Comprehensive characterization of toxins during progression of inhalation anthrax in a non-human primate model.

Inhalation anthrax has three clinical stages: early-prodromal, intermediate-progressive, and late-fulminant. We report the comprehensive characterization of anthrax toxins, including total protective antigen (PA), total lethal factor (LF), total edema factor (EF), and their toxin complexes, lethal toxin and edema toxin in plasma, during the course of inhalation anthrax in 23 cynomolgus macaques. The toxin kinetics were predominantly triphasic with an early rise (phase-1), a plateau/decline (phase-2), and a final rapid rise (phase-3). Eleven animals had shorter survival times, mean±standard deviation of 58.7±7.6 hours (fast progression), 11 animals had longer survival times, 113±34.4 hours (slow progression), and one animal survived. Median (lower-upper quartile) LF levels at the end-of-phase-1 were significantly higher in animals with fast progression [138 (54.9-326) ng/mL], than in those with slow progression [23.8 (15.6-26.3) ng/mL] (p = 0.0002), and the survivor (11.1 ng/mL). The differences were also observed for other toxins and bacteremia. Animals with slow progression had an extended phase-2 plateau, with low variability of LF levels across all time points and animals. Characterization of phase-2 toxin levels defined upper thresholds; critical levels for exiting phase-2 and entering the critical phase-3, 342 ng/mL (PA), 35.8 ng/mL (LF), and 1.10 ng/mL (EF). The thresholds were exceeded earlier in animals with fast progression (38.5±7.4 hours) and later in animals with slow progression (78.7±15.2 hours). Once the threshold was passed, toxin levels rose rapidly in both groups to the terminal stage. The time from threshold to terminal was rapid and similar; 20.8±7.4 hours for fast and 19.9±7.5 hours for slow progression. The three toxemic phases were aligned with the three clinical stages of anthrax for fast and slow progression which showed that anthrax progression is toxin- rather than time-dependent. This first comprehensive evaluation of anthrax toxins provides new insights into disease progression.

Neurobehavioral and Cardiovascular Effects of Potassium Cyanide Administered Orally to Mice.

The Food and Drug Administration Animal Rule requires evaluation of cardiovascular and central nervous system (CNS) effects of new therapeutics. To characterize an adult and juvenile mouse model, neurobehavioral and cardiovascular effects and pathology of a single sublethal but toxic, 8 mg/kg, oral dose of potassium cyanide (KCN) for up to 41 days postdosing were investigated. This study describes the short- and long-term sensory, motor, cognitive, and behavioral changes associated with oral dosing of a sublethal but toxic dose of KCN utilizing functional observation battery and Tier II CNS testing in adult and juvenile mice of both sexes. Selected tissues (histopathology) were evaluated for changes associated with KCN exposure with special attention to brain regions. Telemetry (adult mice only) was used to evaluate cardiovascular and temperature changes. Neurobehavioral capacity, sensorimotor responsivity or spontaneous locomotor activity, and rectal temperature were significantly reduced in adult and juvenile mice at 30 minutes post-8 mg/kg KCN dose. Immediate effects of cyanide included bradycardia, adverse electrocardiogram arrhythmic events, hypotension, and hypothermia with recovery by approximately 1 hour for blood pressure and heart rate effects and by 2 hours for body temperature. Lesions consistent with hypoxia, such as mild acute tubular necrosis in the kidneys corticomedullary junction, were the only histopathological findings and occurred at a very low incidence. The mouse KCN intoxication model indicates rapid and completely reversible effects in adult and juvenile mice following a single oral 8 mg/kg dose. Neurobehavioral and cardiovascular measurements can be used in this animal model as a trigger for treatment.

PI3K activation is associated with intracellular sodium/iodide symporter protein expression in breast cancer.

