• Institute of Biology and Immunology of Reproduction
      "Acad. Kiril Bratanov"

      • Scientific achievements

        More important scientific achievements for the last 10 years

        Stem cells

        • The presence of mesenchymal stem cells /MSC/ has been proved in human endometrium and decidual tissue. These cells can be expanded and cultured in vitro, they possess phenotype characteristics of stromal cells of mesenchymal origin recognized by the expression of CD29, CD73 and CD90, while being negative for markers of haemopoietic cells. The endometrial MSCs possess clonogenic activity they can be differentiated to osteogenic, adipogenic and endothelium-like cells. The hypothesis that the stromal MSCs participate in the remodeling of the endometrium during the menstrual cycle of women as well as during formation of deciduas in pregnancy has been proposed. R.Dimitrov, T.Timeva, D.Kyurkchiev, M.Stamenova, A.Shterev, P.Kostova, V. Zlatkov, I.Kehayov, S.Kyurkchiev. Characterization of clonogenic stromal cells isolated from human endometrium. Reproduction, (2008) 135 551–558; R.Dimitrov, D.Kyurkchiev, T.Timeva, M.Yunakova, M.Stamenova, A. Shterev, S.Kyurkchiev. Human pre-decidual stromal cells have the characteristics of mesenchymal stem cells. Fertil. Steril, 2010, 93 (1), 210-219; S.Kyurkchiev, A.Shterev, R.Dimitrov. Assessment on presence and characteristics of multipotent stromal cells in human endometrium and deciduas. Reproductive Biomedicine Online, 2010, 20 (3), 305 - 313
        • Our investigations about the role of progesterone and other reproductive hormones on the proliferation, differentiation and immunomodulating properties of mesenchymal stem cells have shown the following results: the progesterone have not changed the proliferative properties/characteristics of bone marrow, adipose tissue or early deciduas derived mesenchymal stem cells when added to the culture medium. The combination of estradiol, progesterone and cAMP has induced the decidualization of all MSC, while the strongest effect has been obtained with the ones isolated from early deciduas. D. Kyurkchiev, E. Ivanova-Todorova, S. Kyurkchiev. New target cells of the immunomodulatory effects of progesterone. Reproductive BioMedicine Online. 2010. 
        • Adipose tissue-derived mesenchymal stem cells (ASC) have been used as feeder cells for the elaboration of co-culture system for maintenance of human embryonic stem cells (hESC). For the purpose BG01V cell line have been used. In the condition of long-term co-culture (around 20 passages) with ASC, BG01V cells have preserved their pluripotency and differentiation potential for development of embryoid bodies. According to the collected experimental data it is evident that human ASC can efficiently be used as supporting feeder cells for the growth of BG01V cells in co-cultures. ASC apparently produce and secrete the necessary growth factors which maintain the growth and differentiation potential of BG01V cells. Oreshkova, T., Stoyanova, E., Mourdjeva, M., Kyurkchiev, S. Human adipose tissue-derived mesenchymal stem cells support the growth and plasticity of human embryonic stem cells (BG01V cell line). Comptes Rendus de L'Academie Bulgare des Sciences 63(10), pp. 1477- 1484. 2010 
        • Laboratory protocols for derivation of iPS from ASC and human fibroblast cell line BJ have been developed. By using retroviral system, plasmids encoding the sequences of transcription factors (Oct4, Sox2, Klf4 and c-Myc) have been amplified. Having been introduced into the target cells, a change in the morphology and the expression of specific pluripotency markers have been detected in the target cells. Accumulating data from immunofluorescence labeling, RT-PCR and FACS analyses show that the treated cells have the some characteristics of human embryonic stem cells but do not express all pluripotent markers. 
        • Methods for isolation, in vitro cultivation and characterization of mesenchymal stem cells from human bone marrow, adipose tissue and dental pulp have been developed. By comparison of mesenchymal stem cells from bone marrow (bm-MSC) or adipose tissue (at-MSC) we found that the cells from both sources have almost identical morphological, immunophenotype and cloning characteristics. As to the rate of in vitro proliferation we proved that at-MSCs have higher proliferative ability but lower potential for differentiation to osteogenic cells. The finding that at-MSCs have higher inhibitory activity on the synthesis of immunoglobulins than human B-cells is of particular importance, which presumes their application as immunomodulator in clinical practices. Bochev, I., G. Elmadjian, D. Kyurkchiev, L. Tzvetanov, I. Altankova, P. Tivchev, S. Kyurkchiev. Mesenchymal stem cells from human bone marrow or adipose tissue differently modulate mitogen-stimulated B-cell immunoglobulin production in vitrо. Cell Biol Internatl., 32, 384 – 393. 2008; I.Bochev, S.Kyurkchiev. Immunomodulatory effects of human bone marrow or adipose tissue derived mesenchymal stem cells on T-helper cell cytokine production in vitro. Comptes Rendus de L'Academie Bulgare des Sciences 62 (12), pp. 1553-1558. 2009. 
        • During characterization of cells isolated from umbilical cord vein we found that according to their morphological and immunophenotype characteristic they belong to the family of mesenchymal stem cells (uc-MSC). These cells express survivin, bcl-2, vimentin and endoglin, which is proved at in vivo protein level with antibodies and at in vivo mRNA level through RT-PCR. The finding that after differentiation of these cells to endothelium-like cells there is no expression of survivin is of particular importance. This fact shows that MSCs are suitable for therapeutic use because after differentiation their proliferative potential becomes lower, i.e. there is no danger of uncontrolled growth of the transplanted cells. S.Kestendjieva, D.Kyurkchiev, G.Tsvetkova, T.Mehandjiev, A.Dimitrov, A.Nikolov, S.Kyurkchiev. Characterization of mesenchymal stem cells isolated from human umbilical cord. Cell Biol Internatl., 2008, 32,724 - 732


