Jane Zveiter de Moraes

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Synergistic effect of vascular endothelial growth factor and granulocyte colony‐stimulating factor double gene therapy in mouse limb ischemia

Vascular endothelial growth factor (VEGF) has mostly been tested to treat ischemic diseases, although the outcomes obtained are not satisfactory. Our hypothesis is that the local transient expression of VEGF and stem cell mobilizer granulocyte colony‐stimulating factor (G‐CSF) genes in ischemic limbs can complement their activities and be more efficient for limb recovery.

Granulocyte-macrophage colony-stimulating factor gene based therapy for acute limb ischemia in a mouse model.

Granulocyte‐colony‐stimulating factor (GM‐CSF) is a pleiotropic factor for hematopoiesis that stimulates myeloblasts, monoblasts and mobilization of bone marrow stem cells. Therefore, the GM‐CSF gene is a potential candidate for vessel formation and tissue remodeling in the treatment of ischemic diseases.

The idiotype (Id) cascade in mice elicited the production of anti-R24 Id and anti-anti-Id monoclonal antibodies with antitumor and protective activity against human melanoma

Gangliosides have been considered as potential targets for immunotherapy because they are overexpressed on the surface of melanoma cells. However, immunization with purified gangliosides results in a very poor immune response, usually mediated by IgM antibodies. To overcome this limitation, we immunized mice with R24, a monoclonal antibody (mAb) that recognizes the most tumor‐restricted ganglioside (GD3); our goal was to obtain anti‐idiotype (Id) antibodies bearing the internal image of GD3. Animals produced anti‐Id and anti‐anti‐Id antibodies. Both anti‐Id and anti‐anti‐Id antibodies were able to inhibit mAb R24 binding to GD3. In addition, the anti‐anti‐Id antibodies were shown to recognize GD3 directly. Anti‐Id and anti‐anti‐Id mAb were then selected from two fusion experiments for evaluation. The most interesting finding emerged from the characterization of the anti‐anti‐Id mAb 5.G8. It was shown to recognize two different GD3‐expressing human melanoma cell lines in vitro and to mediate tumor cell cytotoxicity by complement activation and antibody‐dependent cellular cytotoxicity. The biological activity of the anti‐anti‐Id mAb was also tested in a mouse tumor model, in which it was shown to be a powerful growth inhibitor of melanoma cells. Thus, activity of the anti‐anti‐Id mAb 5.G8 matched that of the prototypic anti‐GD3 mAb R24 both in vitro and in vivo. Altogether, our results indicate that the idiotype approach might produce high affinity, specific and very efficient antitumor immune responses. (Cancer Sci 2011; 102: 64–70)

Blocking FGF2 with a new specific monoclonal antibody impairs angiogenesis and experimental metastatic melanoma, suggesting a potential role in adjuvant settings.

Compelling evidence suggests that fibroblast growth factor 2 (FGF2), overexpressed in melanomas, plays an important role in tumor growth, angiogenesis and metastasis. In this study, we evaluated the therapeutic use of a new anti-FGF2 monoclonal antibody (mAb), 3F12E7, using for that the B16-F10 melanoma model. The FGF2 neutralizing effect of this antibody was certified by in vitro assays, which allowed the further track of its possible in vivo application. 3F12E7 mAb could be retained in B16-F10 tumors, as shown by antibody low-pH elution and nuclear medicine studies, and also led to reduction in number and size of metastatic foci in lungs, when treatment starts one day after intravenous injection of B16-F10 cells. Such data were accompanied by decreased CD34(+) tumor vascular density and impaired subcutaneous tumor outgrowth. Treatments starting one week after melanoma cell intravenous injection did not reduce tumor burden, remaining the therapeutic effectiveness restricted to early-adopted regimens. Altogether, the presented anti-FGF2 3F12E7 mAb stands as a promising agent to treat metastatic melanoma tumors in adjuvant settings.

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.

Skin sensitizer identification by IL-8 secretion and CD86 expression on THP-1 cells.

Substantial progress has been made in the development of alternative methods for skin sensitization in the last decade in several countries around the world. Brazil is experiencing an increasing concern about using animals for product development, since the publication of the Law 9605/1998, which prohibits the use of animals when an alternative method is available. In this way, an in vitro test to evaluate allergenic potential is a pressing need.This preliminary study started setting the use of myelomonocytic THP-1 cell line, according to the human cell line activation test (h-CLAT), already under validation process. We found that 48-h chemical exposure was necessary to identify 22 out of 23 sensitizers by the analyses of CD86 expression. In addition, the CD54 expression analyses presented a poor efficiency to discriminate sensitizers from non-sensitizers in our conditions. In view of these results, we looked for changes of pro-inflammatory interleukin profile. The IL-8 secretion analyses after 24-h chemical incubation seemed to be an alternative for CD54 expression assessing.Altogether, our findings showed that the combination of the analyses of CD86 expression and IL-8 secretion allowed predicting allergenicity.

Anti-bevacizumab idiotype antibody vaccination is effective in inducing vascular endothelial growth factor-binding response, impairing tumor outgrowth

Tumors require blood supply and, to overcome this restriction, induce angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in this process, which explains the great number of antiangiogenic therapies targeting VEGF. The research and development of targeted therapy has led to the approval of bevacizumab, a humanized anti‐VEGF monoclonal antibody (mAb), in clinical settings. However, side effects have been reported, usually as a consequence of bolus‐dose administration of the antibody. This limitation could be circumvented through the use of anti‐idiotype (Id) antibodies. In the present study, we evaluated the efficacy of an active VEGF‐binding immune response generated by an anti‐bevacizumab idiotype mAb, 10.D7. The 10.D7 anti‐Id mAb vaccination led to detectable levels of VEGF‐binding anti‐anti‐Id antibodies. In order to examine whether this humoral immune response could have implications for tumor development, 10.D7‐immunized mice were challenged with B16‐F10 tumor cells. Mice immunized with 10.D7 anti‐Id mAb revealed reduced tumor growth when compared to control groups. Histological analyses of tumor sections from 10.D7‐immunized mice showed increased necrotic areas, decreased CD31‐positive vascular density and reduced CD68‐positive cell infiltration. Our results encourage further therapeutic studies, particularly if one considers that the anti‐Id therapeutic vaccination maintains stable levels of VEGF‐binding antibodies, which might be useful in the control of tumor relapse.