Effect of TRPV4 Antagonist GSK2798745 on Chlorine Gas-Induced Acute Lung Injury in a Swine Model.

As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO2/FiO2), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO2/FiO2, but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

Head Kinematics, Blood Biomarkers, and Histology in Large Animal Models of Traumatic Brain Injury and Hemorrhagic Shock.

Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) are each leading causes of mortality and morbidity worldwide, and present additional treatment considerations when they are comorbid (TBI+HS) as a result of competing pathophysiological responses. The current study rigorously quantified injury biomechanics with high precision sensors and examined whether blood-based surrogate markers were altered in general trauma as well as post-neurotrauma. Eighty-nine sexually mature male and female Yucatan swine were subjected to a closed-head TBI+HS (40% of circulating blood volume; n = 68), HS only (n = 9), or sham trauma (n = 12). Markers of systemic (e.g., glucose, lactate) and neural functioning were obtained at baseline, and at 35 and 295 min post-trauma. Opposite and approximately twofold differences existed for both magnitude (device > head) and duration (head > device) of quantified injury biomechanics. Circulating levels of neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and ubiquitin C-terminal hydrolase L1 (UCH-L1) demonstrated differential sensitivity for both general trauma (HS) and neurotrauma (TBI+HS) relative to shams in a temporally dynamic fashion. GFAP and NfL were both strongly associated with changes in systemic markers during general trauma and exhibited consistent time-dependent changes in individual sham animals. Finally, circulating GFAP was associated with histopathological markers of diffuse axonal injury and blood-brain barrier breach, as well as variations in device kinematics following TBI+HS. Current findings therefore highlight the need to directly quantify injury biomechanics with head mounted sensors and suggest that GFAP, NfL, and UCH-L1 are sensitive to multiple forms of trauma rather than having a single pathological indication (e.g., GFAP = astrogliosis).

17α-Ethinyl estradiol-3-sulfate increases survival and hemodynamic functioning in a large animal model of combined traumatic brain injury and hemorrhagic shock: a randomized control trial.

BACKGROUND: Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) represent leading causes of trauma-induced mortality, especially when co-occurring in pre-hospital settings where standard therapies are not readily available. The primary objective of this study was to determine if 17α-ethinyl estradiol-3-sulfate (EE-3-SO4) increases survival, promotes more rapid cardiovascular recovery, or confers neuroprotection relative to Placebo following TBI + HS. METHODS: All methods were approved by required regulatory agencies prior to study initiation. In this fully randomized, blinded preclinical study, eighty (50% females) sexually mature (190.64 ± 21.04 days old; 28.18 ± 2.72 kg) Yucatan swine were used. Sixty-eight animals received a closed-head, accelerative TBI followed by removal of approximately 40% of circulating blood volume. Animals were then intravenously administered EE-3-SO4 formulated in the vehicle at 5.0 mg/mL (dosed at 0.2 mL/kg) or Placebo (0.45% sodium chloride solution) via a continuous pump (0.2 mL/kg over 5 min). Twelve swine were included as uninjured Shams to further characterize model pathology and replicate previous findings. All animals were monitored for up to 5 h in the absence of any other life-saving measures (e.g., mechanical ventilation, fluid resuscitation). RESULTS: A comparison of Placebo-treated relative to Sham animals indicated evidence of acidosis, decreased arterial pressure, increased heart rate, diffuse axonal injury and blood-brain barrier breach. The percentage of animals surviving to 295 min post-injury was significantly higher for the EE-3-SO4 (28/31; 90.3%) relative to Placebo (24/33; 72.7%) cohort. EE-3-SO4 also restored pulse pressure more rapidly post-drug administration, but did not confer any benefits in terms of shock index. Primary blood-based measurements of neuroinflammation and blood brain breach were also null, whereas secondary measurements of diffuse axonal injury suggested a more rapid return to baseline for the EE-3-SO4 group. Survival status was associated with biological sex (female > male), as well as evidence of increased acidosis and neurotrauma independent of EE-3-SO4 or Placebo administration. CONCLUSIONS: EE-3-SO4 is efficacious in promoting survival and more rapidly restoring cardiovascular homeostasis following polytraumatic injuries in pre-hospital environments (rural and military) in the absence of standard therapies. Poly-therapeutic approaches targeting additional mechanisms (increased hemostasis, oxygen-carrying capacity, etc.) should be considered in future studies.