        Search for tumor stem cells specific markers

        • Keratinocytes from basal layer from healthy human epidermis have been isolated. In the obtained primary cultures of human keratinocytes more than 80% from the cells have been CD71dim and CD49bright, which suggests the presence of high number of stem cells with high proliferative potential. After a depletion of СD133+ cells by cell sorting, a loss of the ability of colony formation has been observed.
        • Testing of serum raised against membrane fraction of human keratinocytes have shown the appearance of two protein bands at 56.7 kDa and 49.5 kDa identified by Western blot. When comparing the reactivity of the obtained polyclonal serum against membrane and cytoplasmic fraction of human fibroblasts from cell line BJ and squamous cell carcinoma of the tongue SCC-9, the same bands have been obtained. About 30% from the keratinocytes and 10% from SCC-9 have shown specific membrane staining with granular pattern suggesting receptors grouping. This type of staining has been completely lacking in human fibroblasts from cell line BJ.


        IMMUNOREGULATION IN PREGNANCY

        • A specific monoclonal antibody Mat3A6 against progesterone induced blocking factor has been produced (PIBF). This antibody is with proven unique PIBF specificity that may be used in clinical diagnostics. By using Mat3A6, it has been demonstrated that PIBF is constitutively expressed in mesenchymal stem cells isolated from human decidua. The expression and secretion of this protein is modulated by progesterone. Ivanova-Todorova, E., D. S. Kyurkchiev, A. Nalbanski, T. Timeva, A. Shterev, S. D. Kyurkchiev. Production and characterization of a novel monoclonal antibody against progesterone induced blocking factor (PIBF). J. Reprod Immunol., 78, 94–101, 2008. Ivanova-Todorova, E., Kyurkchiev, D., Mourdjeva, M., Dimitrov, R., Stoyanova, E., Timeva, T., Yunakova, M., Shterev, A., Kyurkchiev, S. Pre-decidual multipotent stromal cells (PreDMSc) constitutively express progesterone induced blocking factor (PIBF). Comptes Rendus de L'Academie Bulgare des Sciences 2009.
        • It has been found that the progesterone induces the expression of HLA-G in human mesenchymal stem cells. The presented results prove that the progesterone induces significant changes in the physiology of human MSC by the induction of expression of factors with immunomodulatory functions. E.Ivanova-Todorova, M.Mourdjeva, D.Kyurkchiev, I.Bochev, E.Stoyanova, R.Dimitrov, T.Timeva, M.Yunakova, D.Bukarev, A.Shterev, P.Tivchev, S.Kyurkchiev. HLA-G Expression Is Up-Regulated by Progesterone in Mesenchymal Stem Cells. Am J Reprod Immunol; 2009. Mourdjeva, M., Ivanova-Todorova, E., Kyurkchiev, D., Bochev, I., Stoyanova, E., Dimitrov, R., Kyurkchiev, S. Progesterone enhances the HLA-G expression in mesenchymal stem cells. Comptes Rendus de L'Academie Bulgare des Sciences,62(12), pp.1559-1566. 2009.
        • It has been found that the female reproductive hormones – progesterone and estradiol accelerate the maturation and precipitate the apoptosis of mature human dendritic cells, i.e. they accelerate the life cycle of the dendritic cells. Both hormones lower the secretion of cytokines IL-10 and IL-27 and only progesterone increases the production of IL-13 but inhibits the secretion of IL-23. Ivanova E., D. Kyurkchiev, I. Altankova, J. Dimitrov, E. Binakova, S. Kyurkchiev. CD83+ monocyte-derived dendritic cells are present in human decidua and progesterone induces their differentiation in vitro. Amer. J. Reprod. Immunol., 53, 1-7, 2005. Kyurkchiev D., E. Ivanova-Todorova, S. Hayrabedyan, I. Altankova, S. Kyurkchiev. Female sex steroid hormones modify some regulatory properties of monocyte-derived dendritic cells. Amer. J. Reprod. Immunol., 58, 425–433, 2007. These results provide new mechanisms of the immunomodulatory effect of the reproductive steroid hormones during pregnancy in human. 
