Seleção in vivo de peptídeos ligantes à articulações inflamadas e seu potencial uso em diagnóstico

Abstract

CHAPTER I: The immune system is a complex of molecules and cells that functions to protect the organism against pathogens. The healthy immune system must maintain the balance between the ability to respond to infectious agents and sustain self-tolerance. However, failures may occur which result in reactions against cells and tissues of the organism and which cause autoimmune diseases such as Rheumatoid Arthritis (RA). This is an autoimmune disease characterized by symmetrical arthritis and additive, of unknown cause, usually beginning after age 30 and affects three times more women than men, with a prevalence of 1.0% in some regions. In rheumatoid arthritis, the synovial membrane becomes infiltrated by various types of inflammatory cells, which synergize to cause destruction of joints, resulting in strong joint pain. The diagnosis of arthritis depends on the association of a number of clinical signs and symptoms, laboratory findings, and radiographic findings. The goal of treatment is to relieve pain, maintain or improve functional capacity, prevent disability and improve their quality of life. Although there is no test that confirms the presence of the disease, there is no specific laboratory tests considered for confirming diagnosis. Murine models are effective to enrich our understanding of human arthritis, as DBA/1J strain animals presenting high susceptibility to development of arthritis resulting from immunization with type II collagen. Phage display is a selection method wherein a library of peptides that are expressed on the surface of phage, with genetic material coding for each peptide located in the viral genome. The targeting peptide ligands the highly inflamed regions are useful to assist in differential diagnosis, assess the extent of inflammation and determining prognosis and response to therapy. Accordingly, the search for novel biomarkers may lead to new methods for the diagnosis and treatment of RA. This review aims to describe some aspects of Rheumatoid Arthritis and how technology Phage Display in vivo can be used to identify ligands to various antigens associated with inflammation. CHAPTER II: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joints persistent inflammation. Pain, swelling and limitation of movement are the typical signs of this inflammation. Arthritis is the name given to a joint inflammation, with a barely known root, and may affect different joints. The diagnosis of RA depends on the association of a number of symptoms and clinical signs, laboratory and radiographic examinations. Thus, studies directed to investigate new biomarkers become of great importance for the diagnosis of RA. The aim of this study was to select and identify the methodology by Phage Display in vivo binder peptides to inflamed regions of mice DBA / 1J with high inflammation induced by collagen type II. For induction of arthritis, mice of isogenic DBA/1J were immunized with 100ug of type II collagen emulsified with complete Freund\'s adjuvant. For the selection of binder peptides to the inflamed regions was performed an in vivo bioppaning using a peptide library Ph.D.-C7C expressed on the surface of filamentous phage M13 for three cycles of selection, the last one being performed with subtraction of nonspecific using healthy joints. The DNA of selected clones was sequenced, translated and several clones were subjected to the tests Immunohistochemistry. Among the 144 clones sequenced, 115 had valid sequences. Among the last ones were identified 20 different sequences. The bioinformatics analysis demonstrated that there is no consensus among the majority of selected peptides. The test Immunohistochemistry allowed the pre-validation of two clones with a potential biomarker for Immunodiagnosis of Rheumatoid Arthritis.