Elena Nikolova

Poly(3-hydroxybutyrate)/caffeic acid electrospun fibrous materials coated with polyelectrolyte complex and their antibacterial activity and in vitro antitumor effect against HeLa cells.

Abstract The purpose of this work was to investigate the possibility for the preparation of new poly(3-hydroxybutyrate) (PHB)/poly(ethylene glycol) (PEG)-based fibrous materials containing natural phenolic compound caffeic acid (CA) of diverse architectures, as well as to study the impact of the fiber composition on the in vitro CA release profile and on the biological properties of the fibrous materials. The application of the one-pot electrospinning enabled the fabrication of nanofibrous materials from PHB and PEG loaded with the CA. Materials with targeted design were obtained by coating with polyelectrolyte complex of alginate (Alg) and N,N,N-trimethylchitosan (TMCh). Three different processing paths were used to obtain coated mats: (i) with CA incorporated in the PHB/PEG core; (ii) with CA embedded in the Alg layer; and (iii) with CA included in the TMCh layer. The in vitro release of CA was modulated by controlling the composition and the architecture of the nanofibrous mats. The performed microbiological screening and MTT cell viability studies revealed that in contrast to the bare mats, the CA-containing nanofibrous materials were effective in suppressing the growth of the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli and displayed good cytotoxicity against human cervical HeLa tumor cells. In addition, the proliferation of murine spleen lymphocytes and peritoneal macrophages was increased by the prepared CA-containing nanofibrous materials. The obtained materials are promising for antibacterial wound dressing applications as well as for application in local treatment of cervical tumors.

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.

Development of Methods for Safe Application of Viral Vectors for GeneEngineering Manipulations

Additional oncogene copy was transferred in normal cells, and additional tumor-suppression gene copy-in malignant cells, respectively, by transfection with the appropriate recombinant DNA-constructs, containing each one the respective gene. The results obtained were compared with the data from insertion of copy from the respective gene in prokaryotic bacteria strains E. coli, by analogical transfection with appropriate gene constructs. In application of low initial infections (high initial dilutions of viral suspensions, respectively) of eventually attenuated by many passages vaccine strains from the heterologous for mammals and mammalian cells avian viral species, only slight ultra-structural changes and signs of partially-formed immature viral particles in the cells, were established, but no cytopathic effect (CPE was observed). In freezing of the so inoculated mammalian cells after addition of cryoprotector Dimethylsulfoxide (DMSO), thawing and re-incubation in fresh cultivation medium, signs of activated cell proliferation were noted. One of the possible explanations could be eventual transfer of nucleotide sequences from viral particles to separate cells because of activated fusion processes on the influence of DMSO in drastic temperature changes. The results obtained proposed a possibility about application of the described methods as available alternatives for both vaccine production and gene-engineering manipulations with cells, compared with the procedures of preparation and application of designed quite expensive recombinant DNA-constructs. Future studies are necessary in this direction.

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.

Distribution patterns of carbohydrates in murine glycocalyx

Enterocytes are unique cells governing an array of processes. They are covered by the gut glycocalyx, which is an extraneous carbohydrate-rich coat and an integral part of the plasma membrane. The intestinal glycocalyx and secreted mucins constitute a glycosylated milieu which has a number of physiological and protective functions. One of the important functions of the glycocalyx is its barrier function against microbial adherence to different membrane glycolipids. Thus, the glycocalyx is an important part of the mucosal immune system in newborns. The aim of our study was to identify the carbohydrates in the small bowel glycocalyx of Balb/c mice at different ages. We used plant lectins with different sugar specificities. Fluorescein-labelled lectins binding different carbohydrate moieties were detected using confocal laser scanning microscopy. Biotinilated lectins were used for transmission electron microscopy observations of the constituents of the gut glycocalyx at different periods of postnatal development in mice. Different carbohydrate moieties that were identified in the murine intestinal glycocalyx followed different distribution patterns and characteristics. Carbohydrates present on the mucus surface depended on tissue localization, cell type and stage of development. The distribution and mucins glycosylation could be of interest in analysing the response of the mucosal barrier to intestinal pathogens causing infection or inflammation.

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.

