M. V. Ostankov

AB0078 Role of experimental research in study of rheumatoid arthritis etiopathogenesis

Background To study the pathogenesis, diagnosis and therapy of rheumatoid arthritis (RA) there are used numerous experimental in vivo and in vitro models. Topical issue of this problem is to study the causes of disorder and rehabilitation of immune tolerance mechanisms in RA. The concept about the role of different cells in central and peripheral tolerance formation in this pathology is often contradictory and not clear. A tolerogenic activity of products of fetoplacental complex has long been studied at the Cryopathophysiology and Immunology Department of the Institute for Problems of Cryobiology and Cryomedicine of NAS of Ukraine. The presence of a wide range of immunotropic substances in placenta is a premise to use placental cell suspension (PCS) for immunocompetent sphere recovery in autoimmune diseases. Objectives The research aim was to determine the possibilities and features of implementation of a tolerogenic activity by native and cryopreserved placental cells in experimental models of RA development: adjuvant arthritis (AA). Methods Research was carried out in CBA/H mice. The PCS was obtained via homogenizing the murine placenta to days 18–19 of gestation. The AA was induced by subplantar administration of the complete Freunds adjuvant. The AA development was expressed as the arthritis index. Either native (nPCS) or cryopreserved PCS were administered intravenously to day 7 after pathology induction. The PCS was cryopreserved under protection of either 10% dimethyl sulfoxide solution (suspension CD) or Propandiosakharol (suspension CP). A number of CD4+CD25+ T reg cells was determined by direct immunofluorescence using monoclonal antibodies (BD Pharmingen TM) with flow cytometry (FACS Calibur, BD) in AA animal spleen. The foxp3 gene expression in murine spleen cells was assessed by multiplex reverse transcription polymerase chain reaction (RT-PCR). In PCR we used the primer pairs to foxp3 gene and that of housekeeping: β-actin. A number of gene transcripts was compared basing on the relative semi-quantitative estimation of amplification products using Agilent Bioanalyzer 2100 (USA). Results A disordered CD4+CD25+ cell accumulation in spleen of experimental animals with AA was established and possible use of PCS for its correction was demonstrated. The foxp3 expression level in AA animal spleen cells reduced on day 28, suggesting a contribution of this factor into AA pathogenesis within a long-term period of pathology development. During this period a decrease in joint swelling correlated with an increased content of T-reg cells and foxp3 expression level in spleen of animals after either nPCS or CD administration. Conclusions Therapeutic effect of either native or cryopreserved PCS in AA animals is manifested on molecular level, as evidenced by an increased foxp3 expression in spleen cells after suspension administration. The effect of introduced cryopreserved placental cells as for this gene activation and, consequently, T-reg, was herewith determined by cryopreservation regimen. References Goltsev A.N., Lutsenko E.D. Ostankov M.V. Effect of different cryopreservation regimens on manifestation of immune modulating activity of placenta at development of adjuvant arthritis. Problems of Cryobiology 2008; 18(4): 456–459. Haque R., Lei F. Foxp3 and Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development. Arthritis Research and Therapy 2010; 12: R.66. Disclosure of Interest None declared

AB0076 Study of physiological activity of cd4+cd25+ cells during adjuvant arthritis prior to and after introduction of cryopreserved placental cells

Background An important role in the development of autoimmune reactions during rheumatoid arthritis (RA) is played by CD4+CD25+ T-regulatory cells (Treg) possessing a suppressive activity. The ability of placental structures to produce the activation factors of Treg-cells function – IL-2, TGF-β, to increase the number of cells with Treg phenotype - CD4+CD25high and CD4+/Foxp3+ during co-culturing in the mixed lymphocyte culture (MLC) can be the precondition of using the placental cell suspension (PCS) to recover the function of Treg-cells in vivo, in particular during adjuvant arthritis (AA) [1]. Application of PCS as well as other cell therapy preparations foresees a mandatory use of cryopreservation as technological stage of the material preparing. Objectives The research aim was to substantiate the possible correction of the state of CD4+CD25+ T-cells in lymphoid organs at AA with cryopreserved placental cells. Methods The research was performed in 16-18 g CBA/H mice. AA was induced with subplantar injection of Freund’s complete adjuvant [2]. PCS (18-19 gestation days) was cryopreserved in cryovials (Nunc, Denmark) under 10% DMSO protection (suspension cryopreserved with dimexide or suspension cryopreserved with propane diol saccharol) by means of programmable freezer UOP-1. PCS was intravenously injected to the 7th day of AA development in a dose of 1’106 cells per mouse. The content of T-reg cells (CD4+CD25+) was examined with flow cytometer (FACS Calibur, BD) in lymph nodes and spleen of animals with AA prior to and after introduction of PCS by the method of direct immune fluorescence using monoclonal antibodies to CD4 and CD25 (BD Pharmingen TM). Results The introduction of native PCS contributed to the reduced arthritis index (AI) and recovery of the content of immune competent cells in lymphoid organs. When determining the relationship between AI and changed content of Treg-cells with CD4+CD25+ phenotype in lymphoid organs of mice we have found a significant rise in the number of these cells to the 7th day of AA development in lymph nodes and to the 21st day in spleen on the background of increased joint oedema in both terms. After introduction of native PCS the dynamics of changing the content of Treg-cells in spleen and lymph nodes was one-way. The introduction of both cryopreserved suspensions rendered therapeutic effect on AA course along with this the effect after introduction of the suspension cryopreserved with dimexide was related to the regulation of Treg-cells pool in spleen and after introduction of the suspension cryopreserved with propane diol saccharol was done to their content in lymph nodes. Conclusions The prospects of using the cryopreserved PCS when treating AA are related to possible correction of the state of Treg-cells population in lymphoid organs. Implementation of therapeutic effect after PCS introduction testifies to its systemic effect when treating AA. References Goltsev A.N., Lutsenko E.D., Ostankov M.V., Bondarovich N.A. Estimation of cytokine profile in adjuvant arthritis model after cryopreserved placenta cells application // Patologiya. – 2011. – Vol.8, N2. – P. 99-101. Mischenko O.Ya., Kotvitska A.A. Pharmacological efficiency of analbene emulsion in the model of adjuvant arthritis in rats // Visnyk Farmatsii. – 2001. – N3. – P. 124-125. Disclosure of Interest None Declared