Thyroid homeostasis in B6C3F1 mice upon sub-chronic exposure to trifluoroiodomethane (CF3I).

Trifluoroiodomethane (CF3I) is a fire suppressant gas with potential for use in low global-warming refrigerant blends. Data from studies in rats suggest that the most sensitive health effect of CF3I is thyroid hormone perturbation, but the rat is a particularly sensitive species for disruption of thyroid homeostasis. Mice appear to be less sensitive than rats but still a conservative model with respect to humans. The purpose of this study was to test tolerance and thyroid response to CF3I in B6C3F1 male mice. Male mice were exposed to CF3I for 6 h per day, for 28 days, via whole body exposure at concentrations of 2500, 5000 and 10,000 ppm. A 16-day recovery period was included to evaluate reversibility. No adverse clinical signs were observed throughout the study, and body weights were unaffected by exposure. CF3I exposure had no effect on thyroid histology. An increase in relative thyroid weight was observed at 10,000 ppm on day 28 but not in a separate group of animals evaluated on day 29, and thyroid weight was not different from controls at 44 days. Slight and sporadic changes in serum triiodothyronine, thyroxine, and thyroid-stimulating hormone were observed but did not follow a consistent pattern with respect to timing, dose, or direction. Overall, exposure at up to 10,000 ppm (1.0%) of CF3I gas for 28 days produced no overt general toxicity and only transient, recoverable effects on thyroid weight and hormones at certain concentrations. On the basis of the effect of CF3I exposure on the thyroid, including evaluation of thyroid histopathology, the no observed adverse effect level for this study is 10,000 ppm. Considering the apparently greater toxicity reported in prior studies in male rats, our data suggest a species difference between rats and mice in terms of susceptibility to CF3I-induced thyroid hormone perturbation.

Reproducibility and Characterization of Head Kinematics During a Large Animal Acceleration Model of Traumatic Brain Injury.

Acceleration parameters have been utilized for the last six decades to investigate pathology in both human and animal models of traumatic brain injury (TBI), design safety equipment, and develop injury thresholds. Previous large animal models have quantified acceleration from impulsive loading forces (i.e., machine/object kinematics) rather than directly measuring head kinematics. No study has evaluated the reproducibility of head kinematics in large animal models. Nine (five males) sexually mature Yucatan swine were exposed to head rotation at a targeted peak angular velocity of 250 rad/s in the coronal plane. The results indicated that the measured peak angular velocity of the skull was 51% of the impulsive load, was experienced over 91% longer duration, and was multi- rather than uni-planar. These findings were replicated in a second experiment with a smaller cohort (N = 4). The reproducibility of skull kinematics data was mostly within acceptable ranges based on published industry standards, although the coefficients of variation (8.9% for peak angular velocity or 12.3% for duration) were higher than the impulsive loading parameters produced by the machine (1.1 vs. 2.5%, respectively). Immunohistochemical markers of diffuse axonal injury and blood-brain barrier breach were not associated with variation in either skull or machine kinematics, suggesting that the observed levels of variance in skull kinematics may not be biologically meaningful with the current sample sizes. The findings highlight the reproducibility of a large animal acceleration model of TBI and the importance of direct measurements of skull kinematics to determine the magnitude of angular velocity, refine injury criteria, and determine critical thresholds.

Survival Rates and Biomarkers in a Large Animal Model of Traumatic Brain Injury Combined With Two Different Levels of Blood Loss.

