By generating isogenic mutants with a chromosomal in-frame deletion in either inlA or inlB, we have identified InlA and InlB as surface-bound proteins of L. monocytogenes with molecular weights of 88,000 and 65,000, respectively. These results were obtained with monoclonal antibodies raised against either protein and corroborated by N-terminal end sequencing of InlA and InlB. By immunoblot analysis, the production of both polypeptides was found to be strongly dependent on growth temperature and, particularly for InlB, on the presence of the PrfA regulator protein. Expression of InlA was not strictly dependent on the presence of the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that the inlA gene was transcribed by several promoters, of which only one is PrfA dependent. This PrfA-dependent inlA promoter, which contains two base substitutions within its putative PrfA DNA-binding palindrome, is responsible for transcription of both inlA and inlB genes. A hitherto unrecognized promoter located 51 bp upstream of the GTG start codon of the inlB gene was also detected. Hence, inlA and inlB are transcribed both individually and in an operon by PrfA-dependent and-independent mechanisms. Tissue culture invasion assays employing various epithelial cell lines demonstrated that both InlA and InlB are required for invasion. In vivo studies using the mouse infection model revealed that both internalin mutants were attenuated for virulence.

The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3). Reproducibility of protein quantitation based on spectral counting was similar for all 3 methods (CV: 26% vs 24% and 24%, respectively). As a proof-of-concept of secretome proteomics for blood-based biomarker discovery, the gradient 1DGE workflow was subsequently applied to identify IGF1R-signaling related proteins in the secretome of mouse embryonic fibroblasts transformed with human IGF1R (MEF/Toff/IGF1R). VEGF and osteopontin were differentially detected by LC-MS/MS and verified in secretomes by ELISA. Follow-up in serum of mice bearing MEF/Toff/IGF1R-induced tumors showed an increase of osteopontin levels paralleling tumor growth, and reduction in the serum of mice in which IGF1R expression was shut off and tumor regressed.

5015 Background: Based on results of animal experiments intermittent androgen blockade was suggested to delay progression of advanced prostate cancer to the hormone refractory stage. We conducted a prospective randomized study to compare intermittent with continuous androgen suppression. Methods: This was a multi-centre, randomised, two-arm study comparing treatment with goserelin + bicalutamide (intermittent, group A) vs. goserelin + bicalutamide (continuous, group B). The primary endpoint was time to clinical and/or biochemical progression of the disease despite androgen suppression. Secondary enpoints were survival time, patient’s quality of life (QoL) and toxicity. Patients eligibility criteria were: histologically confirmed adenocarcinoma of the prostate in clinical stage T1–4N1–3M0 or T1–4N0–3M1 (D1 oder D2). After an induction phase of 24 weeks with MAB, 335 patients whose PSA decreased under 4 ng/ml or = 90% from baseline were randomized. Results: About two-thirds of the patients of both the intermittent and the continuous therapy arm (65% versus 66%, ITT population) experienced a clinical and/or biochemical disease progression due to any reason during this study. The median time to disease progression was longer for patients randomised to the intermittent therapy arm (16.6 months) compared with patients randomised to the continuous therapy arm (11.5 months). This difference however was not statistically significant (log rank test, p=0.1758). The median time to death from any cause was 51.4 month in the intermittent arm compared and 53.8 months in the continuous therapy arm (p = 0.658). There were no differences in the incidence of patients with AEs or SAEs or in any other safety parameter between patients treated intermittently and patients treated continuously. Patients’ self-assessment of their overall health and of their sexual activity appeared to be favourable in the intermittent compared with the continuous therapy arm. 88% of all patients treated intermittently experienced more than 50% of the number of treatment days as treatment-free days. Conclusions: Intermittent therapy in D1 and D2 prostate cancer patients appears to be safe and feasible. Off treatment periods are > 40 % and attribute to patients quality of life. No significant financial relationships to disclose.

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