        • With the help of produced monoclonal antibody 1H6 against phosphatidylserine /PS/ the externalization of PS has been investigated through flow cytometry and immunofluorescence. A model of the dynamics of externalization of PS during apoptosis has been described. The addition of progesterone in physiological doses lowers the externalization of PS in dendritic cells, T-cells and of monocyte-macrophage line U937. Mourdjeva M., D. Kyurkchiev, A. Mandinova, I. Altankova, I. Kehayov, S. Kyurkchiev. Dynamics of membrane translocation of phosphatidylserine during apoptosis detected by a monoclonal antibody. Apoptosis, 10, 209-217, 2005. Mourdjeva M., Z. Popova, D. Kyurkchiev, K. Kontinopoulou, I. Altankova, I. Kehayov, S. Kyurkchiev. Progesterone-modulated Phosohatidylserine Externalization in Apoptosis and Activation of Jurkat Cells. AJRI, 56, 1-9, 2006. The results provide new prospects for investigation of the interaction between reproductive hormones and lymphoid cells, especially in relation to their specific conditions related to autoimmunity, as apoptosis and activation. 
        • Investigations aimed at establishment of cell mechanisms in the induction of autoimmune response against human beta-2 glycoprotein I /B2-GPI/ have been carried out. It has been found that human dendritic cells become differentiated in vitro and preferentially swallow through endocytosis the B2-GPI/cardiolipine complex but not the negative form of B2-GPI. Flow cytometry analysis proved that after endocytosis the dendritic cells represent epitopes which are recognized by specific monoclonal antibodies against human B2-GPI. The results show that the B2-GPI/CL complex is immunogenic and able to induce autoimmune response leading to synthesis of anti-B2-GPI antibodies. The studies show the role of cell-based immune reaction in the pathogenesis of anti-phospholipid syndrome which is one of the reasons for infertility in human. 
        • Epitope specificity of the serum protein beta 2-GPI has been characterized by the employment of monoclonal and polyclonal antibodies and autoimmune and hyperimmune sera. Obtained results have given reason to make the conclusions that the molecule of beta 2-GPI is able to elicit production of different populations of antibodies, only part of which associable with pathological effects. The fact has relevance to the clarification of the role of phospholipid antibodies and in particular has bearing to the role of beta 2-GPI–dependent antibodies during an unsuccessful pregnancy. Kyurkchiev, D., Alexandrova, K., Nalbanski, A., Robeva, R., Stamenova, M., Altankova, I., Kyurkchiev,S Analysis of epitopes of beta-2-glycoprotein recognized by autoantibodies. (2002) Clinical Application of Immunology,1 (1), pp.53-57.
        • Monoclonal antibodies against the molecule of the atrial natriuretic peptide /ANP/ have been obtained. Cell-specific localization of ANP in rat testis of late fetal to puberty age has been established. The peptide is localized in the Leydig, Certoli and germ cells. The cell localization of the peptide in rat ovary during puberty and after treatment with gonadotropic hormone has been followed. The expression of ANP in all steroid-producing cells in the developing oocytes has been proved through immunocytochemistry. A.Russinova, M.Mourdjeva, C. Valkova, S.Kyurkchiev, I.Kehayov. Immunocytochemical Localization Of Atrial Natriuretic Factor (ANF) In Rat Female Reproductive Tract: Evidences For A Potential Hormonal Regulation. Biology of the cell 94 (2002) 91-98 Mourdjeva M, Russinova A, Kyurkchiev S, Kehayov I. Spatial and temporal distribution of atrial natriuretic factor in the rat testis. Biol Cell 2001 Nov 93:5 301-7. Russinova A., M.Mourdjeva, S.Kyurkchiev, I.Kehayov. Immunohistochemical detection of atrial natriuretic factor (ANF) in different ovarian cell types. Endocrine Regulation, 2001, 35, 81-89. M.Bakalska, M.Mourdjeva, A.Russinova, S.Kyurkchiev, I.Kehayov. Localization of ANP in rat testis after leydig cell destruction: evidence for a potential role in regulating gonadal function. Endocrine regulations 1999, Vol. 33, 183-191.