INTRODUCTION: The pathology resulting from concurrent traumatic brain injury (TBI) and hemorrhagic shock (HS; TBI+HS) are leading causes of mortality and morbidity worldwide following trauma. However, the majority of large animal models of TBI+HS have utilized focal/contusional injuries rather than incorporating the types of brain trauma (closed-head injury caused by dynamic acceleration) that typify human injury. OBJECTIVE: To examine survival rates and effects on biomarkers from rotational TBI with two levels of HS. METHODS: Twenty-two sexually mature Yucatan swine (30.39 ±â€Š2.25 kg; 11 females) therefore underwent either Sham trauma procedures (n = 6) or a dynamic acceleration TBI combined with either 55% (n = 8) or 40% (n = 8) blood loss in this serial study. RESULTS: Survival rates were significantly higher for the TBI+40% (87.5%) relative to TBI+55% (12.5%) cohort, with the majority of TBI+55% animals expiring within 2 h post-trauma from apnea. Blood-based neural biomarkers and immunohistochemistry indicated evidence of diffuse axonal injury (increased NFL/Aß42), blood-brain barrier breach (increased immunoglobulin G) and inflammation (increased glial fibrillary acidic protein/ionized calcium-binding adaptor molecule 1) in the injured cohorts relative to Shams. Invasive hemodynamic measurements indicated increased shock index and decreased pulse pressure in both injury cohorts, with evidence of partial recovery for invasive hemodynamic measurements in the TBI+40% cohort. Similarly, although both injury groups demonstrated ionic and blood gas abnormalities immediately postinjury, metabolic acidosis continued to increase in the TBI+55% group ∼85 min postinjury. Somewhat surprisingly, both neural and physiological biomarkers showed significant changes within the Sham cohort across the multi-hour experimental procedure, most likely associated with prolonged anesthesia. CONCLUSION: Current results suggest the TBI+55% model may be more appropriate for severe trauma requiring immediate medical attention/standard fluid resuscitation protocols whereas the TBI+40% model may be useful for studies of prolonged field care.

A systematic review of large animal models of combined traumatic brain injury and hemorrhagic shock.

Traumatic brain injury (TBI) and severe blood loss (SBL) frequently co-occur in human trauma, resulting in high levels of mortality and morbidity. Importantly, each of the individual post-injury cascades is characterized by complex and potentially opposing pathophysiological responses, complicating optimal resuscitation and therapeutic approaches. Large animal models of poly-neurotrauma closely mimic human physiology, but a systematic literature review of published models has been lacking. The current review suggests a relative paucity of large animal poly-neurotrauma studies (N = 52), with meta-statistics revealing trends for animal species (exclusively swine), characteristics (use of single biological sex, use of juveniles) and TBI models. Although most studies have targeted blood loss volumes of 35-45%, the associated mortality rates are much lower relative to Class III/IV human trauma. This discrepancy may result from potentially mitigating experimental factors (e.g., mechanical ventilation prior to or during injury, pausing/resuming blood loss based on physiological parameters, administration of small volume fluid resuscitation) that are rarely associated with human trauma, highlighting the need for additional work in this area.

Inhalation delivery of topotecan is superior to intravenous exposure for suppressing lung cancer in a preclinical model.

Intravenous (IV) topotecan is approved for the treatment of various malignancies including lung cancer but its clinical use is greatly undermined by severe hematopoietic toxicity. We hypothesized that inhalation delivery of topotecan would increase local exposure and efficacy against lung cancer while reducing systemic exposure and toxicity. These hypotheses were tested in a preclinical setting using a novel inhalable formulation of topotecan against the standard IV dose. Respirable dry-powder of topotecan was manufactured through spray-drying technology and the pharmacokinetics of 0.14 and 0.79 mg/kg inhalation doses were compared with 0.7 mg/kg IV dose. The efficacy of four weekly treatments with 1 mg/kg inhaled vs. 2 mg/kg IV topotecan were compared to untreated control using an established orthotopic lung cancer model for a fast (H1975) and moderately growing (A549) human lung tumors in the nude rat. Inhalation delivery increased topotecan exposure of lung tissue by approximately 30-fold, lung and plasma half-life by 5- and 4-folds, respectively, and reduced the maximum plasma concentration by 2-fold than the comparable IV dose. Inhaled topotecan improved the survival of rats with the fast-growing lung tumors from 7 to 80% and reduced the tumor burden of the moderately-growing lung tumors over 5- and 10-folds, respectively, than the 2-times higher IV topotecan and untreated control (p < .00001). These results indicate that inhalation delivery increases topotecan exposure of lung tissue and improves its efficacy against lung cancer while also lowering the effective dose and maximum systemic concentration that is responsible for its dose-limiting toxicity.

