Reexamination In Vitro

Purpose. To introduce additional methods to detect and to quantify single pathogens in the complex biofilm formation on an antibacterial dental material. Materials and Methods. A conventional (ST) and an antibacterial dental composite (B) were manufactured. In vitro: specimens were incubated with a mixture of early colonizers. Bacterial adhesion was analyzed by TaqMan PCR after 8/24 h. In situ: TaqMan PCR and 16S rRNA Next Generation Sequencing (NGS) were performed. Results. In vitro: after 8 h incubation, B was covered by 58.6% of the bacterial amount that was attached to ST. After 24 h, the amount of attached bacteria to ST remained constant on ST only slightly lower on B. In situ: after 8 h the amount of adhering A. viscosus and S. mitis was prominent on ST and reduced on B. NGS revealed that S. sanguinis, S. parasanguinis, and Gemella sanguinis were the mainly attached species with S. sanguinis dominant on ST and S. parasanguinis and G. sanguinis dominant on B. Conclusions. Initial biofilm formation was altered by B. A shift between actinomycetes and streptococci was observed in situ. TaqMan PCR and 16S rRNA NGS revealed comparable results in situ and demonstrated the usefulness of NGS to characterize complex bacterial communities

The full-ORF clone resource of the German cDNA Consortium

<p>Abstract</p> <p>Background</p> <p>With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes.</p> <p>Results</p> <p>Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned.</p> <p>Conclusion</p> <p>The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.</p

Pharmacogenetics of the Central Nervous System—Toxicity and Relapse Affecting the CNS in Pediatric Acute Lymphoblastic Leukemia

Despite improving cure rates in childhood acute lymphoblastic leukemia (ALL), therapeutic side effects and relapse are ongoing challenges. These can also affect the central nervous system (CNS). Our aim was to identify germline gene polymorphisms that influence the risk of CNS events. Sixty single nucleotide polymorphisms (SNPs) in 20 genes were genotyped in a Hungarian non-matched ALL cohort of 36 cases with chemotherapy related acute toxic encephalopathy (ATE) and 544 controls. Five significant SNPs were further analyzed in an extended Austrian-Czech-NOPHO cohort (n = 107 cases, n = 211 controls) but none of the associations could be validated. Overall populations including all nations’ matched cohorts for ATE (n = 426) with seizure subgroup (n = 133) and posterior reversible encephalopathy syndrome (PRES, n = 251) were analyzed, as well. We found that patients with ABCB1 rs1045642, rs1128503 or rs2032582 TT genotypes were more prone to have seizures but those with rs1045642 TT developed PRES less frequently. The same SNPs were also examined in relation to ALL relapse on a case-control matched cohort of 320 patients from all groups. Those with rs1128503 CC or rs2032582 GG genotypes showed higher incidence of CNS relapse. Our results suggest that blood-brain-barrier drug transporter gene-polymorphisms might have an inverse association with seizures and CNS relapse

Pre-BCR signaling defines a novel subgroup in B-cell acute lymphoblastic leukemia and can be targeted by SYK inhibition

