has not been applied to differentiation of Leishmania organisms so far. We have developed a real-time PCR that simultaneously detects, quantitates, and categorizes Leishmania organisms into three relevant groups causing distinct clinical pictures. The analytical sensitivity (detection rate of >95% at 94.1 parasites/ml of blood) was within a range that has been determined previously to facilitate the confirmation of visceral leishmaniasis from peripheral blood. Parasites were successfully detected in 12 different clinical samples (blood, bone marrow, skin, and liver). The Leishmania donovani complex, the Leishmania brasiliensis complex, and species other than these could be clearly discriminated by means of distinct melting temperatures obtained with fluorescence resonance energy transfer probes (melting points, 72.7, 67.1, and 65.0°C, respectively). All three groups could be quantified within equal ranges. As in other real-time PCRs, the variability in the quantification of DNA was small (coefficient of variation [CV], <2%). However, human samples containing low levels of parasites (100 parasites per ml of blood) showed higher variation (CV, 60.89%). Therefore, despite its superior analytical performance, care must be taken when real-time PCR is utilized for therapy monitoring.

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Constitutively activated pathways contribute to apoptosis resistance in chronic lymphocytic leukemia (CLL). Little is known about the metabolism of lipids and function of lipases in CLL cells. Performing gene expression profiling including B-cell receptor (BCR) stimulation of CLL cells in comparison to healthy donor CD5 þ B cells, we found significant overexpression of lipases and phospholipases in CLL cells. In addition, we observed that the recently defined prognostic factor lipoprotein lipase (LPL) is induced by stimulation of BCR in CLL cells but not in CD5 þ normal B cells. CLL cellular lysates exhibited significantly higher lipase activity compared to healthy donor controls. Incubation of primary CLL cells (n ¼ 26) with the lipase inhibitor orlistat resulted in induction of apoptosis, with a halfmaximal dose (IC 50 ) of 2.35 lM. In healthy B cells a significantly higher mean IC 50 of 148.5 lM of orlistat was observed, while no apoptosis was induced in healthy peripheral blood mononuclear cells (PBMCs; Po0.001). Orlistat-mediated cytotoxicity was decreased by BCR stimulation. Finally, the cytotoxic effects of orlistat on primary CLL cells were enhanced by the simultaneous incubation with fludarabine (P ¼ 0.003). In summary, alterations of lipid metabolism are involved in CLL pathogenesis and might represent a novel therapeutic target in CLL.

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