Protective Effect of Inflammasome Activation by Hydrogen Peroxide in a Mouse Model of Septic Shock.

OBJECTIVES: To study the effect of a lack of antioxidant defenses during lethal pneumonia induced by Klebsiella pneumonia, compared to wild-type mice. SETTING: Laboratory experiments. SUBJECTS: C57Bl6 and glutathione peroxidase 1 knockout mice. INTERVENTION: Murine acute pneumonia model induced by Klebsiella pneumonia. MEASUREMENTS AND MAIN RESULTS: We show here that despite a lack of one of the major antioxidant defense enzymes, glutathione peroxidase 1 knockout mice are protected during lethal pneumonia induced by Klebsiella pneumonia, compared to wild-type mice. Furthermore, this protective effect was suppressed when antioxidant defenses were restored. Infected glutathione peroxidase 1 mice showed an early and significant, albeit transient, increase in the activity of the NOD-like receptor family, pyrin domain containing 3 inflammasome when compared with wild-type mice. The key role of the NOD-like receptor family, pyrin domain containing 3 inflammasome during acute pneumonia was confirmed in vivo when the protective effect was suppressed by treating glutathione peroxidase 1 mice with an interleukin-1 receptor antagonist. Additionally we report, in vitro, that increased concentrations of active caspase-1 and interleukin-1ß are related to an increased concentration of hydrogen peroxide in bacterially infected glutathione peroxidase 1 macrophages and that restoring hydrogen peroxide antioxidant defenses suppressed this effect. CONCLUSIONS: Our findings demonstrate that, contrary to current thinking, an early intervention targeting NOD-like receptor family, pyrin domain containing 3 inflammasome activity induces a timely and efficient activation of the innate immune response during acute infection. Our findings also demonstrate a role for hydrogen peroxide in the mechanisms tightly regulating NOD-like receptor family, pyrin domain containing 3 activation.

Predicting Disease Severity and Viral Spread of H5N1 Influenza Virus in Ferrets in the Context of Natural Exposure Routes.

UNLABELLED: Although avian H5N1 influenza virus has yet to develop the capacity for human-to-human spread, the severity of the rare cases of human infection has warranted intensive follow-up of potentially exposed individuals that may require antiviral prophylaxis. For countries where antiviral drugs are limited, the World Health Organization (WHO) has developed a risk categorization for different levels of exposure to environmental, poultry, or human sources of infection. While these take into account the infection source, they do not account for the likely mode of virus entry that the individual may have experienced from that source and how this could affect the disease outcome. Knowledge of the kinetics and spread of virus after natural routes of exposure may further inform the risk of infection, as well as the likely disease severity. Using the ferret model of H5N1 infection, we compared the commonly used but artificial inoculation method that saturates the total respiratory tract (TRT) with virus to upper respiratory tract (URT) and oral routes of delivery, those likely to be encountered by humans in nature. We show that there was no statistically significant difference in survival rate with the different routes of infection, but the disease characteristics were somewhat different. Following URT infection, viral spread to systemic organs was comparatively delayed and more focal than after TRT infection. By both routes, severe disease was associated with early viremia and central nervous system infection. After oral exposure to the virus, mild infections were common suggesting consumption of virus-contaminated liquids may be associated with seroconversion in the absence of severe disease. IMPORTANCE: Risks for human H5N1 infection include direct contact with infected birds and frequenting contaminated environments. We used H5N1 ferret infection models to show that breathing in the virus was more likely to produce clinical infection than swallowing contaminated liquid. We also showed that virus could spread from the respiratory tract to the brain, which was associated with end-stage disease, and very early viremia provided a marker for this. With upper respiratory tract exposure, infection of the brain was common but hard to detect, suggesting that human neurological infections might be typically undetected at autopsy. However, viral spread to systemic sites was slower after exposure to virus by this route than when virus was additionally delivered to the lungs, providing a better therapeutic window. In addition to exposure history, early parameters of infection, such as viremia, could help prioritize antiviral treatments for patients most at risk of succumbing to infection.

Subclinical infection without encephalitis in mice following intranasal exposure to Nipah virus-Malaysia and Nipah virus-Bangladesh.

