Einfluss der Genkopiezahlvariation der beta-Defensine drei und vier auf die Inzidenz der Sepsis

In der vorliegenden Arbeit wurde untersucht, ob eine unterschiedliche Anzahl an Genkopien der humanen beta-Defensine drei und vier einen Einfluss auf die Inzidenz der Sepsis haben. Es zeigte sich kein signifikanter Unterschied der Genkopiezahlen zwischen septischen und gesunden Individuen, so dass für die Entstehung einer Sepsis die Anzahl an Genkopien der Defensine keinen Einfluss zu besitzen scheint. Ferner ergab die Genkopiequantifizierung eine intraindividuelle Variabilität der beta-Defensine drei und vier

Comparison of multiplex ligation-dependent probe amplification and real-time PCR accuracy for gene copy number quantification using the beta-defensin locus

The reliable quantification of gene copy number variations is a precondition for future investigations regarding their functional relevance. To date, there is no generally accepted gold standard method for copy number quantification, and methods in current use have given inconsistent results in selected cohorts. In this study, we compare two methods for copy number quantification. beta-defensin gene copy numbers were determined in parallel in 80 genomic DNA samples by real-time PCR and multiplex ligation-dependent probe amplification (MLPA). The pyrosequencing-based paralog ratio test (PPRT) was used as a standard of comparison in 79 out of 80 samples. Realtime PCR and MPLA results confirmed concordant DEFB4, DEFB103A, and DEFB104A copy numbers within samples. These two methods showed identical results in 32 out of 80 samples; 29 of these 32 samples comprised four or fewer copies. The coefficient of variation of MLPA is lower compared with PCR. In addition, the consistency between MLPA and PPRT is higher than either PCR/MLPA or PCR/PPRT consistency. In summary, these results suggest that MLPA is superior to real-time PCR in beta-defensin copy number quantification

Identification of Oncostatin M as a Novel KIT D816V-Dependent Cytokine in Neoplastic Human Mast Cells.

Abstract Abstract 213 Systemic mastocytosis (SM) is a neoplastic disease of mast cells (MC) and their bone marrow-derived progenitors. The clinical picture in SM is variable ranging from an indolent course to highly aggressive variants with short survival time. The pathologic hallmark in SM is the multifocal dense infiltrate of MC in the bone marrow. Other typical features of SM include alterations of the bone marrow microenvironment such as increased angiogenesis and fibrosis. In a majority of patients, MC display the KIT mutation D816V which affects the activation loop at the entrance to the enzymatic pocket of the KIT kinase. As a consequence, KIT D816V exhibits constitutive tyrosine kinase activity and promotes cytokine-independent differentiation of MC. However, so far, little is known about KIT D816V-dependent expression of pathogenetically relevant molecules in neoplastic MC. Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin-6 family which is produced mainly by activated T cells and monocytes. OSM has been shown to inhibit cell growth in cell lines derived from solid tumors but to stimulate proliferation of fibroblasts and endothelial cells. Recently, it has been reported that OSM produced by activated MC promotes growth of human dermal fibroblasts. Moreover, it has been suggested that OSM stimulates growth of murine bone marrow-derived mast cells in a mast cell/fibroblast coculture. However, expression of OSM in neoplastic MC or a potential pathogenetic role of OSM in SM have not been examined so far. The aim of the present study was to analyze expression of OSM in neoplastic human MC and to determine the role of KIT D816V in OSM expression. As assessed by immunohistochemistry performed on bone marrow sections of patients with SM, typical spindle-shaped neoplastic MC were found to express OSM. Serial section-staining confirmed that tryptase-positive MC co-express OSM. Expression of OSM was found in neoplastic MC in all patients investigated (n=15) and in all variants of SM (indolent SM as well as aggressive variants) with comparable staining intensities. Preincubation of anti-OSM antibody with a specific blocking peptide resulted in a negative stain. In Ba/F3 cells, doxycycline-inducible expression of KIT D816V led to a substantial upregulation of OSM mRNA and OSM protein, whereas expression of wild type KIT did not affect expression of OSM. In addition, the KIT D816V-positive HMC-1.2 mast cell line was found to express OSM at high levels, whereas the KIT D816V-negative HMC-1.1 subclone expressed only baseline levels of OSM. Correspondingly, the KIT D816V-targeting drug midostaurine (PKC412) decreased the expression of OSM in HMC-1.2 cells as well as in KIT D816V-expressing Ba/F3 cells in a dose-dependent manner. To investigate signaling pathways involved in KIT D816V-dependent expression of OSM, we applied pharmacologic inhibitors and dominant negative-acting signaling molecules. We found that KIT D816V-dependent expression of OSM is inhibited by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Expression of dominant negative mutants of signal transducer and activator of transcription 5 (STAT5) did not affect expression of OSM in KIT D816V-expressing cells. In summary, our data identify OSM as a novel cytokine expressed in neoplastic MC in patients with SM and show that KIT D816V directly promotes expression of OSM through activation of the mitogen-activated protein-kinase pathway. OSM may be an important KIT D816V-dependent effector promoting angiogenesis and fibrogenesis/sclerosis in patients with SM. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Interference of Mycoplasma Infection in a Gene Expression Study: It Was the Environment and Not the Gene▿

We show that short-term exposure to doxycycline, as used in tetracycline-inducible gene expression models, protects cells from stress-induced death in cultures infected with Mycoplasma arginini. Coinciding with the expected maximum level of gene activity, antimicrobial effects of tetracyclines might be mistaken for antiapoptotic properties of the expressed gene product