Methodological aspects on anti-nuclear antibody determination in canine autoimmunity and in vitro studies of antigen-specific cellular responses

The presence of circulating antinuclear antibodies, ANA, is the hallmark of several systemic autoimmune diseases and has become an important diagnostic tool. Rat liver sections and the human epithelial cell line HEp-2 were compared as substrates for the detection of canine ANA using the indirect immunofluorescence (HF) technique. The HEp-2 cell substrate was found to be superior to rat liver cryostate sections as ANA substrate for canine sera because of their low reactivity with normal sera and the ease of discernment of ANA fluorescence patterns in positive samples. Positive canine ANA sera thus could be subdivided according to the nuclear fluorescence staining patterns of non-mitotic cells and the staining of chromosomal areas in mitotic cells.Immunoblot reactivity as well as die presence of precipitating canine antibodies, determined by Ouchterlony immunodiffusion (ID), was found to be strictly associated with one of the DF ANA subtypes. Among the ID positive sera different antigenic reactivities were detected, represented by different ID subgroups. Only one of the ID subgroups showed identity with any of the well-defined and clinically important human ANA specificities, demonstrating anti-RNP reactivity. The response against the RNP antigen seems to be conserved between man and dog, as the canine anti-RNP reactivity was directed towards the human major antigenic region of the most prominent RNP antigen. Other prominent canine antoantigens were found not to be identical with the principal human ones, probably reflecting dog-specific subgroups of auioantigenic reactivities, which in turn may indicate different canine subgroups of systemic autoimmune disease.In cider to investigate antigen-specific cellular responses of human peripheral blood mononuclear cells (PBMQ, tetanus toxoid (IT) was used as a model antigen. Tetanus toxoid conjugated to beads was found to be a more efficient stimulate»' of a specific CD4+ Th 1 cell response in a primary stimulation assay of human PBMC as compared with soluble TT. It is suggested that targeting of IT antigen to phagocytic antigen-presenting cells, most probably monocytes, is responsible for the enhanced stimulatory properties observed