Expanding applications of cDNA microarrays such as fine needle aspiration biopsy and laser capture microdissection necessitate the ability to perform arrays with minute starting amounts of RNA. While methods for amplifying RNA have been advocated, the fidelity of array results using amplified material has not been fully validated. Here we demonstrate preserved fidelity in arrays using one or two rounds of mRNA amplification, validated by downstream real-time quantitative PCR. In addition, the quality of the array data was superior to that obtained using total RNA. Based on these results, we recommend routine mRNA amplification for all cDNA microarray-based analysis of gene expression.

Optical imaging is a modality that is cost-effective, rapid, easy to use, and can be readily applied to studying disease processes and biology in vivo. For this study, we used a green fluorescent protein (GFP)- and luciferase-expressing mouse tumor model to compare and contrast the quantitative and qualitative capabilities of a fluorescent reporter gene (GFP) and a bioluminescent reporter gene (luciferase). We describe the relationship between tumor volume, tumor mass, and bioluminescent/fluorescent intensity for both GFP and luciferase. Bioluminescent luciferase imaging was shown to be more sensitive than fluorescent GFP imaging. Luciferase-expressing tumors were detected as early as 1 day after tumor cell inoculation, whereas GFP-expressing tumors were not detected until 7 days later. Both bioluminescent and fluorescent intensity correlated significantly and linearly with tumor volume and tumor weight, as measured by caliper. Compared to bioluminescent imaging, fluorescent imaging does not require the injection of a substrate and may be appropriate for applications where sensitivity is not as critical. Knowing the relative strengths of each imaging modality will be important in guiding the decision to use fluorescence or bioluminescence.

The emergence of angiogenesis as an important target for cancer therapy has led to increased research aimed at understanding the mechanisms underlying the development, maintenance, and destruction of tumor vasculature. Concurrently, molecular imaging technologies have been developed and are being incorporated as integral components of biomedical research due to their ability to noninvasively monitor in vivo molecular events. With the evaluation of numerous anti-angiogenic agents in clinical trials, the adaptation and validation of molecular imaging modalities for monitoring angiogenesis is actively being pursued. The importance of selecting appropriate molecular targets in the study of angiogenesis has become increasingly complex due to the pleiotropy of vascular phenotypes. Furthermore, due to both the relatively low abundance of endothelial cells in tumor tissue and the inherent difficulties of detecting molecular events, molecular imaging of vasculature necessitates continued improvements in achieving higher sensitivity. While several studies have been published that set the groundwork for imaging angiogenesis, much has yet to be accomplished. Various tumor models and transgenic mice provide an excellent resource for developing molecular imaging technologies for the understanding of angiogenesis. This research may play a particularly crucial role in evaluating mechanism and efficacy during pre-clinical testing of anti-angiogenic drugs. Due to practical limitations, however, the implementation of angiogenesis-directed molecular imaging may not extend beyond highly specialized clinical trials. That is, imaging modalities that evaluate angiogenesis at a functional level may prove more appropriate. Despite future technical challenges, molecular imaging will become an important research and clinical tool in evaluating tumor angiogenesis.

Objective. To identify self T cell epitopes associated with proinflammatory immune responses and clinically active juvenile dermatomyositis (juvenile DM). The target of our search for relevant epitopes was represented by amino acid sequences shared between human skeletal myosin and Streptococcus pyogenes M5 protein. The long-term objective of the project is to identify suitable targets for immunotherapy of the disease.Methods. We used computerized algorithms to identify putative agretopes on both the human myosin and Streptococcus M5 proteins. Direct binding assays for homolog peptides were used to confirm such predictions. Antigenicity and functional cross-reactivity were evaluated by cytotoxicity assays and by measurement of cytokine levels. Specific T cells were isolated by T cell capture, and T cell receptor (TCR) V ␤ gene usage was identified by reverse transcriptase-polymerase chain reaction.Results. We identified peptides that are targets of disease-specific cytotoxic T cell responses. T cell reactivity against the self peptides correlates with clinical signs of early, active myositis. Such reactivity is accompanied by production of proinflammatory cytokines, which may contribute to the damage. T cell crossrecognition of bacterial and human homologs was shown functionally as well as by sorting peptide-specific T cells and identifying oligoclonal and largely overlapping TCR V ␤ gene usage.Conclusion. These findings represent the first identification of a self epitope in juvenile DM, providing a potential candidate for antigen-specific immune therapy.