In the present study six assays for the quantification of biofilms formed in 96-well microtiter plates were optimised and evaluated: the crystal violet (CV) assay, the Syto9 assay, the fluorescein diacetate (FDA) assay, the resazurin assay, the XTT assay and the dimethyl methylene blue (DMMB) assay. Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, Propionibacterium acnes and Candida albicans were used as test organisms. In general, these assays showed a broad applicability and a high repeatability for most isolates. In addition, the estimated numbers of CFUs present in the biofilms show limited variations between the different assays. Nevertheless, our data show that some assays are less suitable for the quantification of biofilms of particular isolates (e.g. the CV assay for P. aeruginosa).

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The platelet factor-4 variant, designated PF-4var/CXCL4L1, is a recently described natural non-allelic gene variant of the CXC chemokine platelet factor-4/CXCL4. PF-4var/CXCL4L1 was cloned, and the purified recombinant protein strongly inhibited angiogenesis. Recombinant PF-4var/CXCL4L1 was angiostatically more active (at nanomolar concentration) than PF-4/CXCL4 in various test systems, including wound-healing and migration assays for microvascular endothelial cells and the rat cornea micropocket assay for angiogenesis. Furthermore, PF-4var/CXCL4L1 more efficiently inhibited tumor growth in animal models of melanoma and lung carcinoma than PF-4/CXCL4 at an equimolar concentration. For B16 melanoma in nude mice, a significant reduction in tumor size and the number of small i.t. blood vessels was obtained with i.t. applied PF-4var/CXCL4L1. For A549 adenocarcinoma in severe combined immunodeficient mice, i.t. PF-4var/CXCL4L1 reduced tumor growth and microvasculature more efficiently than PF-4/CXCL4 and prevented metastasis to various organs better than the angiostatic IFN-inducible protein 10/CXCL10. Finally, in the syngeneic model of Lewis lung carcinoma, PF-4var/CXCL4L1 inhibited tumor growth equally well as monokine induced by IFN-; (Mig)/CXCL9, also known to attract effector T lymphocytes. Taken together, PF-4var/ CXCL4L1 is a highly potent antitumoral chemokine preventing development and metastasis of various tumors by inhibition of angiogenesis. These data confirm the clinical potential of locally released chemokines in cancer therapy. [Cancer Res 2007;67(12):5940-8]

BackgroundBurkholderia cepacia complex bacteria are opportunistic pathogens, which can cause severe respiratory tract infections in patients with cystic fibrosis (CF). As treatment of infected CF patients is problematic, multiple preventive measures are taken to reduce the infection risk. Besides a stringent segregation policy to prevent patient-to-patient transmission, clinicians also advise patients to clean and disinfect their respiratory equipment on a regular basis. However, problems regarding the efficacy of several disinfection procedures for the removal and/or killing of B. cepacia complex bacteria have been reported. In order to unravel the molecular mechanisms involved in the resistance of biofilm-grown Burkholderia cenocepacia cells against high concentrations of reactive oxygen species (ROS), the present study focussed on the transcriptional response in sessile B. cenocepacia J2315 cells following exposure to high levels of H2O2 or NaOCl.ResultsThe exposure to H2O2 and NaOCl resulted in an upregulation of the transcription of 315 (4.4%) and 386 (5.4%) genes, respectively. Transcription of 185 (2.6%) and 331 (4.6%) genes was decreased in response to the respective treatments. Many of the upregulated genes in the NaOCl- and H2O2-treated biofilms are involved in oxidative stress as well as general stress response, emphasizing the importance of the efficient neutralization and scavenging of ROS. In addition, multiple upregulated genes encode proteins that are necessary to repair ROS-induced cellular damage. Unexpectedly, a prolonged treatment with H2O2 also resulted in an increased transcription of multiple phage-related genes. A closer inspection of hybridisation signals obtained with probes targeting intergenic regions led to the identification of a putative 6S RNA.ConclusionOur results reveal that the transcription of a large fraction of B. cenocepacia J2315 genes is altered upon exposure of sessile cells to ROS. These observations have highlighted that B. cenocepacia may alter several pathways in response to exposure to ROS and they have led to the identification of many genes not previously implicated in the stress response of this pathogen.

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