The impact of intercellular communication for the generation of complex multicellular prevascularized tissue equivalents

In reconstructive surgery the use of prevascularized soft tissue equivalents is a promising approach for wound coverage of defects after tumor resection or trauma. However, in previous studies to generate soft tissue equivalents on collagen membranes, microcapillaries were restricted to superficial areas. In this study, to understand which factors were involved in the formation of these microcapillaries, the levels of the angiogenic factors vascular endothelial growth factor (VEGF), Interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) in the supernatants of the tissue equivalents were examined at various time points and conditions. Additionally, the influence of these factors on viability, proliferation, migration, and tube formation in monocultures compared to cocultures of fibroblast and endothelial cells was examined. The results showed that VEGF production was decreased in cocultures compared to fibroblast monocultures and the lowest VEGF levels were observed in endothelial cell monocultures. Additionally, the highest levels of IL-8 were observed in cocultures compared to monocultures. Similar results were observed for bFGF with lowest levels seen within the first 24 hr and highest levels in cocultures. VEGF and IL-8 were shown to promote endothelial cell viability, proliferation and migration and angiogenic parameters such as tube density, total tube length, and number of tube branches. Addition of VEGF and IL-8 to cocultures resulted in accelerated and denser formation of capillary-like structures. The results indicate that VEGF, IL-8, and bFGF strongly influence cellular behavior of endothelial cells and this information should be useful in promoting the formation of microcapillary-like structures in complex tissue equivalents

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34+/CD38─ CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4+ SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26+ LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26+ CML LSC engrafted NOD-SCID-IL-2Rγ−/− (NSG) mice with BCR/ABL1+ cells, whereas CD26─ SC from the same patients produced multilineage BCR/ABL1– engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1+ cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy

Small-molecule inhibition of BRD4 as a new potent approach to eliminate leukemic stem- and progenitor cells in acute myeloid leukemia (AML)

Acute myeloid leukemia (AML) is a life-threatening stem cell disease characterized by uncontrolled proliferation and accumulation of myeloblasts. Using an advanced RNAi screen-approach in an AML mouse model we have recently identified the epigenetic ‘reader’ BRD4 as a promising target in AML. In the current study, we asked whether inhibition of BRD4 by a small-molecule inhibitor, JQ1, leads to growth-inhibition and apoptosis in primary human AML stem- and progenitor cells. Primary cell samples were obtained from 37 patients with freshly diagnosed AML (n=23) or refractory AML (n=14). BRD4 was found to be expressed at the mRNA and protein level in unfractionated AML cells as well as in highly enriched CD34+/CD38− and CD34+/CD38+ stem- and progenitor cells in all patients examined. In unfractionated leukemic cells, submicromolar concentrations of JQ1 induced major growth-inhibitory effects (IC50 0.05-0.5 μM) in most samples, including cells derived from relapsed or refractory patients. In addition, JQ1 was found to induce apoptosis in CD34+/CD38− and CD34+/CD38+ stem- and progenitor cells in all donors examined as evidenced by combined surface/Annexin-V staining. Moreover, we were able to show that JQ1 synergizes with ARA-C in inducing growth inhibition in AML cells. Together, the BRD4-targeting drug JQ1 exerts major anti-leukemic effects in a broad range of human AML subtypes, including relapsed and refractory patients and all relevant stem- and progenitor cell compartments, including CD34+/CD38− and CD34+/CD38+ AML cells. These results characterize BRD4-inhibition as a promising new therapeutic approach in AML which should be further investigated in clinical trials

Role of Pl3 Kinase in the differentiation and activation of human basophils and mast cells

