| Popis: |
One of the most prevalent and widespread diseases in western countries still is chronic renal failure. The reasons for that are manifold: besides genetically fixed renal issues, renal failure often is a concomitant disease induced as a consequence of severe intoxications, diabetes or rheumatoide diseases. Due to these complex correlations, therapeutic approaches are often difficult and more of a palliative than a curative character, only to mention immune suppression, organt ransplantations or dialysis. Especially in mesangioproliferative diseases like the IgA Nephropathy a basic mechanistic understanding is of absolutely urgent need,due to very limited access to suitable animal models. Within this work an innovative, unique Phage Display selection procedure was established, from which antibody fragments highly specific for murine mesangial cells were generated and characterized. This cell type represents a key player in mesangioproliferative glomerulonephritis. The C9-(scFv), E4-(scFv) and B4-(scFv)antibody fragments were each fused to a murine IgG2a-Fc portion, leading to an antibody-like chimera protein. Their binding characteristics towards the target cells in different matrices were investigated. The analysis performed during this thesis aimed to show mMC specificity on the basis of a substractive approach. Crossreactivity investigations on healthy competitor tissues like lung, heart, spleen and liver were performed followed by substractive co-localization studies with cell types closely related to the mesangial cells. It was clearly shown that all of the generated scFv-binders are highly specific for mesangial regions and do not bind on other tissues or cell types neighboured in the glomerulus. Nevertheless, the target antigens of the three selected mMC binders were not yet identified. Such investigations are of urgent need to promote therapeutic approaches. Regarding this, mass spectrometry analysis of cross-linked and immunoprecipitated scFv-Fc binders on the target cells is a promising step. So far we were not able to establish such experiments within our group, but this is still of major interest and appropriate steps are initiated. A highly specific recognition of murine mesangial cells within their natural tissue environment was proven for each of the three scFv-Fc fusion proteins. The utilization of these exclusive and newly murine MC specific, antibody-like fusion proteins is a reasonable path forward in diagnostic and descriptive approaches of murine kidney disease models. Fused to the Fc-portion the highly specific mMC binders should be used as an external trigger for the initiation of a new murine animal model for mesangioproliferative glomerulonephritis (GN). Following the disease progress and transformation of the anti-thy 1 nephritis in rats, the idea was to induce mesangial cell lysis, hyper-proliferation of residual cells within the glomerular structure as well as accumulation and agglutination of extracellular matrix within the mesangium by injection of a single portion of scFv-Fc fusion protein. Within the present work we did not succeed to establish such a murine animal model, but prepared the ground for further investigations into this direction. Especially the functionalization of the scFv-Fc proteins via their included recombinant SNAP-tag anchor allows a huge variety of further disease induction approaches. It allows the usage of the generated scFv-Fc fusions as highly cell specific delivery vehicles for different effector moieties. Direct labelling of our proteins with specific cytotoxic agents like Pseudomonas exotoxin A (ETA) or immune stimulating agents like growth factors via the SNAP-tag have the potential to induce site directed cell lysis and macrophage recruitment within the glomerular tuft and therefore mimic the complement derived inflammatory response described for the anti-Thy 1.1 nephritis. Further engineering of our scFv-Fc fusion proteins via the SNAP-tag combined with alterations and modifications in the experimental in-vivo set-up can help to open the wide field of genetically manipulated mouse models and utilize them to gain better understanding of the mechanisms involved in mesangioproliferative glomerulonephritis. |