Magnetic resonance imaging provides sensitive in vivo assessment of experimental ventilator-induced lung injury.

Animal models play a critical role in the study of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). One limitation has been the lack of a suitable method for serial assessment of acute lung injury (ALI) in vivo. In this study, we demonstrate the sensitivity of magnetic resonance imaging (MRI) to assess ALI in real time in rat models of VILI. Sprague-Dawley rats were untreated or treated with intratracheal lipopolysaccharide or PBS. After 48 h, animals were mechanically ventilated for up to 15 h to induce VILI. Free induction decay (FID)-projection images were made hourly. Image data were collected continuously for 30 min and divided into 13 phases of the ventilatory cycle to make cinematic images. Interleaved measurements of respiratory mechanics were performed using a flexiVent ventilator. The degree of lung infiltration was quantified in serial images throughout the progression or resolution of VILI. MRI detected VILI significantly earlier (3.8 ± 1.6 h) than it was detected by altered lung mechanics (9.5 ± 3.9 h, P = 0.0156). Animals with VILI had a significant increase in the Index of Infiltration (P = 0.0027), and early regional lung infiltrates detected by MRI correlated with edema and inflammatory lung injury on histopathology. We were also able to visualize and quantify regression of VILI in real time upon institution of protective mechanical ventilation. Magnetic resonance lung imaging can be utilized to investigate mechanisms underlying the development and propagation of ALI, and to test the therapeutic effects of new treatments and ventilator strategies on the resolution of ALI.

Part 1. Assessment of carcinogenicity and biologic responses in rats after lifetime inhalation of new-technology diesel exhaust in the ACES bioassay.

