Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Histochemical demonstration of oxidoreductase activities in the fat body and symbionts of Blattella germanica (Blattodea) following chlortetracycline-treatment

The metabolic apport of prokaryotic symbionts in the fat body of Blattella germanica was investigated by histoenzymatic methods, using chlortetracycline-treated and normal strains. In the experimental insects, bacteriocytes showed a decreased oxidoreductase activity, whereas the staining intensity of the other cell types was generally unchanged. Electron microscopic observations showed that some bacteria were still present in the bacteriocytes of the treated insects, but exhibited degeneration patterns to a different extent; therefore, they are not likely to carry on any enzymatic activity. Hence, chlortetracycline, an antibiotic that blocks the transovaric transmission of the symbionts, is active also on the endocellular symbionts of the fat body.     

ANALYSIS OF SPATIAL DISTRIBUTIONS OF CELLULAR MOLECULES DURING MECHANICAL STRESSING OF CELL SURFACE RECEPTORS USING CONFOCAL MICROSCOPY

Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.     

Tissue engineering by cell transplantation using degradable polymer substrates.

This paper reviews our research in developing novel matrices for cell transplantation using bioresorbable polymers. We focus on applications to liver and cartilage as paradigms for regeneration of metabolic and structural tissue, but review the approach in the context of cell transplantation as a whole. Important engineering issues in the design of successful devices are the surface chemistry and surface microstructure, which influence the ability of the cells to attach, grow, and function normally; the porosity and macroscopic dimensions, which affect the transport of nutrients to the implanted cells; the shape, which may be necessary for proper function in tissues like cartilage; and the choice of implantation site, which may be dictated by the total mass of the implant and which may influence the dimensions of the device by the available vascularity. Studies show that both liver and cartilage cells can be transplanted in small animals using this approach.