Trotz großer Fortschritte bei der Behandlung von Patienten mit akuter lymphoblastischer Leukämie (ALL) versterben immer noch mehr als die Hälfte (60%) aller Erwachsenen an den Folgen dieser Erkrankung. Auch bei Kindern, bei denen die Heilungsrate in den letzten drei Jahrzehnten auf über 85% angestiegen ist, besteht noch Bedarf nach neuen Therapien. Das Ziel dieser ist es Heilungsraten weiter zu verbessern und gleichzeitig das Auftreten von Chemo- und Strahlentherapie-induzierten Folgeerkrankungen (z.B. Malignome und kognitive Beeinträchtigungen) zu verringern. Der Einsatz gezielter Therapien stellt eine Möglichkeit dar, diesen Zielen näher zu kommen. Es werden dabei Substanzen verabreicht, die einen für die Erkrankung spezifischen Prozess gezielt blockieren. Der Erfolg eines solchen Therapieansatzes setzt jedoch genaues Wissen über die molekularbiologischen Veränderungen der Erkrankung voraus. Erkrankungsbedingte, aberrante Signalwege sind jedoch nicht für alle ALL Untergruppen im Detail erforscht. Im Rahmen der vorliegenden Arbeit beschreiben wir eine neue Subgruppe der B-Zell Vorläufer ALL, die durch die Abhängigkeit von Wachstumssignalen des pre-B Zellrezeptor (pre-BCR) gekennzeichnet ist. Der kombinierte Einsatz molekulargenetischer und pharmakologischer Methoden ermöglicht uns außerdem, die entsprechenden molekularen Mechanismen dieser Abhängigkeit (oder diese Prozesses) aufzuklären. Im Zentrum dieser steht die pre-BCR-mediierte Aktivierung der PI3 Kinase und von AKT. Dies setzt eine Signalkaskade in Gang an deren Ende die Inaktivierung des Tumorsuppressors FOXO1 und die Aufhebung der C-MYC Blockierung steht. Die selektive Blockade dieser Signalwege durch Inhibition der pre- BCR-assoziierten SYK Kinase führt zu reduziertem Tumorwachstum und reduzierter Viabilität in verschiedenen in vitro und in vivo Modellen pre-BCR+ Leukämien. Diese Ergebnisse unterstreichen die therapeutische Relevanz der pre-BCR Signalkaskade in B-ALL und bilden die Grundlage für die mögliche weitere Exploration der Effekte von SYK Inhibitoren in klinischen Studien bei Patienten mit pre-BCR+ ALL.Despite recent advances in B-ALL therapy, with overall survival rates reaching up to 90% in recent clinical trials, there is an urgent need for novel more targeted treatment approaches, particularly for patients failing chemotherapy and for patients at risk to develop chemotherapy-related toxicities. However, the identification of subtype specific therapeutic targets requires a thorough understanding of the underlying molecular mechanisms driving the disease. In this thesis, we describe a novel subgroup of B-ALL, characterized by pre-BCR surface expression and the requirement for active pre-BCR signaling for proliferation and survival. Using publicly available GEP datasets of normal pre-BCR+ B-cell progenitors in combination with GEP datasets from over 400 B-ALL patient samples we show that pre-BCR+ ALL cells inherit pre-BCR-dependency from their non-malignant counterparts the cyto-Ig+, surface IgM- pre-B cell. Furthermore, we employed a comparative approach of genetic pre-BCR pathway interference and pharmacologic inhibition of pre-BCR signaling in order to dissect the underlying molecular mechanisms driving proliferation and survival of pre-BCR+ ALL. This resulted in the identification of a signaling axis, involving the pre-BCR-dependent activation of PI3K, the inactivation of FOXO1 and the upregulation of MYC, which is crucial for the survival of pre-BCR+ ALL cells. Importantly, the inhibition of this signaling axis with SYK inhibitors completely reversed these effects and exhibited promising efficacy in several in vitro and in vivo models of pre-BCR+ ALL. This ultimately provides a rational for the assessment of pre-BCR inhibition as novel therapeutic strategy for subtypes of B-ALL in clinical trials.submitted by Stefan KöhrerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2018(VLID)285268

Effects of vatalanib on tumor growth can be potentiated by mTOR blockade in vivo

.The vascular endothelial growth factor (VEGF) is a central mediator of tumor-induced angiogenesis. Everolimus, a mammalian target of rapamycin (mTOR) inhibitor, decreases VEGF-secretion of cancer cells. Vatalanib is a selective inhibitor of VEGF receptors 1-3. In the present study it was hypothesized that dual inhibition of VEGF signaling by inhibition of VEGF production and VEGF receptor signaling leads to synergistic anti-tumor effects. In vitro, effects of vatalanib and everolimus on cell proliferation, cell cycle, apoptosis and signal transduction were examined in three gastric cancer cell lines. Effects on angiogenesis were assessed using tube formation assays of cultured human umbilical vein endothelial cells (HUVECs). In vivo, the antitumor effect of compounds was studied using a gastric cancer xenograft nude mouse model. VEGF of murine origin (mVEGF) and human cancer cell-derived VEGF (hVEGF) were studied separately by specific ELISAs. Tumor vascularization and proliferation were quantified by immunohistochemistry. In vitro, everolimus but not vatalanib decreased gastric cancer proliferation without inducing apoptosis. Vatalanib abolished endothelial cell tube formation, whereas inhibition of tube formation by everolimus was incomplete. In vivo, the combination of vatalanib with everolimus was superior to single agent treatments and reduced tumor size by about 50% relative to everolimus monotherapy (p < 0.005). Pharmacodynamic analysis of VEGF plasma level showed a decrease of hVEGF by everolimus and indicated a trend towards lower mVEGF level only in the combination group. In line, there was a tendency for lower vascular density and proliferation for combination treatment. We conclude that in a preclinical model of gastric cancer the antitumor activity of vatalanib can be augmented by everolimus

