CHANGES IN THE LEVELS OF AUTOANTIBODIES TO CELLULAR PHOSPHOLIPIDS, CYTOPLASM OF NEUTROPHILS AND NUCLEAR ANTIGENS DURING CHRONIC LAMINITIS IN HORSES
Abstract
Pathogenetic mechanisms involved in the development of laminitis differ based on theories based on inflammatory, vascular, enzymatic, metabolic, or traumatic factors. Regarding the two mechanisms that have enjoyed great favor in the past, i.e., inflammation and digital vascular dysfunction, there is debate as to which is primary or whether they are interdependent and of simultaneous onset, implying that the microcirculation in the distal phalanx always plays a critical role in initiating laminitis According to the latest studies, in chronic laminitis, in certain hyperreactive areas of the dermal lamellae, episodes of subclinical course and exacerbations occur after exposure to antigenic stimulation from vaccinations or environmental allergens, as well as autoimmune components of the inflammatory reaction, which enhances the induction of chemokines for neutrophils, which prolongs inflammation and immunological hyperreactivity. The aim of our research was to determine the levels of autoantibodies to phospholipids, deoxyribonucleic acid, cytoplasm of neutrophils as markers of chronic immune-dependent inflammation of connective tissue and microcirculatory bed in blood serum and hoof skin homogenates in acute pododermatitis and chronic laminitis. The material for the research was blood serum, as well as fragments of the foliar and papillary base of the hoof skin of horses without orthopedic pathology, with acute aseptic pododermatitis and chronic laminitis. In order to increase informativeness, blood for the study was collected from the regional veins of the respective limbs – the subcutaneous vein of the forearm (thoracic limb) and the subcutaneous vein of the lower leg (pelvic limb). Samples of hoof dermis were washed in physiological solution, homogenized in the cold in RVS buffer (pH 7.4), with a 1% solution of Triton X-100 in a ratio of 1:40 and left at +4◦С for 2 hours, then the tissue homogenate was centrifuged at 3000 rpm. within 15 min. after which the supernatant was subjected to cryopreservation. In blood serum and hoof dermis homogenates, the level of antiphospholipid antibodies of the APHL IgG, APHL IgM classes was determined by the method of solid-phase immunoenzymatic ELISA analysis, autoantibodies to native, double-stranded deoxyribonucleic acid (dsDNA) and autonuclear antibodies to single-stranded, denatured deoxyribonucleic acid (ssDNA), as well as anticytoplasmic anti-neutrophil antibodies (ANCA) – automated immunoenzymatic method ELIA Phadia. The content of autoantibodies to APHL, dsDNA, ssDNA and cANCA in tissue samples of hoof dermis homogenates was calculated taking into account the ratio (tissue–RVS buffer). It was established that with chronic laminitis in horses, the level of APHL classes of IgM increases in blood serum and hoof dermis homogenates to 5.43±0.70 IU/ml and 33.95±7.63 IU/ml, respectively, and for the IgG class to 9.43±1.22 IU/ml in blood serum and up to 77.50±10.06 IU/ml. The level of autoantibodies to dsDNA and ssDNA in the blood serum of horses with chronic laminitis increases to the values of 20.18±1.92 IU/ml and 19.55±2.66 IU/ ml, against 5.68±0.82 IU/ml and 5.19 IU/ml in clinically healthy animals, respectively. The concentration of autoantibodies to dsDNA and ssDNA in hoof dermis homogenates of horses with chronic laminitis increases to 270.0±25.11 IU/ml and 305.50±26.48 IU/ml, against 78.80±14.21 IU/ml and 68 ,80±12,22 IU/ml in clinically healthy animals, respectively. Serum anticytoplasmic ANCA antibodies in clinically healthy horses were not detected in 100% of animals, while a positive reaction was detected in 20% of cases of acute pododermatitis, and in 62.5% of cases of chronic laminitis. The perspective of further research is the study of the functioning of the immune system and the pathogenetic mechanisms of the formation of immunedependent inflammation in chronic laminitis in horses.
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