BACKGROUND: The sodium/iodide symporter (NIS) is a membrane glycoprotein mediating active iodide uptake in the thyroid gland and is the molecular basis for radioiodide imaging and therapeutic ablation of thyroid carcinomas. NIS is expressed in the lactating mammary gland and in many human breast tumors, raising interest in similar use for diagnosis and treatment. However, few human breast tumors have clinically evident iodide uptake ability. We previously identified PI3K signaling as important in NIS upregulation in transgenic mouse models of breast cancer, and the PI3K pathway is commonly activated in human breast cancer. METHODS: NIS expression, subcellular localization, and function were analyzed in MCF-7 human breast cancer cells and MCF-7 cells stably or transiently expressing PI3K p110alpha subunit using Western blot of whole cell lysate, cell surface biotinylation Western blot and immunofluorescence, and radioiodide uptake assay, respectively. NIS localization was determined in a human breast cancer tissue microarray using immunohistochemical staining (IHC) and was correlated with pre-existing pAkt IHC data. Statistical analysis consisted of Student's t-test (in vitro studies) or Fisher's Exact Test (in vivo correlational studies). RESULTS: In this study, we demonstrate that PI3K activation in MCF-7 human mammary carcinoma cells leads to expression of underglycosylated NIS lacking cell surface trafficking necessary for iodide uptake ability. PI3K activation also appears to interfere with cell surface trafficking of exogenous NIS as well as all-trans retinoic acid-induced endogenous NIS. A correlation between NIS expression and upregulation of PI3K signaling was found in a human breast cancer tissue microarray. CONCLUSION: Thus, the PI3K pathway likely plays a major role in the discordance between NIS expression and iodide uptake in breast cancer patients. Further study is warranted to realize the application of NIS-mediated radioiodide ablation in breast cancer.

Creation and characterization of a doxycycline-inducible mouse model of thyroid-targeted RET/PTC1 oncogene and luciferase reporter gene coexpression.

BACKGROUND: RET/PTC1 chromosomal rearrangement is associated with papillary thyroid carcinoma formation in children exposed to ionizing radiation. We previously created a transgenic mouse model with thyroid-targeted constitutive RET/PTC1 expression and demonstrated papillary thyroid carcinoma formation. OBJECTIVE: In this study, we aimed to create a doxycycline-inducible mouse model of thyroid RET/PTC1 and luciferase reporter gene coexpression to allow for noninvasive monitoring of transgene expression in mice of various ages and timepoints after induction. DESIGN: Transgenic mice carrying the rtTA gene driven by the thyroglobulin promoter were generated, and crossed with responder mice carrying RET/PTC1 and firefly luciferase genes under control of a bidirectional tetracycline response element. MAIN OUTCOMES: Most bitransgenic mice had thyroid-targeted, doxycycline-independent transgene expression. Only one line had thyroid-targeted, doxycycline-regulated RET/PTC1 and luciferase coexpression, in which doxycycline induction of RET/PTC1 led to Erk phosphorylation and reduced expression of the sodium/iodide symporter (NIS). However, thyroid lesions were not found in any bitransgenic mice examined. CONCLUSIONS: We found that acute RET/PTC1 expression can activate the MEK/Erk pathway and downregulate NIS expression in the mouse thyroid gland. However, a higher level of RET/PTC1 is likely necessary for tumor formation. Thyroid luciferase induction was detectable noninvasively using IVIS in vivo imaging.

Signaling through 3',5'-cyclic adenosine monophosphate and phosphoinositide-3 kinase induces sodium/iodide symporter expression in breast cancer.

The sodium/iodide symporter (NIS) is a membrane transport glycoprotein normally expressed in the thyroid gland and lactating mammary gland. NIS is a target for radioiodide imaging and therapeutic ablation of thyroid carcinomas and has the potential for similar use in breast cancer treatment. To facilitate NIS-mediated radionuclide therapy, it is necessary to identify signaling pathways that lead to increased NIS expression and function in breast cancer. We examined NIS expression in mammary tumors of 14 genetically engineered mouse models to identify genetic manipulations associated with NIS induction. The cAMP and phosphoinositide-3 kinase (PI3K) signaling pathways are associated with NIS up-regulation. We showed that activation of PI3K alone is sufficient to increase NIS expression and radioiodide uptake in MCF-7 human breast cancer cells, whereas cAMP stimulation increases NIS promoter activity and NIS mRNA levels but is not sufficient to increase radioiodide uptake. This study is the first to demonstrate that NIS expression is induced by cAMP and/or PI3K in breast cancer both in vivo and in vitro.

MicroRNA expression in mice infected with seasonal H1N1, swine H1N1 or highly pathogenic H5N1.

