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Final Activity Report Summary - ACUP (Functional analysis of new acute phase proteins)

Within WP1 the role of novel acute phase proteins, mimitin, MCPIP, GADD45alfa was studied in inflammatory processes. These novel proteins are regulated by proinflammatory cytokines and at the same time are important regulators of the course of inflammation. It was shown that mimitin is a mitochondrial protein upregulated by proinflammatory cytokines at the transcriptional and protein levels, with MAP kinases involved in IL-1-dependent induction. This protein interacts with a microtubular protein (MAPIS) and some changes of mimitin gene expression modulate activity of caspases 3/7, thus, mimitin may participate in apoptosis. The other protein, MCPIP was characterised as an RNase regulating half-life of mRNA coding for IL-1 beta.

Gene coding for MCPIP is activated by proinflammatory cytokines, such as IL-1 and TNF and NFkappaB and Elk transcription factors are engaged in its regulation. According to obtained data there is a negative effect of MCPIP on NF-kappaB activation. The mutual regulation between MCPIP and NF-kappaB, its implication in the inflammatory state, makes MCPIP an interesting object to study and a putative target for the future therapeutic approaches.

Several molecules are particularly important for the defence system to function properly, including chemotactic factors and receptors exposed on the surface of immune cells. The former control recruitment of host defence effector cells from blood to afflicted tissues, whereas the latter are needed to communicate between immune cells and their environment. In this work, we focused on proteolytic enzymes such as ADAM17, a member of the ADAM (A Disintegrin And Metalloproteinase) family as regulators of the levels and activity of the receptors on the surface of immune cells, and on cysteine proteases as regulators of activity of chemotactic factors such as chemerin.

To determine if ADAM17 contributes to regulating adaptive immune responses we took advantage of a knock-out mice model - ADAM17 hypomorphic (ex/ex) mice, in which ADAM17 protein expression is reduced by 90-95%. Our data demonstrate that ADAM17 plays an important role in B cell mediated immunity. We also found that cysteine proteases are potent activators of chemerin. The phage display method which is a new technique of obtaining monoclonal antibodies (mAb) was introduced to the research carried out at the Host. This method allows for generating fully human mAb or antibodies against non-immunogenic molecules and does not require sacrificing animals.

The researchers at the Faculty gained a substantial experience in this methodology and are successful not only in the generation of new mAb but also in the training of students and other researchers. The single chain variable fragments (scFv) of antibodies that recognise and inhibit ADAM17, a membrane protease involved in development of chronic inflammatory diseases and promoting tumour progression, are being developed. The topic of the phage display method for obtaining monoclonal antibodies has become a permanent item in two unique courses for the students of biotechnology (a lecture and an advanced practical course).

The activities of WP4 concentrated on analysis of the involvement of mimitin and MCPIP in cancer. Neuroblastoma was chosen as the model of cancer. Expression of mimitin and MCPIP genes in different human neuroblastoma cell lines was evaluated. It was found that in four neuroblastoma cell lines tested, the highest mimitin content was accompanied by N-myc amplification. Therefore, chromatin immunoprecipitation of N-myc transcription factor bound to the mimitin promoter was done revealing possible binding of that transcription factor to the promoter in vivo. Owing to a very low level of MCPIP mRNA detectable only by quantitative RT-PCR, efforts were made to evaluate an effect of overexpression of MCPIP wt and MCPIP mutants on neuroblastoma tumour growth and metastatic potential. Different stably transfected BE(2)C cell clones were characterised in vitro and in vivo. The in vivo studies involving mice models of tumour growth are continued.

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