The Health Effects Institute and its partners conceived and funded a program to characterize the emissions from heavy-duty diesel engines compliant with the 2007 and 2010 on-road emissions standards in the United States and to evaluate indicators of lung toxicity in rats and mice exposed repeatedly to 2007-compliant new-technology diesel exhaust (NTDE*). The a priori hypothesis of this Advanced Collaborative Emissions Study (ACES) was that 2007-compliant on-road diesel emissions "... will not cause an increase in tumor formation or substantial toxic effects in rats and mice at the highest concentration of exhaust that can be used ... although some biological effects may occur." This hypothesis was tested at the Lovelace Respiratory Research Institute (LRRI) by exposing rats by chronic inhalation as a carcinogenicity bioassay. Indicators of pulmonary toxicity in rats were measured after 1, 3, 12, 24, and 28-30 months of exposure. Similar indicators of pulmonary toxicity were measured in mice, as an interspecies comparison of the effects of subchronic exposure, after 1 and 3 months of exposure. A previous HEI report (Mauderly and McDonald 2012) described the operation of the engine and exposure systems and the characteristics of the exposure atmospheres during system commissioning. Another HEI report described the biologic responses in mice and rats after subchronic exposure to NTDE (McDonald et al. 2012). The primary motivation for the present chronic study was to evaluate the effects of NTDE in rats in the context of previous studies that had shown neoplastic lung lesions in rats exposed chronically to traditional technology diesel exhaust (TDE) (i.e., exhaust from diesel engines built before the 2007 U.S. requirements went into effect). The hypothesis was largely based on the marked reduction of diesel particulate matter (DPM) in NTDE compared with emissions from older diesel engine and fuel technologies, although other emissions were also reduced. The DPM component of TDE was considered the primary driver of lung tumorigenesis in rats exposed chronically to historical diesel emissions. Emissions from a 2007-compliant, 500-horsepower-class engine and after treatment system operated on a variable-duty cycle were used to generate the animal inhalation test atmospheres. Four groups were exposed to one of three concentrations (dilutions) of exhaust combined with crankcase emissions, or to clean air as a negative control. Dilutions of exhaust were set to yield average integrated concentrations of 4.2, 0.8, and 0.1 ppm nitrogen dioxide (NO2). Exposure atmospheres were analyzed by daily measurements of key effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors. components and periodic detailed physical-chemical characterizations. Exposures were conducted 16 hours/day (overnight, during the rats' most active period), 5 days/week. Responses to exposure were evaluated via hematology, serum chemistry, bronchoalveolar lavage (BAL), lung cell proliferation, histopathology, and pulmonary function. The exposures were accomplished as planned, with average integrated exposure concentrations within 20% of the target dilutions. The major components from exhaust were the gaseous inorganic compounds, nitrogen monoxide (NO), NO2, and carbon monoxide (CO). Minor components included low concentrations of DPM and volatile and semi-volatile organic compounds (VOCs and SVOCs). Among the more than 100 biologic response variables evaluated, the majority showed no significant difference from control as a result of exposure to NTDE. The major outcome of this study was the absence of pre-neoplastic lung lesions, primary lung neoplasia, or neoplasia of any type attributable to NTDE exposure. The lung lesions that did occur were minimal to mild, occurred only at the highest exposure level, and were characterized by an increased number and prominence of basophilic epithelial cells (considered reactive or regenerative) lining distal terminal bronchioles, alveolar ducts, and adjacent alveoli (termed in this report "Hyperplasia; Epithelial; Periacinar"), which often had a minimal increase in subjacent fibrous stroma (termed "Fibrosis; Interstitial; Periacinar"). Slight epithelial metaplastic change to a cuboidal morphology, often demonstrating cilia, was also noted in some animals (termed "Bronchiolization"). In addition to the epithelial proliferation, there was occasionally a subtle accumulation of pulmonary alveolar macrophages (termed "Accumulation; Macrophage") in affected areas. The findings in the lung progressed slightly from 3 to 12 months, without further progression between 12 months and the final sacrifice at 28 or 30 months. In addition to the histologic findings, there were biochemical changes in the lung tissue and lavage fluid that indicated mild inflammation and oxidative stress. Generally, these findings were observed only at the highest exposure level. There was also a mild progressive decrease in pulmonary function, which was more consistent in females than males. Limited nasal epithelial changes resulted from NTDE exposure, including increases in minor olfactory epithelial degeneration, hyperplasia, and/or metaplasia. Increases in these findings were present primarily at the highest exposure level, and their minor and variable nature renders their biologic significance uncertain. Overall, the findings of this study demonstrated markedly less severe biologic responses to NTDE than observed previously in rats exposed similarly to TDE. Further, the effects of NTDE in the present study were generally consistent with those observed previously in rats exposed chronically to NO2 alone. This suggests that NO2 may have been the primary driver of the biologic responses to NTDE in the present study. There was little evidence of effects characteristic of rats exposed chronically to high concentrations of DPM in TDE, such as an extensive accumulation of DPM within alveolar macrophages and inflammation leading to neoplastic transformation of epithelia and lung tumors.

Local and systemic tolerability of a 2'O-methoxyethyl antisense oligonucleotide targeting interleukin-4 receptor-α delivery by inhalation in mouse and monkey.

Antisense oligonucleotides (ASOs) bind and facilitate degradation of RNA and inhibit protein expression in pathways not easily targeted with small molecules or antibodies. Interleukin (IL)-4 and IL-13 potentiate signaling through the shared IL-4 receptor-α (IL-4Rα) subunit of their receptors. ASO targeting of IL-4Rα mRNA in a mouse model of asthma led to attenuation of airway hyperactivity, demonstrating potential benefit in asthma patients. This study focused on tolerability of inhaled IL-4Rα-targeting ASOs. Toxicity studies were performed with mouse- (ISIS 23189) and human-specific (ISIS 369645) sequences administered by inhalation. Four week (monkey) or 13 week (mouse) repeat doses at levels of up to 15 mg/kg/exposure (exp) and 50 mg/kg/exp, respectively, demonstrated dose-dependent effects limited to increases in macrophage size and number in lung and tracheobronchial lymph nodes. The changes were largely non-specific, reflecting adaptive responses that occur during active exposure and deposition of ASO and other material in the lung. Reversibility was observed at a rate consistent with the kinetics of tissue clearance of ASO. Systemic bioavailability was minimal, and no systemic toxicity was observed at exposure levels appreciably above pharmacological doses and doses proposed for clinical trials.

Effects of simulated downwind coal combustion emissions on pre-existing allergic airway responses in mice.

