Making the Case for Absorbed Radiation Response Biodosimetry - Utility of a High-Throughput Biodosimetry System.

There is an unmet need to provide medical personnel with a Food and Drug Administration (FDA)-approved biodosimetry method for quantifying individualized absorbed dose response to inform treatment decisions for a very large patient population potentially exposed to ionizing radiation in the event of a nuclear incident. Validation of biodosimetry devices requires comparison of absorbed dose estimates to delivered dose as an indication of accuracy; however, comparison to delivered dose does not account for biological variability or an individual's radiosensitivity. As there is no FDA-cleared gene-expression-based biodosimetry method for determining biological response to radiation, results from accuracy comparisons to delivered dose yield relatively wide tolerance intervals or uncertainty. The Arizona State University Biodesign Institute is developing a high-throughput, automated real-time polymerase chain reaction (RT-PCR)-based biodosimetry system that provides absorbed dose estimates for patients exposed to 0-10 Gy from blood collected 1-7 days postirradiation. While the absorbed dose estimates result from a calibration against the actual exposed dose, the reported dose estimate is a measure of response to absorbed dose based on the exposure models used in developing the system. A central concern with biodosimetry test evaluation is how variability in the dose estimate results could affect medical decision-making, and if the biodosimetry test system performance is quantitatively sufficient to inform effective treatment. A risk:benefit analysis of the expected system performance in the proposed intended use environment was performed to address the potential medical utility of this biodosimetry system. Uncertainty analysis is based on biomarker variability in non-human primate (NHP) models. Monte Carlo simulation was employed to test multiple groups of biomarkers and their potential variation in response to determine uncertainty associated with dose estimate results. Dose estimate uncertainty ranges from ±1.2-1.7 Gy depending on the exposure dose over a range of 2-10 Gy. The risk:benefit of individualized absorbed dose estimates within the context of medical interventions after a nuclear incident is considered and the application of the biodosimetry system is evaluated in this framework. NHP dose-response relationships, as measured by clinical outcome end points, show expected biological and radiosensitivity responses in the primate populations tested and corroborate the biological variability observed in the reported absorbed dose estimate. Performance is examined in relationship to current clinical management and treatment recommendations, with evaluation of potential patient risk in over- and underestimating absorbed dose.

In vitro analysis of the invasive phenotype of SUM 149, an inflammatory breast cancer cell line.

BACKGROUND: Inflammatory breast cancer (IBC) is the most lethal form of locally invasive breast cancer known. However, very little information is available on the cellular mechanisms responsible for manifestation of the IBC phenotype. To understand the unique phenotype of IBC, we compared the motile and adhesive interactions of an IBC cell line, SUM 149, to the non-IBC cell line SUM 102. RESULTS: Our results demonstrate that both IBC and non-IBC cell lines exhibit similar adhesive properties to basal lamina, but SUM 149 showed a marked increase in adhesion to collagen I. In vitro haptotaxis assays demonstrate that SUM 149 was less invasive, while wound healing assays show a less in vitro migratory phenotype for SUM 149 cells relative to SUM 102 cells. We also demonstrate a role for Rho and E-cadherin in the unique invasive phenotype of IBC. Immunoblotting reveals higher E-cadherin and RhoA expression in the IBC cell line but similar RhoC expression. Rhodamine phalloidin staining demonstrates increased formation of actin stress fibers and larger focal adhesions in SUM 149 relative to the SUM 102 cell line. CONCLUSION: The observed unique actin and cellular architecture as well as the invasive and adhesive responses to the extracellular matrix of SUM 149 IBC cells suggest that the preference of IBC cells for connective tissue, possibly a mediator important for the vasculogenic mimicry via tubulogenesis seen in IBC pathological specimens. Overexpression of E-cadherin and RhoA may contribute to passive dissemination of IBC by promoting cell-cell adhesion and actin cytoskeletal structures that maintain tissue integrity. Therefore, we believe that these findings indicate a passive metastatic mechanism by which IBC cells invade the circulatory system as tumor emboli rather than by active migratory mechanisms.

Luminex(®) multiplex bead suspension arrays for the detection and serotyping of Salmonella spp.

