We have developed a novel TGF-β detection system, using genetically modified macrophages as cytokine biosensors, capable of detecting bioactive TGF-β in vivo and in vitro. Stimulation by TGF-β1 increases secretion of plasminogen activator inhibitor-1 (PAI-1) by activation of the promoter. An adenovirus containing the first 800bp of the PAI-1 promoter fused to the reporter gene β-galactosidase (Ad-PAI1800 βgal) was used to transfect primary cultures of rat bone marrow derived macrophages (BMDM). After transfection the BMDM were stimulated with TGF-β, 2 or 3 or left unstimulated. X-gal histochemical assay was performed 72-hours post-transfection to detect β-galactosidase expression. Macrophages transfected with Ad-PAI1800βgal and stimulated with TGF-β1, 2 or 3 showed a dose-dependent production of β-galacosidase. AdPAI1800βgal BMDM also expressed β-galactosidase when co-cultured with NR8383 rat alveolar macrophages transfected with recombinant adenovirus expressing active TGF-β1. Genetically modified macrophages could detect expression of TGF-β1 in vivo. AdPAI1800βgal transfected BMDM were co-injected with AdTGF-βl transfected NR8383 macrophages into the renal artery of rats with nephrotoxic nephritis (NTN). Isolated glomeruli showed X-gal positive macrophages on assay. AdPAI1800βgal BMDM were also injected into the renal artery of rats 7 days after the induction of Thy 1.1 nephritis, 100% of isolated glomeruli contained X-gal positive macrophages on assay. Signalling macrophages were capable of detecting TGF-β1 expression in sections of kidney tissue containing Ad-TGF-β1 transfected NR8383 macrophages. BMDM showing β-galactosidase were readily detected over the transfected glomeruli. Pre-treatment of Ad-PAI1800βgal transfected BMDM with IFN-γ prevented production of the reporter gene. However when stimulation with TGF-β occurred 20 hours after IFN-γ macrophage programmed unresponsiveness was abrogated. Macrophage responsiveness to TGF-β stimulation in Thy 1.1 nephritic kidneys was also prevented by pre-treatment with IFN-γ. This system demonstrates the use of genetically modified macrophages as both <i>in vivo </i>and <i>in vitro </i>sensors of the bioactive TGF-β providing a novel approach to assess cytokine activity.

Ulm, Univ. Diss., 2006.

Stuttgart, Univ., Diss., 2002.

Bladder cancer (BlCa) is the second most common genitourinary cancer, affecting both men and women. Most (70%) cases present at the superficial stage; 20% of these recur with muscle-invasive disease. Major genetic alterations associated with BlCa include: loss/gain in expression or mutations in Retinoblastoma (RB) gene, human epidermal growth factor receptors (HERs), H-ras, p53 and FGFR3. Only p53 mutations are well correlated with invasive BlCa; other changes show variable correlations with disease status. To understand the progression of BlCa, a model of nine human BlCa cell sublines derived from a single parent but differing in in vivo characteristics, has been developed previously. These cells represent a heterogenous population from a single tumour and a model of different stages of BlCa progression, from non-tumourigenic to invasive. Two sublines were selected for further investigation: C3 (non-tumourigenic) and B8 (invasive). These were transfected with green (C3-GSP-2) and red fluorescent reporters (B8-RSP-gck) respectively to investigate the effects of their co-injection in vivo, specifically, promotion of C3 tumour growth by B8 cells. Surprisingly, B8 tumour growth was inhibited by C3 cells in vivo at different cell numbers and proportions of cells injected. Microarray analysis of C3 and B8 cells revealed differential expression of 1367 genes with dramatic differences in the transforming growth factor-?? and integrin-mediated pathways. Gene expression of BMP2, INHBB, FST, NOG, ID4 and TGF- ??1, in the TGF- ?? pathway was further analysed with qRT-PCR in all nine sublines. Expression of BMP2 was significantly related to tumourigenic potential (p=0.0238, Mann-Whitney) and INHBB to invasive ability (p=0.0476, Mann-Whitney). The BlCa model did not include a metastatic component. To broaden the model, cell lines were established from an invaded lymph-node (B8-RSP-LN) and a bone-metastasis (B8-RSP-BN) after subcutaneous and intra-cardiac injection of B8-RSP-gck cells. No significant differences were observed in the migratory capability and anchorage-independent colony formation of these metastatic cells compared with B8 cells. Evaluation of expression of the panel of TGF-beta genes (BMP2, INHBB, FST, NOG, ID4 and TGF- ??1) and metastasis-related genes (MMP9, MMP2 and KAI1) indicated that expression of BMP2, FST, ID4 and MMP9 was decreased or lost in the metastatic sublines.

Bladder cancer (BlCa) is the second most common genitourinary cancer, affecting both men and women. Most (70%) cases present at the superficial stage; 20% of these recur with muscle-invasive disease. Major genetic alterations associated with BlCa include: loss/gain in expression or mutations in Retinoblastoma (RB) gene, human epidermal growth factor receptors (HERs), H-ras, p53 and FGFR3. Only p53 mutations are well correlated with invasive BlCa; other changes show variable correlations with disease status. To understand the progression of BlCa, a model of nine human BlCa cell sublines derived from a single parent but differing in in vivo characteristics, has been developed previously. These cells represent a heterogenous population from a single tumour and a model of different stages of BlCa progression, from non-tumourigenic to invasive. Two sublines were selected for further investigation: C3 (non-tumourigenic) and B8 (invasive). These were transfected with green (C3-GSP-2) and red fluorescent reporters (B8-RSP-gck) respectively to investigate the effects of their co-injection in vivo, specifically, promotion of C3 tumour growth by B8 cells. Surprisingly, B8 tumour growth was inhibited by C3 cells in vivo at different cell numbers and proportions of cells injected. Microarray analysis of C3 and B8 cells revealed differential expression of 1367 genes with dramatic differences in the transforming growth factor-?? and integrin-mediated pathways. Gene expression of BMP2, INHBB, FST, NOG, ID4 and TGF- ??1, in the TGF- ?? pathway was further analysed with qRT-PCR in all nine sublines. Expression of BMP2 was significantly related to tumourigenic potential (p=0.0238, Mann-Whitney) and INHBB to invasive ability (p=0.0476, Mann-Whitney). The BlCa model did not include a metastatic component. To broaden the model, cell lines were established from an invaded lymph-node (B8-RSP-LN) and a bone-metastasis (B8-RSP-BN) after subcutaneous and intra-cardiac injection of B8-RSP-gck cells. No significant differences were observed in the migratory capability and anchorage-independent colony formation of these metastatic cells compared with B8 cells. Evaluation of expression of the panel of TGF-beta genes (BMP2, INHBB, FST, NOG, ID4 and TGF- ??1) and metastasis-related genes (MMP9, MMP2 and KAI1) indicated that expression of BMP2, FST, ID4 and MMP9 was decreased or lost in the metastatic sublines.

Univ., Diss.--München, 1996.

Würzburg, Univ., Diss., 2009. / Zsfassung in engl. Sprache.

University, Diss., 2006--Giessen. / Zeichendarst. im Sachtitel teilw. nicht vorlagegemäß wiedergegeben.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

No abstract available