CONTEXT: Coal-fired power plant emissions can contribute a significant portion of the ambient air pollution in many parts of the world. OBJECTIVE: We hypothesized that exposure to simulated downwind coal combustion emissions (SDCCE) may exacerbate pre-existing allergic airway responses. METHODS: Mice were sensitized and challenged with ovalbumin (OVA). Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day for 3 days to air (control, C) or SDCCE containing particulate matter (PM) at low (L; 100 µg/m³), medium (M; 300 µg/m³), or high (H; 1000 µg/m³) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after SDCCE exposure, mice received another OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air/SDCCE. Measurement of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed ~24 h after the last exposure. RESULTS: SDCCE significantly increased BAL macrophages and eosinophils in OVA-sensitized mice from the post-OVA protocol. However, there was no effect of SDCCE on BAL macrophages or eosinophils in OVA-sensitized mice from the pre-OVA protocol. BAL neutrophils were elevated following SDCCE in both protocols in nonsensitized mice. These changes were not altered by filtering out the PM. In the post-OVA protocol, SDCCE decreased OVA-specific IgG1 in OVA-sensitized mice but increased levels of total IgE, OVA-specific IgE and OVA-specific IgG1 and IgG(2a) in non-sensitized animals. In the pre-OVA protocol, SDCCE increased OVA-specific IgE in both sensitized and non-sensitized animals. Additionally, BAL IL-4, IL-13, and IFN-γ levels were elevated in sensitized mice. CONCLUSION: These results suggest that acute exposure to either the particulate or gaseous phase of SDCCE can exacerbate various features of allergic airway responses depending on the timing of exposure in relation to allergen challenge.

Delta inulin polysaccharide adjuvant enhances the ability of split-virion H5N1 vaccine to protect against lethal challenge in ferrets.

BACKGROUND: The reduced immunogenicity of the H5 hemagglutinin (HA), compared to seasonal HA serotypes, has stimulated searches for effective adjuvants to improve H5 vaccine efficacy. This study examined the immunogenicity and protective efficacy in ferrets immunized with a split-virion H5N1 vaccine combined with Advax™, a novel delta inulin-based polysaccharide adjuvant technology that has previously demonstrated ability to augment humoral and cellular immunity to co-administered antigens. METHODS: Ferrets were vaccinated twice 21 days apart with 7.5 µg or 22.5 µg of a split-virion preparation of A/Vietnam/1203/2004 with or without adjuvant. An additional group received just one immunization with 22.5 µg HA plus adjuvant. Serum antibodies were measured by hemagglutination inhibition and microneutralization assays. Vaccinated animals were challenged intranasally 21 days after the last immunization with 10(6) EID(50) of the homologous strain. Morbidity was assessed by observed behavior, weight loss, temperature, cytopenias, histopathology, and viral load. RESULTS: No serum neutralization antibody was detected after two immunizations with unadjuvanted vaccine. Two immunizations with high or low dose adjuvanted vaccine stimulated high neutralizing antibody titers. Survival was 100% in all groups receiving adjuvanted-vaccine including the single dose group, compared to 67% survival with unadjuvanted vaccine, and 0% survival in saline or adjuvant-alone controls. Minimal morbidity was seen in all animals receiving adjuvanted vaccine, and was limited to rhinorrhea and mild thrombocytopenia, without fever, weight loss, or reduced activity. H5N1 virus was cleared from the nasal wash by day 4 post-challenge only in animals receiving adjuvanted vaccine which also prevented viral invasion of the brain in most animals. CONCLUSIONS: In this initial study, Advax™ adjuvant formulations improved the protective efficacy of a split-virion H5N1 vaccine as measured by significantly enhanced immunogenicity, survival, and reduced morbidity.

Health effects of subchronic inhalation exposure to simulated downwind coal combustion emissions.