Effects of vatalanib on tumor growth can be potentiated by mTOR blockade in vivo

Background: Vatalanib is a selective inhibitor of vascular endothelial growth factor (VEGF) receptors 1-3. Vatalanib increases pro-angiogenic serum VEGF in response to VEGF receptor blockade which is considered to limit vatalinib´s anti-tumor activity. Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) signaling and decreases VEGF-secretion of cells. It was hypothesized that inhibition of VEGF production by everolimus potentiates the anti-tumor activity of vatalanib. Methods: In vitro, the effects of vatalanib and everolimus on gastric cancer cell proliferation, cell cycle, apoptosis and signal transduction were studied. Effects on angiogenesis were mimicked using tube formation assays of cultured human umbilical vein endothelial cells (HUVECs). In vivo, the anti-tumor effect of compounds was studied using a gastric cancer xenograft nude mouse model. VEGF production from the murine tumor host and human gastric cancer cells was analyzed by species specific ELISAs. Tumor vascularization and proliferation were studied by immunohistochemistry. Results: In vitro, everolimus but not vatalanib decreased gastric cancer proliferation without affecting apoptosis. Vatalanib completely abolished endothelial cell tube formation, whereas inhibition of tube formation by everolimus was incomplete. In vivo, the combination of vatalanib with everolimus was superior to single agent treatment. Treatment with vatalanib + everolimus significantly decreased mVEGF levels but not hVEGF. Treatment with everolimus decreased hVEGF production significantly. There was a trend for lower vascular density and proliferation for combination treatment. Conclusion: We conclude that anti-tumor activity of vatalanib can be augmented by everolimus in a preclinical model of gastric cancer, an effect potentially mediated by suppression of mVEGF production in the tumor microenvironment

Reexamination in vitro and in situ of an antibacterially modified experimental dental resin composite with molecular methods : a pilot study

Purpose. To introduce additional methods to detect and to quantify single pathogens in the complex biofilm formation on an antibacterial dental material. Materials and Methods. A conventional (ST) and an antibacterial dental composite (B) were manufactured. In vitro: specimens were incubated with a mixture of early colonizers. Bacterial adhesion was analyzed by TaqMan PCR after 8/24 h. In situ: TaqMan PCR and 16S rRNA Next Generation Sequencing (NGS) were performed. Results. In vitro: after 8 h incubation, B was covered by 58.6% of the bacterial amount that was attached to ST. After 24 h, the amount of attached bacteria to ST remained constant on ST only slightly lower on B. In situ: after 8 h the amount of adhering A. viscosus and S. mitis was prominent on ST and reduced on B. NGS revealed that S. sanguinis, S. parasanguinis, and Gemella sanguinis were the mainly attached species with S. sanguinis dominant on ST and S. parasanguinis and G. sanguinis dominant on B. Conclusions. Initial biofilm formation was altered by B. A shift between actinomycetes and streptococci was observed in situ. TaqMan PCR and 16S rRNA NGS revealed comparable results in situ and demonstrated the usefulness of NGS to characterize complex bacterial communities

Copy Number Changes and Allele Distribution Patterns of Chromosome 21 in B Cell Precursor Acute Lymphoblastic Leukemia

Chromosome 21 is the most affected chromosome in childhood acute lymphoblastic leukemia. Many of its numerical and structural abnormalities define diagnostically and clinically important subgroups. To obtain an overview about their types and their approximate genetic subgroup-specific incidence and distribution, we performed cytogenetic, FISH and array analyses in a total of 578 ALL patients (including 26 with a constitutional trisomy 21). The latter is the preferred method to assess genome-wide large and fine-scale copy number abnormalities (CNA) together with their corresponding allele distribution patterns. We identified a total of 258 cases (49%) with chromosome 21-associated CNA, a number that is perhaps lower-than-expected because ETV6-RUNX1-positive cases (11%) were significantly underrepresented in this array-analyzed cohort. Our most interesting observations relate to hyperdiploid leukemias with tetra- and pentasomies of chromosome 21 that develop in constitutionally trisomic patients. Utilizing comparative short tandem repeat analyses, we were able to prove that switches in the array-derived allele patterns are in fact meiotic recombination sites, which only become evident in patients with inborn trisomies that result from a meiosis 1 error. The detailed analysis of such cases may eventually provide important clues about the respective maldistribution mechanisms and the operative relevance of chromosome 21-specific regions in hyperdiploid leukemias