To better understand the cellular and molecular responses to overexposure to millimeter waves, alterations in the gene expression profile and histology of skin after exposure to 35 GHz radiofrequency radiation were investigated. Rats were subjected to sham exposure, to 42 degrees C environmental heat, or to 35 GHz millimeter waves at 75 mW/cm(2). Skin samples were collected at 6 and 24 h after exposure for Affymetrix GeneChip analysis. The skin was harvested from a separate group of rats at 3-6 h or 24-48 h after exposure for histopathology analysis. Microscopic findings observed in the dermis of rats exposed to 35 GHz millimeter waves included aggregation of neutrophils in vessels, degeneration of stromal cells, and breakdown of collagen. Changes were detected in 56 genes at 6 h and 58 genes at 24 h in the millimeter-wave-exposed rats. Genes associated with regulation of transcription, protein folding, oxidative stress, immune response, and tissue matrix turnover were affected at both times. At 24 h, more genes related to extracellular matrix structure and chemokine activity were altered. Up-regulation of Hspa1a, Timp1, S100a9, Ccl2 and Angptl4 at 24 h by 35 GHz millimeter-wave exposure was confirmed by real-time RT-PCR. These results obtained from histopathology, microarrays and RT-PCR indicate that prolonged exposure to 35 GHz millimeter waves causes thermally related stress and injury in skin while triggering repair processes involving inflammation and tissue matrix recovery.

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.

Breast cancer incidence and mortality increase with age. A better understanding of the biological behavior of metastatic and nonmetastatic breast tumors in older subjects may help to develop improved breast cancer therapies. In this study, we used syngeneic metastatic (4TO7cg) and nonmetastatic (64pT) mouse breast tumor models at three age levels to evaluate various characteristics that are considered to be important for effective anti-breast cancer immunotherapy. These included tumor size and growth, metastases, vascularization, gene expression levels of the tumor-associated antigen (TAA) Mage-b (homologous to human MAGE-B) in primary breast tumors and metastases, and the presence of CD4(+) and CD8(+) T cells in the inguinal lymph nodes at the site of the tumor. The primary breast tumors and metastases were generated by injection of mouse mammary tumor cell lines 4TO7cg or 64pT into a mammary fat pad of normal 3-, 9-, or 21/24-month old BALB/c mice. In the nonmetastatic breast tumor model, significantly smaller tumors were observed in old compared with young mice. This was associated with a significant increase in the percentage of CD8(+) T cells in inguinal lymph nodes and significantly higher Mage-b expression levels in the primary tumors at old age. In the metastatic (4TO7cg) breast tumor model, a less pronounced, not statistically significant, smaller tumor size was found in the old mice, without a difference in the percentage of CD8(+) T cells or Mage-b expression levels. However, in this mouse model almost all metastases showed high levels of Mage-b expression (2- to 3-fold higher than the primary tumors in the same animals) regardless of age. These results indicate that the metastatic and nonmetastatic breast tumor models could be useful model systems to analyze how breast cancer vaccines for humans can be tailored to old age.

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.