        ENDOMETRIOSIS

        • Immunocytochemistry and immune-enzyme methods have been applied for establishment the expression of angiogenic factors and specific tumor markers in endometrial tissue. Developed in the department antibodies against IL-1 alpha, FGF-1 and ovary carcinoma has been used. Polyclonal antibodies against S100A13 and endoglin have been used for immunocytochemistry localization of angiogeic factors. Hayrabedyan S., S. Kyurkchiev, I. Kehayov. Endoglin (CD105) and S100A13 as markers of active angiogenesis in endometriosis. Reprod. Biol., 5, /1/, 51-67, 2005. Hayrabedyan S., S. Kyurkchiev, I. Kehayov. FGF-1 and S100A13 possibly contribute to angiogenesis in endometriosis. J. Reprod. Immunol., 67, /1-2/, 87-101, 2006. Hayrabedyan, S., S. Kyurkchiev, I. Kehayov. Calcium-binding protein S100A13 is overexpressed in endometriosis - C. r. Acad. bulg Sci, 2008, 61(2), 281-288.
        • Analysis of obtained data of the peritoneal fluid content in women with and without endometriosis have revealed quality and quantity differences between patients and healthy donors, which is indicative of the existence of molecular markers with the prospective use for non-invasive diagnostic of the disease. Dimitrov, R., Nalbansky, A., Mollova, M., Stamenova, M., Kyurkchiev, S Autoantibodies in sera from women with endometriosis against components of peritoneal fluid. (2004) Clinical Application of Immunology, 3(1), pp.306-309. Zasheva, D., Dimitrov, R., Stamenova, M. Endometriosis and the role of the integrins in the pathogenesis of the endometriosis. (2007) Akusherstvo i ginekologia, 46 (5), pp. 37-48.
        • Presence of autoantibodies in sera obtained from patients with endometriosis against components of their corresponding peritoneal fluid has been documented. Data have been obtained showing the unfavourable influence of peritoneal fluid received from infertile women on spermatozoa characteristics with significance for their fertilization potential. Dimitrov, R., Nalbansky, A., Mollova, M., Stamenova, M., Kyurkchiev, S Autoantibodies in sera from women with endometriosis against components of peritoneal fluid. (2004) Clinical Application of Immunology, 3(1), pp.306-309.
        • It has been found that peritoneal fluid received from women with endometriosis can exert negative effect of on the process of decidualization of pre-decidual stromal cells in vitro. This finding is in support of the possibility for the existence of mechanism affecting the receptive capacity of women with endometriosis by components of the peritoneal fluid and may be associated with the infertility etiology of these patients. 
        • It has been found that peritoneal fluid received from women with endometriosis can exert negative effect of on sperm cells’ parameters in correlation to the fertility potential of the sperm. 
        • It has been found that peritoneal fluid received from women with endometriosis can exert negative effect of on the process of decidualization of pre-decidual stromal cells in vitro. This finding is in support of the possibility for the existence of mechanism affecting the receptive capacity of women with endometriosis by components of the peritoneal fluid and may be associated with the infertility etiology of these patients.