Die Phosphoinositol 3 Kinase spielt eine wichtige Rolle in der Aktivierung und im Wachstum von Mastzellen und basophilen Granulozyten. Aus diesem Grund untersuchten wir die Effekte des neuen Phosphoinositol 3 Kinase/mTOR blockers NVP-BEZ235 auf die IgE-abhängige Aktivierung und das Wachstum von Basophilen und Mastzellen. Die Zell-Proliferation wurde mittels Thymidineinbau bestimmt. Die Beurteilung und Quantifizierung der NVP-BEZ235- induzierten Apoptose erfolgte lichtmikroskopisch und durchflußzytometrisch über Caspase 3- und AnnexinV/PI Färbungen, sowie mit Hilfe eines TUNEL Assay. Überdies wurde die Wirkung von NVP-BEZ235 auf die IgE-abhängige Histamin-Freisetzung und die IgE-abhängige Hochregulation der Oberflächenantigene CD63 und CD203c untersucht. In einem weiteren Schritt analysierten wir die Effekte von NVP-BEZ235 auf die Aktivierung von bestimmten Signalmolekülen mittels Durchflußzytometrie und Western Blotting. Letztlich wurden auch die in vivo Effekte von NVP-BEZ235 in einem Mausmodell untersucht. Es zeigte sich, dass NVP-BEZ235 den IgE-abhängigen Histamin-Release in Basophilen hemmt, und zwar sowohl in Normalspendern als auch in Allergikern, mit IC50 Werten von 0.5 bis 1 M. Weiters konnte durch NVP-BEZ235 die anti-IgE-induzierte Hochregulation von CD203c in humanen Basophilen und die Hochregulation von CD63 in den Mastzellen gehemmt werden. Ebenso hemmt NVP-BEZ235 die IgE-abhängige Mediator-Freisetzung in den Mastzellen. Zusätzlich wurde durch NVP-BEZ235 das IL-3-abhängige Wachstum der Basophilen und die SCF-abhängige Entwicklung der Mastzellen aus ihren Vorläuferzellen unterdrückt. Überdies blockierte NVP-BEZ235 das Wachstum der leukämischen Basophilen-Zelllinien KU812 und der Mastzell-Leukämie-Zellinie HMC-1. Obwohl wachstums-inhibierende Effekte von NVP-BEZ235 mit ähnlichen IC50 Werten in beiden HMC-1 Subklonen gezeigt werden konnten, scheint die Wirkung von NVP-BEZ235 in der HMC-1.1, welche keine KIT D816V Mutation exprimiert, stärker zu sein. NVP-BEZ235 blockierte auch die Aktivierung der Signalmoleküle Akt, S6 und STAT5 in den HMC-1 Zellen, während der mTOR Blocker RAD001 nur die Aktivierung von S6 inhibierte. Letztlich konnte gezeigt werden, dass das in vivo Wachstum der HMC-1 Zellen in einem Xenotransplantantations-Modell in NMR1-Foxn1nu Mäusen durch NVP-BEZ235 nachhaltig gehemmt wird. Der mTOR Inhibitor RAD001 zeigte auch hier keine vergleichsame Wirksamkeit. Zusammen-fassend kann gesagt werden, dass NVP-BEZ235 ein potenter Inhibitor des Wachstums und der Aktivierung von basophilen Granulozyten und Mastzellen ist. Ob sich daraus klinisch-therapeutische Konzepte ableiten lassen, werden zukünftige Studien zeigen.The phosphoinositide 3 kinase (PI3 kinase) and the mammalian target of rapamycin (mTOR) have been implicated as critical signaling molecules in the activation and growth of mast cells (MC) and basophils (BA). We examined the effects of the PI3 kinase/mTOR-targeting drug NVP-BEZ235 on immunoglobulin E (IgE)-dependent activation and growth of BA and cultured cord blood-derived MC. Growth of MC and BA were calculated by measuring 3H-thymidine uptake, and apoptosis by morphologic analyses, Caspase 3 staining, AnnexinV/propidium (PI) staining and TUNEL assay. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. The effects of NVP-BEZ235 on expression of activated signal transduction molecules were analyzed by flow cytometry and Western blotting. NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA in healthy individuals as well as in allergic patients (IC50 0.5-1 M). NVP-BEZ235 was also found to counteract anti-IgE-induced upregulation of CD203c in BA, and to block IgE-dependent upregulation of CD63 as well as IgE-dependent mediator-secretion in MC. Additionally, NVP-BEZ235 was found to suppress interleukin-3 (IL-3)-dependent differentiation of BA and stem cell factor (SCF)-dependent development of MC from their progenitors. Finally, NVP-BEZ235 inhibited growth in the BA leukemia cell line KU812 and in the MC leukemia cell line HMC-1. Although growth-inhibitory effects of NVP-BEZ235 showed similar IC50 values (0.01 M) in both HMC-1 subclones, strong apoptosis-inducing effects were only seen in HMC-1.1 cells lacking KIT D816V, but not in HMC-1.2 cells expressing KIT D816V. Apoptosis-inducing effects of NVP-BEZ235 were not mimicked by the mTOR-blocker RAD001. NVP-BEZ235 was found to inhibit the activation of the downstream signaling molecules Akt, S6, and STAT5 in HMC-1 cells. By contrast, as expected, RAD001 was only capable of suppressing the activation of S6. Finally, we examined the in vivo effects of NVP-BEZ235 in a xenotransplantation model employing HMC-1 cells and NMR1-Foxn1nu mice. In these experiments, NVP-BEZ235 was found to exert strong growth-inhibitory effects on the in vivo growth of neoplastic MC, whereas RAD001 showed no substantial effect in this model. In summary, NVP-BEZ235 inhibits IgE-dependent activation in BA and MC, and produces growth-inhibitory effects in neoplastic BA and MC. Whether these drug effects have clinical implications remains to be determined.submitted by Katharina BlattAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2011OeBB(VLID)171506