BACKGROUND: Nipah virus and Hendra virus are closely related and following natural or experimental exposure induce similar clinical disease. In humans, encephalitis is the most serious outcome of infection and, hitherto, research into the pathogenesis of henipavirus encephalitis has been limited by the lack of a suitable model. Recently we reported a wild-type mouse model of Hendra virus (HeV) encephalitis that should facilitate detailed investigations of its neuropathogenesis, including mechanisms of disease recrudescence. In this study we investigated the possibility of developing a similar model of Nipah virus encephalitis. FINDINGS: Aged and young adult wild type mice did not develop clinical disease including encephalitis following intranasal exposure to either the Malaysia (NiV-MY) or Bangladesh (NiV-BD) strains of Nipah virus. However viral RNA was detected in lung tissue of mice at euthanasia (21 days following exposure) accompanied by a non-neutralizing antibody response. In a subsequent time course trial this viral RNA was shown to be reflective of an earlier self-limiting and subclinical lower respiratory tract infection through successful virus re-isolation and antigen detection in lung. There was no evidence for viremia or infection of other organs, including brain. CONCLUSIONS: Mice develop a subclinical self-limiting lower respiratory tract infection but not encephalitis following intranasal exposure to NiV-BD or NiV-MY. These results contrast with those reported for HeV under similar exposure conditions in mice, demonstrating a significant biological difference in host clinical response to exposure with these viruses. This finding provides a new platform from which to explore the viral and/or host factors that determine the neuroinvasive ability of henipaviruses.

Vaccination of ferrets with a recombinant G glycoprotein subunit vaccine provides protection against Nipah virus disease for over 12 months.

BACKGROUND: Nipah virus (NiV) is a zoonotic virus belonging to the henipavirus genus in the family Paramyxoviridae. Since NiV was first identified in 1999, outbreaks have continued to occur in humans in Bangladesh and India on an almost annual basis with case fatality rates reported between 40% and 100%. METHODS: Ferrets were vaccinated with 4, 20 or 100 µg HeVsG formulated with the human use approved adjuvant, CpG, in a prime-boost regime. One half of the ferrets were exposed to NiV at 20 days post boost vaccination and the other at 434 days post vaccination. The presence of virus or viral genome was assessed in ferret fluids and tissues using real-time PCR, virus isolation, histopathology, and immunohistochemistry; serology was also carried out. Non-immunised ferrets were also exposed to virus to confirm the pathogenicity of the inoculum. RESULTS: Ferrets exposed to Nipah virus 20 days post vaccination remained clinically healthy. Virus or viral genome was not detected in any tissues or fluids of the vaccinated ferrets; lesions and antigen were not identified on immunohistological examination of tissues; and there was no increase in antibody titre during the observation period, consistent with failure of virus replication. Of the ferrets challenged 434 days post vaccination, all five remained well throughout the study period; viral genome - but not virus - was recovered from nasal secretions of one ferret given 20 µg HeVsG and bronchial lymph nodes of the other. There was no increase in antibody titre during the observation period, consistent with lack of stimulation of a humoral memory response. CONCLUSIONS: We have previously shown that ferrets vaccinated with 4, 20 or 100 µg HeVsG formulated with CpG adjuvant, which is currently in several human clinical trials, were protected from HeV disease. Here we show, under similar conditions of use, that the vaccine also provides protection against NiV-induced disease. Such protection persists for at least 12 months post-vaccination, with data supporting only localised and self-limiting virus replication in 2 of 5 animals. These results augur well for acceptability of the vaccine to industry.

A new model for Hendra virus encephalitis in the mouse.

Hendra virus (HeV) infection in humans is characterized by an influenza like illness, which may progress to pneumonia or encephalitis and lead to death. The pathogenesis of HeV infection is poorly understood, and the lack of a mouse model has limited the opportunities for pathogenetic research. In this project we reassessed the role of mice as an animal model for HeV infection and found that mice are susceptible to HeV infection after intranasal exposure, with aged mice reliably developing encephalitic disease. We propose an anterograde route of neuroinvasion to the brain, possibly along olfactory nerves. This is supported by evidence for the development of encephalitis in the absence of viremia and the sequential distribution of viral antigen along pathways of olfaction in the brain of intranasally challenged animals. In our studies mice developed transient lower respiratory tract infection without progressing to viremia and systemic vasculitis that is common to other animal models. These studies report a new animal model of HeV encephalitis that will allow more detailed studies of the neuropathogenesis of HeV infection, particularly the mode of viral spread and possible sequestration within the central nervous system; investigation of mechanisms that moderate the development of viremia and systemic disease; and inform the development of improved treatment options for human patients.