Influenza virus infections in humans remain a healthcare concern, and the need for vaccines, therapeutics and prophylactics remains a high priority. Understanding the molecular events associated with influenza-virus-induced pathology may lead to the identification of clinical disease biomarkers and novel antiviral targets. MicroRNAs (miRNAs) are well-conserved endogenous non-coding RNAs known to regulate post-transcriptional gene expression as well as play a major role in many biological processes and pathways. Animal studies have demonstrated that miRNAs are involved in viral disease and controlling inflammation. In this study, we examined the differences in the miRNA expression profiles associated with the lung in mice infected with influenza viruses that varied in virulence and pathogenicity. A statistical model was employed that utilized changes in miRNA expression to identify the virus that was used to infect the mice. This study identified a unique fingerprint of viral pathogenicity associated with seasonal H1N1, swine H1N1 and highly pathogenic H5N1 in the mouse model, and may lead to the identification of novel therapeutic and prophylactic targets.

Development of an inhalational Bacillus anthracis exposure therapeutic model in cynomolgus macaques.

Appropriate animal models are required to test medical countermeasures to bioterrorist threats. To that end, we characterized a nonhuman primate (NHP) inhalational anthrax therapeutic model for use in testing anthrax therapeutic medical countermeasures according to the U.S. Food and Drug Administration Animal Rule. A clinical profile was recorded for each NHP exposed to a lethal dose of Bacillus anthracis Ames spores. Specific diagnostic parameters were detected relatively early in disease progression, i.e., by blood culture (∼37 h postchallenge) and the presence of circulating protective antigen (PA) detected by electrochemiluminescence (ECL) ∼38 h postchallenge, whereas nonspecific clinical signs of disease, i.e., changes in body temperature, hematologic parameters (ca. 52 to 66 h), and clinical observations, were delayed. To determine whether the presentation of antigenemia (PA in the blood) was an appropriate trigger for therapeutic intervention, a monoclonal antibody specific for PA was administered to 12 additional animals after the circulating levels of PA were detected by ECL. Seventy-five percent of the monoclonal antibody-treated animals survived compared to 17% of the untreated controls, suggesting that intervention at the onset of antigenemia is an appropriate treatment trigger for this model. Moreover, the onset of antigenemia correlated with bacteremia, and NHPs were treated in a therapeutic manner. Interestingly, brain lesions were observed by histopathology in the treated nonsurviving animals, whereas this observation was absent from 90% of the nonsurviving untreated animals. Our results support the use of the cynomolgus macaque as an appropriate therapeutic animal model for assessing the efficacy of medical countermeasures developed against anthrax when administered after a confirmation of infection.

Lack of toxicity of a STAT3 decoy oligonucleotide.

BACKGROUND: STAT3 overexpression has been detected in several cancers including head and neck squamous cell carcinoma (HNSCC). Previous studies using intratumoral administration of a STAT3 decoy oligonucleotide that abrogates STAT3-mediated gene transcription in preclinical cancer models have demonstrated antitumor efficacy. This study was conducted to observe the toxicity and biologic effects of the STAT3 decoy in a non-human primate model, in anticipation of initiating a clinical trial in HNSCC patients. METHODS: Three study groups (two monkeys/sex/group) were administered a single intramuscular injection of low dose of STAT3 decoy (0.8 mg total dose/monkey), high dose of STAT3 decoy (3.2 mg total dose/monkey) or vehicle control (PBS alone) on day 1 and necropsies were performed on days 2 and 15 (one monkey/sex/group/day). Low and high doses of the decoy were administered in the muscle in a volume of 0.9 ml. Tissue and blood were harvested for toxicology and biologic analyses. RESULTS: Upon observation, the STAT3 decoy-treated animals exhibited behavior that was similar to the vehicle control group. Individual animal body weights remained within 1% of pretreatment weights throughout the study. Hematological parameters were not significantly different between the control and the treatment groups. Clinical chemistry fluctuations were considered within normal limits and were not attributed to the STAT3 decoy. Assessment of complement activation breakdown product (Bb) levels demonstrated no activation of the alternative pathway of complement in any animal at any dose level. At necropsy, there were no gross or microscopic findings attributed to STAT3 decoy in any organ examined. STAT3 target gene expression at the injection site revealed decreased Bcl-X(L) and cyclin D1 expression levels in the animals treated with high dose of STAT3 decoy compared to the animals injected with low dose of STAT3 decoy or the vehicle as control. CONCLUSION: Based on these findings, the no-observable-adverse-effect-level (NOAEL) was greater than 3.2 mg/kg when administered as a single dose to male and female Cynomolgus monkeys. Plans are underway to test the safety and biologic effects of intratumoral administration of the STAT3 decoy in HNSCC patients.