http://hdl.handle.net/10033/6874 Wed, 22 May 2013 22:56:41 GMT 2013-05-22T22:56:41Z http://hdl.handle.net/10033/270452 Title: High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination. Authors: Pohla, Heike; Buchner, Alexander; Stadlbauer, Birgit; Frankenberger, Bernhard; Stevanovic, Stefan; Walter, Steffen; Frank, Ronald; Schwachula, Tim; Olek, Sven; Kopp, Joachim; Willimsky, Gerald; Stief, Christian G; Hofstetter, Alfons; Pezzutto, Antonio; Blankenstein, Thomas; Oberneder, Ralph; Schendel, Dolores J Abstract: Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10033/270452 2013-01-01T00:00:00Z http://hdl.handle.net/10033/267812 Title: Analysis of gene expression data from non-small cell lung carcinoma cell lines reveals distinct sub-classes from those identified at the phenotype level. Authors: Dalby, Andrew R; Emam, Ibrahim; Franke, Raimo Abstract: Microarray data from cell lines of Non-Small Cell Lung Carcinoma (NSCLC) can be used to look for differences in gene expression between the cell lines derived from different tumour samples, and to investigate if these differences can be used to cluster the cell lines into distinct groups. Dividing the cell lines into classes can help to improve diagnosis and the development of screens for new drug candidates. The micro-array data is first subjected to quality control analysis and then subsequently normalised using three alternate methods to reduce the chances of differences being artefacts resulting from the normalisation process. The final clustering into sub-classes was carried out in a conservative manner such that sub-classes were consistent across all three normalisation methods. If there is structure in the cell line population it was expected that this would agree with histological classifications, but this was not found to be the case. To check the biological consistency of the sub-classes the set of most strongly differentially expressed genes was be identified for each pair of clusters to check if the genes that most strongly define sub-classes have biological functions consistent with NSCLC. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10033/267812 2012-01-01T00:00:00Z http://hdl.handle.net/10033/245751 Title: Characterization of biphenyl dioxygenase sequences and activities encoded by the metagenomes of highly polychlorobiphenyl-contaminated soils. Authors: Standfuss-Gabisch, Christine; Al-Halbouni, Djamila; Hofer, Bernd Abstract: Total extracted DNA from two heavily polychlorobiphenyl-contaminated soils was analyzed with respect to biphenyl dioxygenase sequences and activities. This was done by PCR amplification and cloning of a DNA segment encoding the active site of the enzyme. The translated sequences obtained fell into three similarity clusters (I to III). Sequence identities were high within but moderate or low between the clusters. Members of clusters I and II showed high sequence similarities with well-known biphenyl dioxygenases. Cluster III showed low (43%) sequence identity with a biphenyl dioxygenase from Rhodococcus jostii RHA1. Amplicons from the three clusters were used to reconstitute and express complete biphenyl dioxygenase operons. In most cases, the resulting hybrid dioxygenases were detected in cell extracts of the recombinant hosts. At least 83% of these enzymes were catalytically active. Several amino acid exchanges were identified that critically affected activity. Chlorobiphenyl turnover by the enzymes containing the prototype sequences of clusters I and II was characterized with 10 congeners that were major, minor, or not constituents of the contaminated soils. No direct correlations were observed between on-site concentrations and rates of productive dioxygenations of these chlorobiphenyls. The prototype enzymes displayed markedly different substrate and product ranges. The cluster II dioxygenase possessed a broader substrate spectrum toward the assayed congeners, whereas the cluster I enzyme was superior in the attack of ortho-chlorinated aromatic rings. These results demonstrate the feasibility of the applied approach to functionally characterize dioxygenase activities of soil metagenomes via amplification of incomplete genes. Sun, 01 Apr 2012 00:00:00 GMT http://hdl.handle.net/10033/245751 2012-04-01T00:00:00Z http://hdl.handle.net/10033/237371 Title: Phosphatidylinositol 3'-kinase activity is critical for initiating the oxidative burst and bacterial destruction during CEACAM3-mediated phagocytosis. Authors: Buntru, Alexander; Kopp, Kathrin; Voges, Maike; Frank, Ronald; Bachmann, Verena; Hauck, Christof R Abstract: Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is an immunoglobulin-related receptor expressed on human granulocytes. CEACAM3 functions as a single chain phagocytotic receptor recognizing gram-negative bacteria such as Neisseria gonorrhoeae, which possess CEACAM-binding adhesins on their surface. The cytoplasmic domain of CEACAM3 contains an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is phosphorylated upon receptor engagement. Here we show that the SH2 domains of the regulatory subunit of phosphatidylinositol 3'-kinase (PI3K) bind to tyrosine residue 230 of CEACAM3 in a phosphorylation-dependent manner. PI3K is rapidly recruited and directly associates with CEACAM3 upon bacterial binding as shown by FRET analysis. Although PI3K activity is not required for efficient uptake of the bacteria by CEACAM3-transfected cells or primary human granulocytes, it is critical for the stimulated production of reactive oxygen species by infected phagocytes and the intracellular degradation of CEACAM-binding bacteria. Together, our results highlight the ability of CEACAM3 to coordinate signaling events that not only mediate bacterial uptake, but also trigger the killing of internalized pathogens. Fri, 18 Mar 2011 00:00:00 GMT http://hdl.handle.net/10033/237371 2011-03-18T00:00:00Z