Synergistic growth-inhibitory effects of ponatinib and midostaurin (PKC412) on neoplastic mast cells carrying KIT D816V

Patients with advanced systemic mastocytosis, including mast cell leukemia, have a poor prognosis. In these patients, neoplastic mast cells usually harbor the KIT mutant D816V that confers resistance against tyrosine kinase inhibitors. We examined the effects of the multi-kinase blocker ponatinib on neoplastic mast cells and investigated whether ponatinib acts synergistically with other antineoplastic drugs. Ponatinib was found to inhibit the kinase activity of KIT G560V and KIT D816V in the human mast cell leukemia cell line HMC-1. In addition, ponatinib was found to block Lyn- and STAT5 activity in neoplastic mast cells. Ponatinib induced growth inhibition and apoptosis in HMC-1.1 cells (KIT G560V(+)) and HMC-1.2 cells (KIT G560V(+)/KIT D816V(+)) as well as in primary neoplastic mast cells. The effects of ponatinib were dose-dependent, but higher IC(50)-values were obtained in HMC-1 cells harboring KIT D816V than in those lacking KIT D816V. In drug combination experiments, ponatinib was found to synergize with midostaurin in producing growth inhibition and apoptosis in HMC-1 cells and primary neoplastic mast cells. The ponatinib+midostaurin combination induced substantial inhibition of KIT-, Lyn-, and STAT5 activity, but did not suppress Btk. We then applied a Btk short interfering RNA and found that Btk knockdown sensitizes HMC-1 cells against ponatinib. Finally, we were able to show that ponatinib synergizes with the Btk-targeting drug dasatinib to produce growth inhibition in HMC-1 cells. In conclusion, ponatinib exerts major growth-inhibitory effects on neoplastic mast cells in advanced systemic mastocytosis and synergizes with midostaurin and dasatinib in inducing growth arrest in neoplastic mast cells

Journal of Hematology & Oncology / Cancer stem cells in basic science and in translational oncology : can we translate into clinical application?

Since their description and identification in leukemias and solid tumors, cancer stem cells (CSC) have been the subject of intensive research in translational oncology. Indeed, recent advances have led to the identification of CSC markers, CSC targets, and the preclinical and clinical evaluation of the CSC-eradicating (curative) potential of various drugs. However, although diverse CSC markers and targets have been identified, several questions remain, such as the origin and evolution of CSC, mechanisms underlying resistance of CSC against various targeted drugs, and the biochemical basis and function of stroma cell-CSC interactions in the so-called ‘stem cell niche. Additional aspects that have to be taken into account when considering CSC elimination as primary treatment-goal are the genomic plasticity and extensive subclone formation of CSC. Notably, various cell fractions with different combinations of molecular aberrations and varying proliferative potential may display CSC function in a given neoplasm, and the related molecular complexity of the genome in CSC subsets is considered to contribute essentially to disease evolution and acquired drug resistance. In the current article, we discuss new developments in the field of CSC research and whether these new concepts can be exploited in clinical practice in the future.(VLID)486718

Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy

BackgroundGrass pollen is one of the most important sources of respiratory allergies worldwide.ObjectiveThis study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach.MethodsFusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity.ResultsTen hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation.ConclusionA recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy

FLAG-induced remission in a patient with acute mast cell leukemia (MCL) exhibiting t(7;10)(q22;q26) and KIT D816H

Mast cell leukemia (MCL) is a life-threatening disease associated with high mortality and drug-resistance. Only few patients survive more than 12 months. We report on a 55-year-old female patient with acute MCL diagnosed in May 2012. The disease was characterized by a rapid increase in white blood cells and mast cells (MC) in the peripheral blood, and a rapid increase of serum tryptase levels. The KIT D816H mutation was detected in the blood and bone marrow (BM). Induction chemotherapy with high-dose ARA-C and fludarabine (FLAG) was administered. Unexpectedly, the patient entered a hematologic remission with almost complete disappearance of neoplastic MC and a decrease of serum tryptase levels to normal range after 2 cycles of FLAG. Consecutively, the patient was prepared for allogeneic stem cell transplantation. However, shortly after the third cycle of FLAG, tryptase levels increased again, immature MC appeared in the blood, and the patient died from cerebral bleeding. Together, this case shows that intensive chemotherapy regimens, like FLAG, may induce remission in acute MCL. However, treatment responses are short-lived and the overall outcome remains dismal in these patients. We propose to separate this acute type of MCL from more subacute or chronic variants of MCL