Importanza prognostica delle citochine e loro ruolo nell'immunomodulazione: risultati di studi sperimentali in vivo e in vitro

Citokines are proteins produced by several cell types and secreted in response to various stimuli. These molecules are able to modify the behaviour of other cells inducing activities like growth, differentiation and apoptosis. In the last years, veterinary scientists have investigated the role played by these factors; in fact, cytokines can act as intercellular communicative signals in immune response, cell damage repair and hematopoiesis. Up to date, various cytokines have been identified and in depth comprehension of their effects in physiology, pathology and therapy is an interesting field of research. This thesis aims to understand the role played by these mediators during natural or experimentally induced pathologies. In particular, it has been evaluated the genic and protein expressions of a large number of cytokines during several diseases and starting from different matrix. Considering the heterogeneity of materials used in experimentations, multiple methods and protocols of nucleic acids and proteins extractions have been standardized. Results on cytokines expression obtained from various in vitro and in vivo experimental studies have shown how important these mediators are in regulation and modulation of the host immune response also in veterinary medicine. In particular, the analysis of inflammatory and septic markers, like cytokines, has allowed a better understanding in the pathogenesis during horse Recurrent Airway Obstruction, foal sepsis, Bovine Viral Diarrhea Virus infection and dog Parvovirus infection and the effects of these agents on the host immune system. As experimentations with mice have shown, some pathologies of the respiratory and nervous system can be reduced or even erased by blocking cytokines inflammatory production. The in vitro cytokines expression evaluation in cells which are in vivo involved in the response to exogenous (like pathogens) or endogenous (as it happens during autoimmune diseases) inflammatory stimuli could represent a model for studying citokines effects during the host immune response. This has been analyzed using lymphocytes cultured with several St. aureus strains isolated from bovine mastitic milk and different colostrum products. In the first experiment different cytokines were expressed depending on enterotoxins produced, justifying a different behaviour of the microrganism in the mammal gland. In the second one, bone marrow cells derived incubated with murine lymphocytes with colostrum products have shown various cluster of differentiation expression , different proliferation and a modified cytokines profile. A better understanding of cytokine expression mechanisms will increase the know-how on immune response activated by several pathogen agents. In particular, blocking the cytokine production, the inhibition or catalyzation of the receptor binding mechanism and the modulation of signal transduction mechanism will represent a novel therapeutic strategy in veterinary medicine.

In vitro characterisation and expansion of human regulatory T cells for their in vivo application in the induction of tolerance in haematopoietic stem cell and solid organ transplantation

Solid organ transplantation (SOT) is considered the treatment of choice for many end-stage organ diseases. Thus far, short term results are excellent, with patient survival rates greater than 90% one year post-surgery, but there are several problems with the long term acceptance and use of immunosuppressive drugs. Hematopoietic Stem Cells Transplantation (HSCT) concerns the infusion of haematopoietic stem cells to re-establish acquired and congenital disorders of the hematopoietic system. The main side effect is the Graft versus Host Disease (GvHD) where donor T cells can cause pathology involving the damage of host tissues. Patients undergoing acute or chronic GvHD receive immunosuppressive regimen that is responsible for several side effects. The use of immunosuppressive drugs in the setting of SOT and GvHD has markedly reduced the incidence of acute rejection and the tissue damage in GvHD however, the numerous adverse side effects observed boost the development of alternative strategies to improve the long-term outcome. To this effect, the use of CD4+CD25+FOXP3+ regulatory T cells (Treg) as a cellular therapy is an attractive approach for autoimmunity disease, GvHD and limiting immune responses to allograft after transplantation. Treg have a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. Results of my thesis provide the characterization and cell processing of Tregs from healthy controls and patients in waiting list for liver transplantation, followed by the development of an efficient expansion-protocol and the investigation of the impact of the main immunosuppressive drugs on viability, proliferative capacity and function of expanded cells after expansion. The conclusion is that ex vivo expansion is necessary to infuse a high Treg dose and although many other factors in vivo can contribute to the success of Treg therapy, the infusion of Tregs during the administration of the highest dose of immunosuppressants should be carefully considered.