CONTEXT: There have been no animal studies of the health effects of repeated inhalation of mixtures representing downwind pollution from coal combustion. Environmental exposures typically follow atmospheric processing and mixing with pollutants from other sources. OBJECTIVE: This was the fourth study by the National Environmental Respiratory Center to create a database for responses of animal models to combustion-derived pollutant mixtures, to identify causal pollutants-regardless of source. METHODS: F344 and SHR rats and A/J, C57BL/6, and BALB/c mice were exposed 6 h/day 7 days/week for 1 week to 6 months to three concentrations of a mixture simulating key components of "downwind" coal combustion emissions, to the highest concentration filtered to remove particulate matter (PM), or to clean air. Emissions from low-sulfur subbituminous coal were modified to create a mixture recommended by an expert workshop. Sulfur dioxide, nitrogen oxides, and PM were the dominant components. Nonanimal-derived PM mass concentrations of nominally 0, 100, 300, and 1000 µg/m(3) were mostly partially neutralized sulfate. RESULTS: Only 17 of 270 species-gender-time-outcome comparisons were significantly affected by exposure; some models showed no effects. There was strong evidence that PM participated meaningfully in only three responses. CONCLUSION: On a total mass or PM mass basis, this mixture was less toxic overall than diesel and gasoline exhausts or wood smoke. The largely sulfate PM contributed to few effects and was the sole cause of none. The study did not allow identification of causal pollutants, but the potential role of NOx in some effects is suggested by the literature.

Chronic carcinogenicity study of gasoline vapor condensate (GVC) and GVC containing methyl tertiary-butyl ether in F344 rats.

Chronic inhalation studies were conducted to compare the toxicity and potential carcinogenicity of evaporative emissions from unleaded gasoline (GVC) and gasoline containing the oxygenate methyl tertiary-butyl ether (MTBE; GMVC). The test materials were manufactured to mimic vapors people would be exposed to during refueling at gas stations. Fifty F344 rats per gender per exposure level per test article were exposed 6 h/d, 5 d/wk for 104 wk in whole body chambers. Target total vapor concentrations were 0, 2, 10, or 20 g/m³ for the control, low-, mid-, and high-level exposures, respectively. Endpoints included survival, body weights, clinical observations, organs weights, and histopathology. GVC and GMVC exerted no marked effects on survival or clinical observations and few effects on organ weights. Terminal body weights were reduced in all mid- and high-level GVC groups and high-level GMVC groups. The major proliferative lesions attributable to gasoline exposure with or without MTBE were renal tubule adenomas and carcinomas in male rats. GMV exposure led to elevated testicular mesothelioma incidence and an increased trend for thyroid carcinomas in males. GVMC inhalation caused an increased trend for testicular tumors with exposure concentration. Mid- and high-level exposures of GVC and GMVC led to elevated incidences of nasal respiratory epithelial degeneration. Overall, in these chronic studies conducted under identical conditions, the health effects in F344 rats following 2 yr of GVC or GMVC exposure were comparable in the production of renal adenomas and carcinomas in male rats and similar in other endpoints.

Levofloxacin cures experimental pneumonic plague in African green monkeys.

BACKGROUND: Yersinia pestis, the agent of plague, is considered a potential bioweapon due to rapid lethality when delivered as an aerosol. Levofloxacin was tested for primary pneumonic plague treatment in a nonhuman primate model mimicking human disease. METHODS AND RESULTS: Twenty-four African Green monkeys (AGMs, Chlorocebus aethiops) were challenged via head-only aerosol inhalation with 3-145 (mean = 65) 50% lethal (LD(50)) doses of Y. pestis strain CO92. Telemetered body temperature >39 °C initiated intravenous infusions to seven 5% dextrose controls or 17 levofloxacin treated animals. Levofloxacin was administered as a "humanized" dose regimen of alternating 8 mg/kg and 2 mg/kg 30-min infusions every 24-h, continuing until animal death or 20 total infusions, followed by 14 days of observation. Fever appeared at 53-165 h and radiographs found multilobar pneumonia in all exposed animals. All control animals died of severe pneumonic plague within five days of aerosol exposure. All 16 animals infused with levofloxacin for 10 days survived. Levofloxacin treatment abolished bacteremia within 24 h in animals with confirmed pre-infusion bacteremia, and reduced tachypnea and leukocytosis but not fever during the first 2 days of infusions. CONCLUSION: Levofloxacin cures established pneumonic plague when treatment is initiated after the onset of fever in the lethal aerosol-challenged AGM nonhuman primate model, and can be considered for treatment of other forms of plague. Levofloxacin may also be considered for primary presumptive-use, multi-agent antibiotic in bioterrorism events prior to identification of the pathogen.