Antitumor activities and on-target toxicities mediated by a TRAIL receptor agonist following cotreatment with panobinostat.

The recent development of novel targeted anticancer therapeutics such as histone deacetylase inhibitors (HDACi) and activators of the TRAIL pathway provide opportunities for the introduction of new treatment regimens in oncology. HDACi and recombinant TRAIL or agonistic anti-TRAIL receptor antibodies have been shown to induce synergistic tumor cell apoptosis and some therapeutic activity in vivo. Herein, we have used syngeneic preclinical models of human solid cancers to demonstrate that the HDACi panobinostat can sensitize tumor cells to apoptosis mediated by the anti-mouse TRAIL receptor antibody MD5-1. We demonstrate that the combination of panobinostat and MD5-1 can eradicate tumors grown subcutaneously and orthotopically in immunocompetent mice, while single agent treatment has minimal effect. However, escalation of the dose of panobinostat to enhance antitumor activity resulted in on-target MD5-1-mediated gastrointestinal toxicities that were fatal to the treated mice. Studies performed in mice with knockout of the TRAIL receptor showed that these mice could tolerate doses of the panobinostat/MD5-1 combination that were lethal in wild type mice resulting in superior tumor clearance. Given that clinical studies using HDACi and activators of the TRAIL pathway have been initiated, our preclinical data highlight the potential toxicities that could limit the use of such a treatment regimen. Our studies also demonstrate the power of using syngeneic in vivo tumor models as physiologically relevant preclinical systems to test the antitumor effects and identify potential side effects of novel anticancer regimens.

Notch1 functions as a tumor suppressor in a model of K-ras-induced pancreatic ductal adenocarcinoma.

K-ras is the most commonly mutated oncogene in pancreatic cancer and its activation in murine models is sufficient to recapitulate the spectrum of lesions seen in human pancreatic ductal adenocarcinoma (PDAC). Recent studies suggest that Notch receptor signaling becomes reactivated in a subset of PDACs, leading to the hypothesis that Notch1 functions as an oncogene in this setting. To determine whether Notch1 is required for K-ras-induced tumorigenesis, we used a mouse model in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in the pancreas. Unexpectedly, the loss of Notch1 in this model resulted in increased tumor incidence and progression, implying that Notch1 can function as a tumor suppressor gene in PDAC.

Evaluation of risk factors for morbidity and mortality after pylorectomy and gastroduodenostomy in dogs.

OBJECTIVES: To (1) identify and describe the type and frequency of postoperative complications after pylorectomy and gastroduodenostomy in dogs and (2) identify preoperative and intraoperative risk factors, including the presence of neoplasia, prognostic for patient mortality after surgery. STUDY DESIGN: Case series. ANIMALS: Dogs (n=24) treated by pylorectomy and gastroduodenostomy. METHODS: Medical records (2000-2007) for 2 teaching hospitals of dogs treated that had pylorectomy and gastroduodenostomy were reviewed. Pre-, intra-, and postoperative data were obtained from the medical record. RESULTS: Of the 24 dogs, 75% survived 14 days, but 10 (41%) died by 3 months. Overall median survival time (MST) was 578 days. On log-rank univariate analysis, preoperative weight loss (P=.001) and malignant neoplasia (P=.01) were associated with decreased survival time. Dogs with malignant neoplasia had a MST of 33 days. Common postoperative morbidity included hypoalbuminemia (62.5%) and anemia (58.3%). CONCLUSIONS: Pylorectomy with gastroduodenostomy has a good short-term outcome but long-term survival time is poor in dogs with malignant neoplasia. CLINICAL RELEVANCE: Overall, most dogs treated with pylorectomy and gastroduodenostomy survived the postoperative period; however, preoperative weight loss and malignant neoplasia were associated with decreased survival time. Because dogs with malignant neoplasia have markedly shortened survival times, pertinent preoperative, diagnostics steps should be exhausted to identify underlying neoplasia.

Contrast harmonic ultrasonography of splenic masses and associated liver nodules in dogs.

