Protein changes in macrophages induced by plasma from rats exposed to 35 GHz millimeter waves.

A macrophage assay and proteomic screening were used to investigate the biological activity of soluble factors in the plasma of millimeter wave-exposed rats. NR8383 rat macrophages were incubated for 24 h with 10% plasma from male Sprague-Dawley rats that had been exposed to sham conditions, or exposed to 42 °C environmental heat or 35 GHz millimeter waves at 75 mW/cm² until core temperature reached 41.0 °C. Two-dimensional polyacrylamide gel electrophoresis, image analysis, and Western blotting were used to analyze approximately 600 protein spots in the cell lysates for changes in protein abundance and levels of 3-nitrotyrosine, a marker of macrophage stimulation. Proteins of interest were identified using peptide mass fingerprinting. Compared to plasma from sham-exposed rats, plasma from environmental heat- or millimeter wave-exposed rats increased the expression of 11 proteins, and levels of 3-nitrotyrosine in seven proteins, in the NR8383 cells. These altered proteins are associated with inflammation, oxidative stress, and energy metabolism. Findings of this study indicate both environmental heat and 35 GHz millimeter wave exposure elicit the release of macrophage-activating mediators into the plasma of rats.

Prevention of metastases with a Mage-b DNA vaccine in a mouse breast tumor model: potential for breast cancer therapy.

Anti-tumor vaccines are a relatively non-toxic alternative to conventional chemotherapeutic strategies to control breast cancer. Immunization with tumor-associated antigens (TAAs) triggers anti-tumor cytotoxic T lymphocytes (CTL), which can limit tumor progression. Here we report on the development and effectiveness of a TAA-based DNA vaccine encoding Mage-b1/2, the mouse homologue of the human MAGE-B1/2. As model system, we used immune competent Balb/c mice with syngeneic non-metastatic (64pT) or metastatic (4TO7cg) breast tumors. First, the presence of Mage-btranscripts in the 64pT and 4TO7cg breast tumors and metastases was demonstrated by RT-PCR, Southern blotting, and DNA sequencing. A DNA-based vaccine was developed from transcripts of one of the 64pT tumors, encoding the complete Mage-b1/2 protein, and subsequently tested for its preventive efficacy in both breast tumor models. Mice were immunized two times intramuscularly with the vaccine (pcDNA3.1-Mage-b1/2-V5), the control vector (pcDNA3.1-V5), or saline. Two weeks after the last immunization, the syngeneic 4TO7cg or 64pT tumor cell lines were injected in a mammary fat pad. Mice were monitored during the next 4 weeks for tumor formation, latency and size, and subsequently sacrificed for analysis. While the Mage-b1/2 vaccine had only a minor effect on the latency and growth of primary tumors, a significant and reproducible reduction in the number of 4TO7cg metastases was observed (vaccine versus control vector, p=0.0329; vaccine versus saline, p=0.0128). The observed protective efficacy of the Mage-b DNA vaccine correlated with high levels of vaccine-induced IFNgamma in spleen and lymph nodes upon re-stimulation in vitro. These results demonstrate the potential of TAA-based DNA vaccines in controlling metastatic disease in breast cancer patients.

Potential mouse tumor model for pre-clinical testing of mage-specific breast cancer vaccines.

Currently, there is a lack of suitable pre-clinical mouse models for testing and optimization of experimental cancer vaccines. Here, in situ developed mammary tumors of MMTV-v-Ha-ras and MMTV-c-myc transgenic mice and normal mammary, liver, spleen, and testis were screened for expression of tumor-associated antigens (TAA) Mage-b1/2/3 by reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blot hybridization. Mage-b1/2/3 are homologues of the human TAA MAGE-B1/2/3. Expression of these human MAGE genes has been found in tumors of various histological types, including breast cancer. Mage-specific RT-PCR products (using primers that amplify all three Mage-b1/2/3) were detected in mammary tumors of the MMTV-v-Ha-ras and MMTV-c-myc transgenic mice and in testis, but not in other normal tissues. RT-PCR products obtained from the mammary tumors (using primers that amplify the complete protein-encoding region of Mage-b1/2/3) were cloned and sequenced, and appeared to be most homologous with Mage-b3. Comparison of the Mage-b3 gene in mammary tumors and normal tissues suggest that somatic mutations did not occur in the Mage-b3 gene of the ras- and myc-induced mammary tumors. In addition, no differences were found between the Mage-b3 cDNA of testis, the only normal tissue that expresses Mage-b3, and Mage-b3 in genomic DNA of normal kidney, where Mage-b3 is silent. The MMTV-v-Ha-ras and MMTV-c-myc transgenic mice of this study are the first immune competent mouse models with in situ developed mammary tumors in which the expression of Mage-b3 TAA has been demonstrated. This makes them potentially suitable as a mouse model for pre-clinical testing of Mage-specific cancer vaccines in vivo.