Use of fluorescence microspectral method for studying bone marrow EGFP+ cell transplantation in 5-fluorouracil-treated mice

In this paper the experimental results of bone marrow transplantation from C57BL/6-Tg(ACTB-EGFP)1Osb/J transgenic mice into C57BL/6 mice subjected to 5-fluorouracil treatment are represented. It has been shown that EGFP⁺ cell engraftment in bone marrow, spleen and thymus of host mice after 5-Fu treatment significantly increased. More long-term engraftment was recorded after transplantation between closely related donors and 5-fluorouracil treatment hosts. We have also obtained data on differences in the dynamics of EGFP⁺ cell engraftment in host investigated organs. To assess the effect of the donor’s bone marrow cells on the host immune system, functional activity of the synthetic apparatus (synthetic activity) of cells in bone marrow, spleen, thymus and blood have been investigated with fluorescence microspectral method. The results obtained allow of improving techniques for bone marrow transplantation without host irradiation in order to minimize the adverse effects.     

Regeneration of photoreceptor organs in freshwater planarians at different levels of accumulation of natural methylmercury compounds

The effects of natural methylmercury compounds on regeneration of photoreceptor organs were studied in three freshwater planarians: Polycelis tenuis, Dugesia lugubris, and D. tigrina. Accumulation of methyl mercury in the planarian body suppressed regeneration of P. tenuis with numerous photoreceptor organs to a greater extent than in two other planarians that have only two eyes. High methyl mercury concentrations inhibited the restoration of photoreceptor organs in asexual and sexual D. tigrina races.     

Regeneration of photoreceptor organs in freshwater planarians at different levels of accumulation of natural methylmercury compounds

The effects of natural methylmercury compounds on regeneration of photoreceptor organs were studied in three freshwater planarians: Polycelis tenuis, Dugesia lugubris, and D. tigrina. Accumulation of methyl mercury in the planarian body suppressed regeneration of P. tenuis with numerous photoreceptor organs to a greater extent than in two other planarians that have only two eyes. High methyl mercury concentrations inhibited the restoration of photoreceptor organs in asexual and sexual D. tigrina races     

The Effect of Cryopreservation of Bone Marrow Cells from Donor Mice that Carry the <Emphasis Type="Italic">egfp</Emphasis> Gene,on the Lifespan of Mice after Syngeneic Transplantation

The effect of cryopreservation of bone marrow cells on the lifespan of mice after syngeneic transplantation has been studied in nonirradiated mice and 7 Gy-irradiated mice. Mice with the enhanced green fluorescent protein gene were the donors. Bone marrow cells were cryopreserved according to the method used in clinical practice in the field of bone marrow autotransplantation in the treatment for patients with cancer. Dimethyl sulfoxide dissolved in polyglucin at the final concentration of 5% acted as a cryoprotectant agent. Transplantation of the thawed stem cells was carried out without washing out the cryoprotectant. No side effects associated with the cryoprotectant toxicity were observed. It has been shown that staining of bonemarrow cells with trypan blue is a more selective technique to evaluate the extent of cell damage after cryopreservation. The mean lifespan of nonirradiated recipient mice was not statistically different from that of the intact control group. In irradiated recipient mice, the mean lifespan increased by 51 ± 2% compared to the group of irradiated controls. The analysis of a blood sample taken from the tail vein of irradiated mice revealed lifelong engraftment of donor-derived cells in the hematopoietic system of the recipient mice. Thus, model experiments on the syngeneic strain of mice showed that cryopreserved bone-marrow cells can be effectively used for cell therapy in autotransplantation in patients after X-ray radiation therapy.     

A Study of the Regenerative Potential of Bone Marrow Cells of Donor Mice that Carry the <Emphasis Type="Italic">egfp</Emphasis> Gene in Irradiated Mice

A study of the regenerative potential of bone marrow cells of donor mice that express the enhanced green fluorescent protein was conducted in mice irradiated at a dose of 7 Gy. Expression of this protein allowed us to carry out monitoring of the presence of donor cells in recipient blood over the entire lifespan of the recipient. The lifespan of young recipients increased by 93% after transplantation; for old recipients it increased by 15%. Total acceptance of the bone marrow, spleen, thymus, and blood of the recipient with donor bone marrow cells was demonstrated over the entire life of the recipient. Only the donor colonies were detected with the studied irradiation dose and number of transplanted cells (11.7 ± 0.4) · 10⁶ on the spleen surface. The percentage of bone marrow and spleen cells that expressed the CD117 and CD34 stem cell markers in the recipient mice was above the control level for a long period of time after the irradiation. More than half of the cells with CD117, CD34, CD90.2, and CD45R/B220 phenotypes in the studied organs were donor cells. Further detailed study of the peculiarities of the engraftment of bone marrow cells, both without preliminary treatment of recipients and after the effects of extreme factors, will allow improvement of the methods of cell therapy.     

Larvae of related Diptera species from thermally contrasting habitats exhibit continuous up-regulation of heat shock proteins and high thermotolerance

A population of Stratiomys japonica, a species belonging to the family Stratiomyidae (Diptera), common name ‘soldier flies’, occurs in a hot volcanic spring, which is apparently among the most inhospitable environments for animals because of chemical and thermal conditions. Larvae of this species, which naturally often experience temperatures more than 40 °C, have constitutively high concentrations of the normally inducible heat-shock protein Hsp70, but very low level of corresponding mRNA. Larvae of three other species of the same family, Stratiomys singularior, Nemotelus bipunctatus and Oxycera pardalina, are confined to different type semi-aquatic habitats with contrasting thermal regime. However, all of them shared the same pattern of Hsp70 expression. Interestingly, heat-shock treatment of S. japonica larvae activates heat-shock factor and significantly induces Hsp70 synthesis, whereas larvae of O. pardalina, a species from constant cold environment, produce significantly less Hsp70 in response to heat shock. Adults of the four species also exhibit lower, but detectable levels of Hsp70 without heat shock. Larvae of all species studied have very high tolerance to temperature stress in comparison with other Diptera species investigated, probably representing an inherent adaptive feature of all Stratiomyidae enabling successful colonization of highly variable and extreme habitats.     

Action of nonviral gene delivery vectors on human complement system: low anticomplementary activity of lipoplexes based on lacZ plasmid and phospholipid/oligocation liposomes

A simple test-system has been developed for the first time in order to detect the ability of effectors (lipoplexes) to activate the complement system in an antibody-independent manner to serve as acceptors of nascent C4b and to inhibit formation of the key enzyme of complement, C3-convertase. The effect of plasmid DNA (pCMV-SPORT-LacZ), negatively charged cardiolipin (CL), neutral phosphatidylcholine (PC) vesicles and their lipoplexes, on the complement system was studied using the method developed. It was revealed that PC vesicles did not affect the complement system, while CL vesicles manifested low activation. The influence of plasmid DNA and its lipoplex based on PC liposomes as well on the complement system was very low. PC/LacZ lipoplex (143 microg/ml) acted on the complement system like 5.36 microg/ml heat aggregated IgG (agg) (the level of no pathological ruptures), whereas CL/LacZ lipoplex (143 microg/ml) acted similar to 10.7 microg/ml IgG (agg). Thus, weak activation of the complement system with CL lipoplex, and even weaker for the PC lipoplex testified to the use of neutral and positively charged lipoplexes preferably in gene therapy protocols. The technique can also be used for testing the influence of injectable gene therapy vectors on the complement system.