Velichka Pavlova

Developmental study of tripeptidyl peptidase I activity in the mouse central nervous system and peripheral organs

Tripeptidyl peptidase I (TPPI) — a lysosomal serine protease — is encoded by the CLN2 gene, mutations that cause late-infantile neuronal ceroid lipofuscinosis (LINCL) connected with profound neuronal loss, severe clinical symptoms and early death at puberty. Developmental studies of TPPI activity levels and distribution have been done in the human and rat central nervous systems (CNS) and visceral organs. Similar studies have not been performed in mouse. In this paper, we follow up on the developmental changes in the enzyme activity and localization pattern in the CNS and visceral organs of mouse over the main periods of life — embryonic, neonate, suckling, infantile, juvenile, adult and aged — using biochemical assays and enzyme histochemistry. In the studied peripheral organs (liver, kidney, spleen, pancreas and lung) TPPI is present at birth but further its pattern is not consistent in different organs over different life periods. TPPI activity starts to be expressed in the brain at the 10th embryonic day but in most neuronal types it appears at the early infantile period, increases during infancy, reaches high activity levels in the juvenile period and is highest in adult and aged animals. Thus, in mice TPPI activity becomes crucial for the neuronal functions later in development (juvenile period) than in humans and does not decrease with aging. These results are essential as a basis for comparison between normal and pathological TPPI patterns in mice. They can be valuable in view of the use of animal models for studying LINCL and other neurodegenerative disorders.

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.

Demyelination and neurodegenerative changes in serum IgG antibodies to GM1, GD1a and GM3 gangliosides in a patient on dialysis

During the last two decades, an enormous effort has been made to discover biological markers of neuronal damage to effective response to therapy. Gangliosides a family of acidic glycosphingolipids are highly represented in the nervous system. Their spectra show considerable changes in pathological conditions. The complex of anti-ganglioside antibodies may be useful diagnostic and prognostic tool of markers for demyelination (GM1), neurodegeneration (GD1a)and(GM3) correlates with the loss of integrity of the blood brain barrier (BBB). Additionally, patients with renal failure may manifest a variety of neurologic disorders.Dialysis itself is associated with at least three distinct disorders of the central nervous system, including the dialysis disequilibrium syndrome, dialysis dementia, and progressive intellectual dysfunction. Our case studyinvolves a patient on dialysis, a 72-years old woman, as an example of long-term toxicity. She was on dialysis for 1,5 years. The values of IgG anti-GM1, anti-GD1a and anti-GM3 antibodies titers in the blood serum were detected by ELISA. Significantly elevated serum IgG anti-GM1 antibodies titers were detected in our patient. The value of the titer of IgG antibodies against GM1 showed the presence of weak demyelination, increasing with dialysis treatment but not neurodegeneration. On the other hand, other biomarkers were indicative of the preserved integrity of the BBB since IgG titers of antiGD1a and anti-GM3 antibodies were in norm.The patient had many signs that show and prove symptoms of encephalopathy.Neurologic findings in the patients with uremic encephalopathy (UE) range from normal to a comatose state. Neurologic diseases which are specifically related to uremia and/or dialysis occur with increased frequency in patients with end-stage renal disease on chronic hemodialysis. These include subdural hematoma, electrolyte disorders, vitamin deficiencies, drug intoxication, hypertensive encephalopathy, and acute trace element intoxication. Our observations show that there is a relationship between kidney problems and central nervous system (CNS). Correspondence to: Vera Kolyovska, Assistant professor, Institute of Experimental Morphology, Pathology and Anthropology with Museum, Department of Experimental Morphology; Address: Bulgaria, Sofia 1113, Acad. G. Bonchev Str., Bl. 25, Tel: + 359 2 979 2397, E-mail: verakol@abv.bg

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.

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.