Humoral immune response after genetic immunization is consistently improved by electroporation.

Aiming to evaluate some parameters to influence the immune response to DNA vaccination, we compare three protocols of DNA immunization (i.m. injections, i.m. injections followed by electroporation, and the effect of i.p. injection of stably antigen-transfected cells before DNA administration), using three different antigens. Statistical analyses showed that electroporation after intramuscular injections provided an immune response comparable to that obtained by pre-treatment with antigen-transfected cells and similar to that obtained by protein immunization. The results allowed us selecting a protocol that worked well for all three antigens and reinforced the idea that high level of gene expression is essential to get good immunization.

Hydrodynamics- and ultrasound-based transfection of heart with naked plasmid DNA.

A novel, efficient transfection method, based on ultrasound and hydrodynamics, has been developed to transfect heart tissue with plasmid DNA. An ultrasound probe was aimed at the heart of anesthetized rats for 30 sec, at an intensity of 1 MHz and 2 W/cm2. The aorta was clamped and a phosphate-buffered saline (PBS) solution containing pSV-LacZ was quickly injected into the left ventricle. Each animal was maintained in this condition for 20 sec, and then the clamp was opened and the needle was removed. Electrocardiography, performed after 4 weeks, showed mild or no sign of ischemia in all groups. Visual evaluation of heart tissue samples from rats that received 100 microg of pSV-LacZ in 100 microl had only a few blue cells, indicating transfection, and those that received only PBS had no blue cells. However, all heart tissue samples from rats transfected with 100 to 500 microg of pSV-LacZ in 200 microl, or with 200 to 500 microg of pSV-LacZ in 100 micro had many blue cells. The base and epicardium of the heart tissue samples had many more blue cells than did the rest of the samples. Histological results, based on staining with hematoxylin and eosin, showed similar results between control and transfected groups. Therefore, we concluded that gene delivery by plasmid vector in association with ultrasound and hydrodynamics was highly effective in transfecting rat heart.

Expression of the selectable marker gene bsrm in BALB/MK cells induces apoptosis by overproduction of hydrogen peroxide.

Transduction of the retroviral vector LBmSN, which expresses the blasticidin S resistance gene bsrm in the murine keratinocyte cell line BALB/MK, induces death in these cells. Cell death is caused by a factor called DOKEB (death factor obtained from keratinocytes expressing bsrm), which is released before the cells' death. In this report we describe and discuss the purification and characterization of DOKEB. Our results were as follows. (i) The 5-day-old medium from the modified BALB/MK cells with LBmSN was used for purification and characterization by filtration and chromatography: DOKEB was a stable and highly hydrophilic compound, with a molecular mass less than that of 1 amino acid. (ii) The conditioned medium containing DOKEB was reactive against thiobarbituric acid and dichlorofluorescein diacetate. (iii) DOKEB activity was neutralized by the incubation of the conditioned medium with catalase. Therefore, our conclusion is that the BALB/MK cells expressing bsrm produce a large amount of hydrogen peroxide, which catalyzes the process of apoptosis of those cells.

Carcinoembryonic antigen (CEA) mimicry by an anti-idiotypic scFv isolated from anti-Id 6.C4 hybridoma.

Since carcinoembryonic antigen (CEA) is expressed during embryonic life, it is not immunogenic in humans. The use of anti-idiotypic (Id) antibodies as a surrogate of antigen in the immunization has been considered a promising strategy for breaking tolerance to some tumor associated antigens. We have described an anti-Id monoclonal antibody (MAb), designated 6.C4, which is able to mimic CEA functionally. The anti-Id MAb 6.C4 was shown to elicit antibodies that recognized CEA in vitro and in vivo. In the present study, we sought to verify whether a single chain (scFv) antibody obtained, the scFv 6.C4, would retain the ability to mimic CEA. Two scFv containing the variable heavy and light chain domains of 6.C4 were constructed with a 15-amino acid linker: one with and another without signal peptide. DNA immunization of mice with both forms of scFv individually elicited antibodies able to recognize CEA.

Over expression of the selectable marker blasticidin S deaminase gene is toxic to human keratinocytes and murine BALB/MK cells.

BACKGROUND: The blasticidin S resistance gene (bsr) is a selectable marker used for gene transfer experiments. The bsr gene encodes for blasticidin S (BS) deaminase, which has a specific activity upon BS. Therefore, its expression is supposed to be harmless in cells. The work reported on herein consisted of experiments to verify a possible toxicity of bsr on mammalian cells, which include several cell lines and primary cultures. RESULTS: Murine keratinocyte BALB/MK and human primary keratinocyte cells transduced with the retroviral vector LBmSN, which has an improved expression system of bsr, namely bsrm, died in five days after the transduction. Meanwhile the control vector LBSN, which expresses bsr, did not provoke cell death. The lethal activity of bsrm was observed only in human keratinocytes and BALB/MK cells among the cell types tested here. Death appears to be mediated by a factor, which is secreted by the BALB/MK transduced cells. CONCLUSION: By our study we demonstrated that the expression of bsrm gene is toxic to human keratinocytes and BALB/MK cells. It is likely over expression of BS deaminase gene is responsible for the death.