Achievement of balanced oncogenes and tumor- suppressor genes activity in normal and malignant cells in vitro and in vivo

The main goal is connected with providing, on the one hand, of active tumor-suppressor genes for prevention of eventual malignant transformations, and, on the other hand, of functionally active oncogenes for prevention of early aging and death, bothxa0in vitroxa0andxa0in vivo. Modulation of an adequate immune control was also necessary, and in this way any eventual unwished side effects from the genetic manipulations applied, could be escaped. Gene transfer in laboratory-cultivated mouse embryonic stem cells (mESCs) was made by use of appropriate recombinant DNA-constructs, which contained the promoter for gene, coding Elongation Factor 1-alpha (EF1-α), isolated from adeno-associated virus (AAV) (Parvoviridae); gene Dcn1, isolated from 3T3 fibroblasts of laboratory mice Balb/c, as well as gene for neomycin resistance, isolated from bacterial DNA-plasmid. Besides those indicated in the scientific literature inactivation of oncogene Dcn1 in the process of normal cell differentiation,xa0its presence in the genome was supported and confirmed by our results from electrophorhesis of genomic DNA from normal mature epithelial cells of adult Balb/c laboratory mice. Furthermore, electrophorhetic profiles of genetic material from wild type (WT) on oncogene Dcn1 and “knock-down” (KD) on it inbred lines experimental mice differed not only on this oncogene, but also on the tumor-suppressor gene HACE1 in both categories of laboratory rodents.xa0Similarly transfected Hela and RIN-5F malignant cells were thenxa0in vitro-co-cultivated with myeloid cell precursors, derived from populations of non-transfected laboratory-cultivated mESCs, in the presence of Doxyciclin, known from many literature data as activator of tumor-suppressor genes from STAT-family expression. Our results were also confirmed by the noticed differences in the degree of myeloid differentiation of derived precursor cells in theirxa0in vitro-co-cultivation with containing additional copies of tumor-suppressor genes malignant cells from both lines described, in comparison with the data, obtained in their laboratory co-cultivation with non-treated human cervical carcinoma Hela cells. Differences were also observed inxa0in vitro-co-cultivation with the derived by us normal mESCs, containing additional copy of oncogene Dcn1 by the described above transfection with recombinant DNA-constructs. On the other hand, the derived normal cells with inserted additional copy of oncogene Dcn1 have indicated good safety and immunogenity. These cells have also indicated preserved normal cell characteristics, as well as eventual over-expression of the experimentally-activated oncogene Dcn1 in them. n n Key words:xa0Oncogenes, tumor-suppressor genes, myeloid cell precursors, recombinant gene constructs, cell transfection.

EXPERIMENTAL MODEL FOR SAFE GENE TRANSFER BY RECOMBINANT GENE CONSTRUCTS

ABSTRACT For gene transfer in laboratory-cultivated mouse embryonic stem cells (mESCs), previously designed recombinant gene constructs with respective genes inserted in them are necessary. For this aim, recombinant DNA-genomes from adeno-associated virus (AAV) (Parvoviridae), containing promoter of gene, coding Elongation Factor 1-alpha (EF1-α); isolated from 3T3 fibroblasts of adult laboratory mice Balb/c inserted oncogene Dcn1, (in its role of regulator on the tumor-suppressor gene p53 by specific pathways of indirect inhibition), as well as gene for neomycin resistance, isolated from bacterial DNA-plasmid, are used for gene transfection by electroporation. On the other hand, eventual subsequent super-activation of tumor-suppressor genes both in vitro and in vivo is also necessary.

WHEY PROTEIN CONCENTRATE AS A PROTECTIVE AGENT AGAINST DOXORUBICIN-INDUCED CARDIOTOXICITY IN MICE

ABSTRACT Whey proteins are considered as functional food ingredients which have high cysteine content and promote the biosynthesis of glutathione—the primary intracellular antioxidant. Doxorubicin (DOX) is one of the most active antitumor antibiotics in current use. The therapeutic value of DOX, however, is limited by its toxicity. Oxidative stress is one of the underlying mechanisms of DOX toxicity in noncancerous (nontargeted) tissues. We investigated the protective effect of whey protein concentrate against DOX toxicity and oxidative stress. The administration of DOX (20 mg/kg i.p.) to BALB/c mice caused significant decrease of tissue glutathione level in the heart and severe histopathological changes, examined by light and transmission electron microscopy. These biochemical and histological alterations were effectively attenuated on pretreatment with whey protein concentrate. We therefore concluded that the protective action of whey is due to the enhancement of tissue glutathione level which might have important cytoprotective effects on oxidative stress, induced by DOX treatment.