Carcinogenic interactions between a single inhalation of 239PuO2 and chronic exposure to cigarette smoke in rats.

Rats were exposed once by inhalation to plutonium-239 dioxide ((239)PuO(2)), resulting in chronic alpha-particle irradiation of the lung, and exposed chronically to cigarette smoke to examine carcinogenic interactions between the two exposures. F344 rats were exposed to (239)PuO(2) to achieve an initial lung burden of 0.5 kBq and then exposed 6 h/day, 5 days/week to cigarette smoke at 100 or 250 mg particulate matter/m(3) for up to 30 months. Exposure to cigarette smoke increased the cumulative radiation dose to lung by slowing the clearance of (239)PuO(2). (239)PuO(2) alone did not affect survival, but the higher cigarette smoke exposure shortened survival in females. Combined exposure to (239)PuO(2) and cigarette smoke acted synergistically to shorten survival in both genders. The combined effects of cigarette smoke and (239)PuO(2) were approximately additive for lung hyperplasia and adenomas but were strongly synergistic for carcinomas. Differences between observed incidences and incidences predicted by survival-adjusted models accounting for increased radiation dose revealed a substantial component of synergy for carcinomas above that attributable to the radiation dose effect. The synergy for malignant lung tumors is consistent with findings from uranium miners and nuclear weapons production workers. These results bolster confidence in the epidemiological findings and have implications for risk assessment.

Interstitial myocardial fibrosis in a captive chimpanzee (Pan troglodytes) population.

The clinical and necropsy records of 36 (25 male and 11 female) chimpanzees age 10 to 40 y old that died over a 6-y period (2001 to 2006) were reviewed. All animals had annual physical exams that included electrocardiograms and serial blood pressures. Nine of the 36 animals had a complete cardiac evaluation by a board certified veterinary cardiologist, and 7 of the 36 animals (19%) were diagnosed with some form of cardiomyopathy. Systemic hypertension was noted in 3 cases. Cardiac arrhythmias (ventricular ectopy) were seen in 15 (12 male and 3 female) of the 36 animals (42%). Sudden cardiac death (SCD) occurred in 13 (11 male and 2 female) chimps (36%) and was the leading cause of death (n = 13), followed by renal failure (n = 9) and septicemia (n = 3). Histologic examination of the hearts revealed interstitial myocardial fibrosis (IMF) in 29 chimpanzees (81%), and all of the animals that died suddenly due to cardiac causes had IMF to varying degrees. More data will be needed to identify the possible causes of IMF in captive chimpanzees, and IMF may be associated with arrhythmias and SCD in these animals.

A review of the histopathology of cigarette smoke-induced lung cancer in rats and mice.

In the past several years an increased number of lung tumors has been reported in laboratory studies of rats and mice after lifetime exposure to mainstream cigarette smoke. Proliferative epithelial lesions are present in the lungs of both species and are apparent antecedent lesions to benign and malignant tumors. Both species have alveolar epithelia hyperplasia, alveolar adenomas, and alveolar carcinomas. The incidence of all three are more in the rats. In addition, mice also have bronchiolar epithelial hyperplasia and bronchial papillomas not found in rats. Rats have a low incidence of squamous cyst that is not found in mice. Lung tumors in rats and mice are found at the end of the life span and rarely metastasize. The characteristics of the lung tumors, and the proliferative changes associated with the tumors, are important in helping understand the mechanisms of lung cancer induction. These studies in rats and mice allow new approaches to the study of cigarette smoke-induced changes in the lung.

Lung toxicity of ambient particulate matter from southeastern U.S. sites with different contributing sources: relationships between composition and effects.

BACKGROUND: Exposure to air pollution and, more specifically, particulate matter (PM) is associated with adverse health effects. However, the specific PM characteristics responsible for biological effects have not been defined. OBJECTIVES: In this project we examined the composition, sources, and relative toxicity of samples of PM with aerodynamic diameter