Preparation of Tc99m-Labeled Pseudomonas Bacteriophage without Adversely Impacting Infectivity or Biodistribution.

Bacteriophages (phages) are ubiquitous viruses which have adapted to infect and replicate within target bacteria, their only known hosts, in a strain specific fashion with minimal cross infectivity. The recent steep rise in antibiotic resistance throughout the world has renewed interest in adapting phages for the imaging and treatment of bacterial infection in humans. In this article, we describe the current limitations surrounding the radiolabeling of phage for the imaging and treatment of bacterial infection and methods to overcome these difficulties. Specifically, we examined the effects of hydrazinonicotinamide conjugation and removal of bacterial DNA on the infectivity, biodistribution, and radionuclide imaging of a phage lytic for a clinically relevant strain of Pseudomonas aeruginosa, a common Gram-negative bacterial pathogen often resistant to multiple antibiotics. We found that all but the briefest reaction of concentrated phage with hydrazinonicotinamide (≤3 min) resulted in nearly complete loss of infectivity. Furthermore, we determined that digestion and removal of bacterial DNA was needed to avoid high nonspecific uptake of hydrazinonicotinamide-labeled phage within the liver and spleen as well as prolonged circulation in the blood. We also demonstrate the surprisingly wide soft tissue and organ biodistribution and rapid pharmacokinetics of 99mTc-hydrazinonicotinamide-labeled phage in normal mice as well as its imaging characteristics and efficacy in wounded mice infected with bioluminescent Pseudomonas aeruginosa. In conclusion, the preservation of phage infectivity and removal of all bacterial containments including DNA are critical methodologic considerations in the labeling of phages for imaging and therapy.

Microarray analysis of cellular thermotolerance.

BACKGROUND AND OBJECTIVES: Previously, we have shown that a 43°C pretreatment can provide thermotolerance to a following, more severe, thermal stress at 45°C. Using cells that lack the Hsp70 gene, we have also shown that there is still some thermotolerance in the absence of HSP70 protein. The purpose of this study was to determine which genes play a role in thermotolerance by measuring viability and proliferation of the cells at 2 days after heating. Specifically, we wanted to understand which pathways may be responsible for protecting cells in the absence of HSP70. STUDY DESIGN/MATERIALS AND METHODS: Murine embryonic fibroblast cells with and without Hsp70 (MEF(+/+) and MEF(-/-), respectively) were exposed to a mild heat shock of 43°C for 30 minutes in a constant temperature water bath. After 3 hours of recovery, RNA was harvested from three heated samples alongside three untreated controls using a MicroRNeasy kit with DNAse treatment. RNA quality was verified by an Agilent Bioanalyzer. The RNA was then converted to cDNA and hybridized to Affymetrix gene expression DNA microarrays. The genes that showed a twofold change (up or down) relative to unheated controls were filtered by t-test for significance at a threshold of P < 0.05 using Genespring software. Data were verified by qRT-PCR. Genes were then categorized based upon their ontology. RESULTS: While many genes were similarly upregulated, the main difference between cell types was an increase in transcription factors and nucleic acid binding proteins. Several genes known to be involved in the heat response were upregulated more than twofold (Hsp70, Hsp40, Hsp110, Hsp25, Atf3), however, another well studied heat responsive gene Hsp90 only increased by 1.5-fold under these conditions despite its role in thermotolerance. CONCLUSIONS: The data herein presents genetic pathways which are candidates for further study of pretreatment protocols in laser irradiation.

Role of HSP70 in cellular thermotolerance.

BACKGROUND AND OBJECTIVE: Thermal pretreatment has been shown to condition tissue to a more severe secondary heat stress. In this research we examined the particular contribution of heat shock protein 70 (HSP70) in thermal preconditioning. STUDY DESIGN/MATERIALS AND METHODS: For optimization of preshock exposures, a bioluminescent Hsp70-luciferase reporter system in NIH3T3 cells tracked the activation of the Hsp70 gene. Cells in 96-well plates were pretreated in a 43 degrees C water bath for 30 minutes, followed 4 hours later with a severe heat shock at 45 degrees C for 50 minutes. Bioluminescence was measured at 2, 4, 6, 8, and 10 hours after preshock only (PS) and at 4 hours after preshock with heatshock (PS+HS). Viability was assessed 48 hours later with a fluorescent viability dye. Preshock induced thermotolerance was then evaluated in hsp70-containing Murine Embryo Fibroblast (+/+) cells and Hsp70-deficient MEF cells (-/-) through an Arrhenius damage model across varying temperatures (44.5-46 degrees C). RESULTS: A time gap of 4 hours between preconditioning and the thermal insult was shown to be the most effective for thermotolerance with statistical confidence of P<0.05. The benefit of preshocking was largely abrogated in Hsp70-deficient cells. The Arrhenius data showed that preshocking leads to increases in the activation energies, E(a), and increases in frequency factors, A. The frequency factor increase was significantly greater in Hsp70-deficient cells. CONCLUSION: The data shows that HSP70 contributes significantly to cellular thermotolerance but there are other pathways that provide residual thermotolerance in cells deficient in Hsp70.

Potential applications of conventional and molecular imaging to biodefense research.

Imaging methods that visualize the structure and function of the living body are widely used in patient care and biomedical research, but their full potential has not yet been applied to the study and treatment of the severe illnesses caused by pathogens of biodefense concern. "Conventional" imaging techniques (e.g., radiography, computed tomography, ultrasound, or magnetic resonance imaging) delineate anatomic changes in tissues, whereas "molecular" methods employ magnetic resonance, positron emission tomography, single-photon emission computed tomography, or optical (fluorescence or bioluminescence) imaging to detect biochemical reactions that accompany pathogen replication or host responses. We review the basic principles of these methods, describe the diseases caused by 6 pathogens classified as category A or B bioterror agents (anthrax, plague, tularemia, filoviral hemorrhagic fever, smallpox, and aerosolized equine encephalitis virus infection), and discuss how imaging could be used to study their pathogenesis in laboratory animals and to diagnose and monitor infection in humans.

Seeing it through: translational validation of new medical imaging modalities.

Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to "bridge the gap" between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.