In this chapter we describe two commercially available bead-based molecular assays for detection, identification and serotyping of Salmonella. The xTAG(®) Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplex test for the simultaneous detection of nucleic acids from Salmonella plus 14 other gastroenteritis-causing bacteria, viruses, and parasites from stool specimens. xTAG GPP uses the Luminex(®) xTAG universal array technology for the identification of specific target sequences combined with the xMAP(®) bead multiplexing platform for detection of the targets that were present in the starting sample. The xMAP Salmonella Serotyping Assay (SSA) is a multiplex nucleic acid-based direct hybridization assay for molecular identification of the serotype of Salmonella isolates. In xMAP SSA, target sequences amplified from cultured Salmonella isolates are captured by hybridization to sequence-specific capture probes which have been coupled to the multiplexed bead sets. Herein we provide detailed protocols for each of these assays and present data which describe their performance characteristics for detection and serotyping Salmonella.

In situ analysis of breast cancer progression in murine models using a macroscopic fluorescence imaging system.

BACKGROUND AND OBJECTIVE: The goal of this study was to use an inexpensive macroscopic imaging system to monitor tumor progression in mouse models in real-time with minimal intervention. STUDY DESIGN/MATERIALS AND METHODS: Illumination is provided via a xenon arc lamp and a fiber optic probe which delivers white light or quasi-monochromatic excitation via specific bandpass filters. Fluorescence emission from SCID and nude mice following mammary fat pad injection of red fluorescence protein (RFP)-expressing human breast cancer cell lines was recorded and quantified using a single lens reflex (SLR) digital camera. RESULTS: This simple system enabled the verification of successful tumor take and temporal quantification of tumor progression in mouse models. CONCLUSION: The macroscopic fluorescence imaging system represents an inexpensive and portable tool to facilitate non-invasive in situ cancer detection with the potential to monitor fluorescent tumor formation and investigation of the efficacy of potential cancer therapeutics.

Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion.

Reactive oxygen species induce myocardial damage after ischemia and reperfusion in experimental animal models. Numerous studies have investigated the deleterious effects of ischemia-reperfusion (I/R)-induced oxidant production using various pharmacological interventions. More recently, in vitro studies have incorporated gene-targeted mice to decipher the role of antioxidant enzymes in myocardial reperfusion injury. We examined the role of cellular antioxidant enzymes in the pathogenesis of myocardial I/R (MI/R) injury in vivo in gene-targeted mice. Neither deficiency nor overexpression of Cu-Zn superoxide dismutase (SOD) altered the extent of myocardial necrosis. Overexpression of glutathione peroxidase did not affect the degree of myocardial injury. Conversely, overexpression of manganese (Mn)SOD significantly attenuated myocardial necrosis after MI/R. Transthoracic echocardiography was performed on MnSOD-overexpressing and wild-type mice that were subjected to a more prolonged period of reperfusion. Cardiac output was significantly depressed in the nontransgenic but not the transgenic MnSOD-treated mice. Anterior wall motion was significantly impaired in the nontransgenic mice. These findings demonstrate an important role for MnSOD but not Cu/ZnSOD or glutathione peroxidase in mice after in vivo MI/R.

Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes.

The Mre11/Rad50 complex is a critical component of the cellular response to DNA double-strand breaks, in organisms ranging from archaebacteria to humans. In mammalian cells, Mre11/Rad50 (M/R) associates with a third component, Nbs1, that regulates its activities and is targeted by signaling pathways that initiate DNA damage-induced checkpoint responses. Mutations in the genes that encode Nbs1 and Mre11 are responsible for the human radiation sensitivity disorders Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, which are characterized by defective checkpoint responses and high levels of chromosomal abnormalities. Here we demonstrate nucleotide-dependent DNA binding by the human M/R complex that requires the Nbs1 protein and is specific for double-strand DNA duplexes. Efficient DNA binding is only observed with non-hydrolyzable analogs of ATP, suggesting that ATP hydrolysis normally effects DNA release. The alleles of MRE11 associated with ATLD and the C-terminal Nbs1 polypeptide associated with NBS were expressed with the other components and found to form triple complexes except in the case of ATLD 3/4, which exhibits variability in Nbs1 association. The ATLD 1/2, ATLD 3/4, and p70 M/R/N complexes exhibit nucleotide-dependent DNA binding and exonuclease activity equivalent to the wild-type enzyme, although the ATLD complexes both show reduced activity in endonuclease assays. Sedimentation equilibrium analysis of the recombinant human complexes indicates that Mre11 is a stable dimer, Mre11 and Nbs1 form a 1:1 complex, and both M/R and M/R/N form large multimeric assemblies of approximately 1.2 MDa. Models of M/R/N stoichiometry in light of this and previous data are discussed.