Iskra Sainova

Experimental model for safe in vitro- and in vivo- influence of internal and external factors of cell differentiation

Gene transfer in laboratory-cultivated mouse embryonic stem cells (mESCs) was made by appropriate recombinant DNA-constructs. Electrophoretic profiles of genetic material from wild type on oncogene Dcn1 and “knock-down” on it inbred experimental mice differed not only in it, but also in tumorsuppressor gene HACE1 between both categories of laboratory rodents. The results obtained were compared with previous data, received from malignant rat insulinoma RIN-5F cells, transfected by recombinant gene constructs with inserted copy of “secretagogin” gene, by their in vitro-co-cultivation with malignant cell precursors, derived from populations of non-transfected laboratory-cultivated mESCs in the presence of Doxyciclin, probably by activation of tumor-suppressor genes of STATfamily. Furthermore, the so induced “secretagogin” over-expression could exert protective function on the transfected Rin-5F cells, which was confirmed by noticed differences in the degree of myeloid differentiation of derived precursor cells in their in vitro-co-cultivation with containing additional copy of “secretagogin” gene Rin-5F malignant rat insulinoma cells, in comparison with the results, obtained in their co-cultivation with human cervical carcinoma Hela cells in our laboratory. On the other hand, the derived normal cells with inserted additional copy of oncogene indicated good safety and immunogenity.

Presence and Levels of Anti-GM3 Antibodies and GM3 Ganglioside Moleculesin Probes from Experimental Cellular in Vitro-Models

Background: The current study was directed to determine the values of both GM3 ganglioside and specific anti-GM3 antibodies in in vitro-incubated normal cells, malignant cell and mixed cultures of co-cultivated cells from both types as well. So a better understanding of the molecular mechanisms, underlining differences between various cellular types and changes during the process of cell differentiation was necessary. Methods: Total lysates from cultivated normal cells from mouse embryos, mouse malignant myeloma line and mixed of both were prepared and passed through GSHAgarose Columns. Molecules with affinity to the reduced form of Glutathione (GSH) were separated. The levels of ganglioside GM3 and of specific antibodies to it were assessed by enzyme-linked immuno-sorbent assay (ELISA) technique. Results: In the normal cells’ lysate, supplemented with molecules, possessing affinity to GSH the lowest values were assessed. These differences could be due to contention of many non-possessing such affinity molecules in equal volume of biological material, as well as with the presence of various bonded forms of GM3. Conclusions: The data obtained confirm literature data about the increased levels of GM3 as a marker for malignancy. We propose derivation of lymphoid-like cells from the embryonic progenitors. Another hypothesis could be the production of immunoglobulins (IgG antibodies) from non-lymphoid cells in appropriate conditions.

A Pilot Study of Methods about Detection of Inter-Molecular Interactions As Individual Units of Intra-Cellular Structures, Intra- And Extra-Cellular Cascade Regulatory Pathways

Different methods for investigation on the mechanisms of participation of cell proteins in direct and/or indirect intraand extra-cellular inter-molecular (protein-lipid, protein-protein, protein-RNA, proteinDNA interactions, etc.), by cascade regulatory pathways were developed and tested. These bio-molecules (in particular microtubule proteins and cyclins) have been found as parts from different complex structures from the nuclear, cytoskeleton and membrane cell fractions, as mitotic spindle, endocytosis vesicles, cell organelles, membrane structures, which are important for basic cell functions. The techniques, developed in the current study, could be useful for identification of the proteins/peptides, participating in the composition of these structures, but also of the interactions between them, on molecular and intra-molecular levels. For confirmation of the results obtained and the reliability of the applied methods, routine techniques as scanning electron microscopy (SEM) and transmission electron microscopy (TEM) assays were used. In all cases, the main goal is directed to search of molecules, participating in the control of cell growth and proliferation, as well as of such, which determine the further cell fate during cell differentiation and maturation.

Differentiation of stem and progenitor cells from bone marrow in activated dendritic cells and lymphocytes with anti-malignant properties

Dendritic cells (DCs) have been characterized as powerful antigen-presenting cells (APCs), which possess the abilities for immune modulation and are used in composition of anti-malignant vaccines and gene-engineered products. By appropriate cultivation, modifications of DCs have shown abilities for an enhanced expression of specific effector molecules. Studies on their biology are focused on their role as main immune response modulators. These properties characterize them as promising candidates for construction of novel safe vaccines and gene-engineered products. In this direction, attention is directed to development of methods and techniques for transduction of in vitroand/or ex vivo-cultivated DCs with previously designed recombinant viral vectors with inserted genes, coding respective malignant antigens. Studies on the biology of lymphocytes are mainly focused on their role in cellular and humoral immune response. Their cultivation and differentiation in the presence of appropriate antigens, on one hand, and by appropriate modifications, on the other hand, have shown the abilities for an enhanced expression of specific effective molecules. These properties have characterized them as promising candidates for construction of novel safe vaccines and geneengineering products.

Differentiation of stem and progenitor cells in activated immune cells with anti-malignant properties

Studies on the biology of immune cells are mainly focused on their role as immune activators and modulators. In their appropriate cultivation and/or modifications, they have shown abilities for an enhanced expression of specific effective molecules. These properties have characterized them as promising candidates for construction of novel safe vaccines and gene-engineering products on their basis. In this aspect, in the last years, attention is directed towards the development of new safe therapeutic methods and techniques with immune cells. In this connection, according to results obtained, in vitro -co-cultivation derived by myeloid and lymphoid precursors with positive and negative on the additionally-inserted copy of the oncogene Dcn1 transfected cells, no immune reaction is observed against both genetically-manipulated cell types, which could be accepted as a proof for their safety. On the other hand, signs of increased degree for both myeloid and lymphoid differentiation in the presence of transfected cells, positive on additionally-inserted copy of the oncogen are indicated.