        TUMORS

        • The activity of the construct survivin-siRNA on prostate carcinoma cells PC3 has been investigated. The relative protein levels have been obtained by immunoblot of three independent experiments. The gene expression of survivin-siRNA and the control siRNA have been analyzed by real time qPCR. Both experimental settings of real time qPCR and immunoblot have shown significant decrease of mRNA and protein expression after transfection with specific siRNA of PC-3 cells. M.Mourdjeva, A.Mandinova, S.Kyurkchiev, I.Kehayov. Experimental data for inhibition of the expression of surviving by small interfering RNA molecules (siRNAs). C.r.Acad Bulg Sci, 2009.
        • The expression of low molecular mass stress proteins (alpha В-crystalline and Hsp27) in reproductive tissue and different tumors has been studied by means of immune fluorescence, Western and immunoblot, Northern blot and real time qPCR. The expression of both proteins has been demonstrated in human placenta, rat testis and human ovary. Differences have been documented showing specific patterns for alpha В- crystalline in benign and malignant tumors – high degree of expression in benign and loss of expression in malignant tumors. Mineva, I., Gartner, W., Hauser, P., Kainz, A., Löffler, M., Wolf, G., Oberbauer, R., Weissel, M., Wagner, L. Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing (2005) Cell Stress and Chaperones, 10 (3), pp. 171-184; Mineva, I., Stamenova, M., Gartner, W., Wagner, L Expression of the small heat shock protein alphaB-crystallin in term human placenta. (2008) American Journal of Reproductive Immunology,60 (5), pp. 440-448.
        • It has been proven through examination of patients with prostate carcinoma, subjected to specific DNA vaccination, that resulting from this procedure the patients develop humoral immune response which is manifested by the production of antibodies against prostate-specific membrane antigen. These results support the idea of the DNA vaccination as a possible therapeutic approach. Todorova K., S. Zoubak, M. Mincheff, S. Kyurkchiev. Biochemical Nature and mapping of PSMA epitopes recognized by human antibodies induced after immunization with gene-based vaccines. Anticancer research, 25, 4727-4732, 2005; Todorova K., I. Ignatova, S. Tchakarov, I. Altankova, S. Zoubak, S. Kyurkchiev, M. Mincheff. Humoral immune response in prostate cancer patients after immunization with gene-based vaccines that encode for a protein that is proteasomally degraded. Cancer Immunity, 5, 1-9, 2005.
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