ALBERT

Description: Overexpression of P-glycoprotein (PgP) is critical for resistance of malignant cells against cytotoxic agents. However, the role of PgP overexpression for the sensitivity of chronic myeloid leukemia (CML) to imatinib remains controversial. We therefore constructed a transposon-based (Sleeping beauty) RNAi system for the stable down-regulation of PgP in imatinib/doxorubicin resistant K562/Dox cell lines. RT-PCR demonstrated the stable integration of the transposon into the genome. Real-time PCR revealed a rapid and almost complete degradation of MDR1 mRNA 72 h after nucleofection. PgP knockdown on protein level was much more efficient in the stable setting as compared to the transient approach. This observation points out to the requirement of stable knockdown approaches for siRNA-mediated downregulation of proteins with long half-life time. The functional impact of stable PgP knock-down was demonstrated by a reduced efflux of rhodamine and doxorubicin. In parallel, MDR1 knockdown cells were re-sensitized to doxorubicin as well as to imatinib-induced cell death. This observation supports the notion that up-regulation of MDR1 in CML might contribute to imatinib resistance in vivo. In summary we describe a very efficient tool for the stable knock-down of PgP by transposon-mediated siRNA delivery in CML cells and propose that the down-regulation of PgP in CML can restore sensitivity to imatinib.

Description: The expandability of CD4+CD25+ regulatory T-cells (Treg) has been shown in vitro and in vivo. Activation of telomerase activity is a prerequisite for clonal expansion and telomere maintenance in T-cells. There is currently no data available on the expression and function of telomerase in proliferating Treg. Analyses of telomere length by flow-FISH, real-time PCR and Southern blotting revealed that Treg isolated from healthy human volunteers have significantly shortened telomeres when compared to CD4+CD25− T-cells. However, telomere length is not further shortened in Treg isolated from the peripheral blood of cancer patients, despite the observation that the regulatory T-cell pool of these patients was significantly enlarged. To gain further insight into maintenance of telomere length of Treg, we induced in vitro proliferation of Treg by stimulation with anti-CD3 and IL-2. This led to a rapid increase of telomerase activity, as determined by PCR-ELISA. However, when we focused on the proliferating fraction of Treg using a sorting strategy based on the dilution of CFSE, we could show a significant telomere shortening in Treg with high proliferative and immmuno-suppressive capacity. Of note, proliferating CFSElow Treg are characterized by high telomerase activity, which however seems to be insufficient to avoid further telomere shortening under conditions of strong in vitro stimulation. In contrast, under conditions of in vivo expansion of Treg in cancer patients, the induction of telomerase activity is likely to compensate for further telomere erosion. These data might be of importance when considering the application of in vitro expanded Treg for the treatment of GvHD or autoimmune diseases, as telomere shortening might be associated with genomic instability.

Description: Imanitib mesylate (Gleevec®) exhibits potent anti-leukemic effects in vitro and in vivo. Despite of it`s well known anti-leukemic effects, the potential of Imatinib in the treatment of inflammation remains elusive so far. Our current report provides strong evidence that Imatinib indeed exerts anti-inflammatory effects. It potently inhibits LPS- and ConA-induced TNF-α and IFN-γ production of human myeloid cells in vitro (PBMNC, CD14-selected monocytes and monocyte-derived macrophages). Of note, the production of the anti-inflammatory cytokine IL-10 was only slightly affected by Imatinib. In line with this observation, the activation of NF-κB, which has recently been shown to be critically involved in TNF-α but not IL-10 expression, was significantly impaired by Imatinib. In addition, Imatinib reduced either LPS or ConA-induced intracellular phophotyrosine content. For in vivo testing of the anti-inflammatory role of Imatinib in a murine model of inflammation, we injected BALB/c mice with either 75 mg/kg body weight Imatinib or solvent before administration of ConA. The latter has recently been shown to induce T-cell, macrophage and TNF-α-dependent inflammatory damage of the liver. Imatinib pre-treatment prevented the development of acute hepatic injury, which was paralleled by reduced intrahepatic TNF-α mRNA and circulating TNF-α protein levels. Improvement of liver pathology by Imatinib was further assessed by light microscopy and TUNEL staining. Of note, Imatinib protected mice also from GalN/LPS- but not from GalN/TNF-induced liver pathology, which corroborates our in vitro findings that Imatinib potently inhibits TNF-α production of myeloid cells. These findings point out to a potent anti-inflammatory role of the tyrosine kinase inhibitor Imatinib, which might be of therapeutic benefit for the treatment of TNF-α-mediated immune-pathologies, such as inflammatory bowel disease (IBD), aGvHD or immune-mediated liver injury.

Description: Various cell sources, culture protocols and characerization steps are being used in the research field of endothelial progenitors and circulating endothelial cells. We generated EPC by plating the mononuclear fraction onto a collagen I-coated well of a six-well-plate in endothelial cell medium. After 6 to 12 days, 1–10 clones with cobblestone morphology appeared and grew rapidly to confluency within a few days. The rapid proliferation of EPC decelerated after 30–40 days, remaining on the same level for additional 30 days and finally stopped after 70–80 days of culture. Immunofluorescence staining and FACS analysis revealed an endothelial phenotype: EC stained positive for vWF, CD31, CD34, KDR, CD105, CD146, bound UEA-1 lectin and took up acetylated LDL. Of note, CD14 and CD45, which are surrogate markers of monocytes and leukocytes, respectively were negative. The expression of ICAM-1 was upregulated by incubation with TNF-a. In addition, EC formed tubes on Matrigel and expressed VEGF-R1 as well as VEGF-R2-mRNA proven by RT-PCR. To gain deeper insight into the regulation of cellular senescence, we performed western blot analyses of cell cycle regulating proteins. In contrast to early passage cells, growth arrested cells from late passages up-regulated p15, p16, p21, whereas p27 and Id1 were significantly dowregulated. Moreover, late-passage cells highly expressed SA-ß-galactosidase. The obvious replicative senescence together with the regular karyogram indicates a relative safety aspect of their application in vivo. To study their homing behaviour to ischemic tissue, we established a model of myocardial infarction, which was induced by ligation of the left anterior descending coronary artery in Rowett nude rats. 5–10x106 PKH red-labeled EPC were injected into the tail vein. Intramyocardial injection served as positive control, while administation of EPC without prior infarction represented the negative control. Rats were sacrificed on day 14 after the operation and cryosections of their hearts were prepared. PKH-positive cells were almost exclusively found in the infarcted left ventricle after i.v.-application, thus showing a high specifity for ischemic myocardium. The results were confirmed by immunohistochemical staining of human CD31. In summary, EPC generated according to the described protocol represent a possible tool for safe cell-based therapy strategies.