OBJECTIVE: To determine whether contrast harmonic ultrasonography (CHUS) can be used in dogs to distinguish splenic hemangiosarcoma from hematoma and to accurately detect and characterize liver nodules. DESIGN: Cross-sectional study. ANIMALS: 20 dogs with a splenic mass. PROCEDURES: Routine abdominal ultrasonography was followed by CHUS of hepatic and splenic lesions. Qualitative evaluation included location, enhancement pattern, and vascularity of lesions. Quantitative evaluation included peak mean pixel intensity, interval to peak intensity, area under the curve (spleen), and liver-to-lesion intensity ratio (liver). Histologic findings were compared with CHUS lesion characteristics. RESULTS: Histologic evaluation of the spleen was performed in 19 dogs, resulting in diagnoses of hemangiosarcoma (n=11), hematoma (7), and undifferentiated sarcoma (1). Benign and malignant processes in the spleen were indistinguishable via CHUS. Histologic evaluation of the liver was performed in 18 dogs, resulting in a diagnosis of hemangiosarcoma in 5 dogs. None of the dogs with splenic hematomas had evidence of hepatic lesions by means of conventional or contrast ultrasonography, and none had histologic evidence of liver metastases. In 3 of 18 dogs, isoenhancing liver nodules were detected and all were histologically benign. Five dogs had liver nodules that remained hypoechoic after contrast agent was injected; all had histologic evidence of metastatic hemangiosarcoma. Results of CHUS were used to characterize hepatic metastases with 100% sensitivity and specificity. CONCLUSIONS AND CLINICAL RELEVANCE: Contrast harmonic ultrasonography was a noninvasive and accurate means of differentiating metastatic versus benign hepatic disease in dogs with splenic hemangiosarcoma but was not useful in distinguishing splenic hemangiosarcoma from hematoma.

Physiologic alterations in the murine model after nasal fungal antigenic exposure.

OBJECTIVE: To determine whether a recently developed murine model of fungus-induced sinonasal inflammation demonstrated alterations in ciliary activity and expression of inflammatory cytokines. STUDY DESIGN: A prospective randomized controlled study of rhinosinusitis after fungal antigenic sensitization was performed with intraperitoneal aspergillus antigen injection followed by intranasal antigen challenge for 4 weeks. Saline solution was used in a parallel fashion for control animals. SUBJECTS AND METHODS: Six mice were used to validate the model. Additional 15 mice were used for ciliary beat frequency (CBF) analysis and cytokine expression with multiplex technology. Mean values for degree of inflammation, secretory hyperplasia, CBF, and cytokine expression were compared. RESULTS: Histologic analyses demonstrated dense chronic inflammation in aspergillus-challenged animals versus sparse inflammatory cells in controls. Significant differences in mean of aspergillus-challenged versus control animals were observed in degree of inflammation (P < 0.01), secretory hyperplasia (P < 0.01), CBF (P < 0.00002), IL-1alpha (P < 0.0002), IL-1beta (P < 0.0003), IL-4 (P < 0.02), TNF-alpha (P < 0.02), and RANTES (P < 0.01). CONCLUSION: Alteration in baseline CBF accompanied by increased expression of specific inflammatory cytokines was observed in aspergillus-challenged mice.

Evaluation of rapid staining techniques for cytologic diagnosis of intracranial lesions.

OBJECTIVE: To evaluate 4 rapid supravital stains and 3 preparation techniques for use in the intraoperative diagnosis of intracranial lesions. ANIMALS: 10 dogs and 1 cat euthanatized for intracranial lesions. PROCEDURE: Specimens were taken from lesions and slides prepared, using 3 techniques: touch impression, medium-pressure impression, or smear preparation. Preparations were then stained with 4 stains: modified Wright stain, May-Grünwald-Giemsa, toluidine blue, and zynostain and examined in a blinded randomized fashion. Cytologic diagnosis was compared with histopathologic diagnosis and classified on the basis of identification of the pathologic process and specific diagnosis into the following categories: complete correlation, partial correlation, or no correlation. RESULTS: An overall diagnostic accuracy of 81% (107/132) was achieved on the basis of a combination of partial and complete correlation. Of the stains examined, modified Wright stain appeared to be most accurate, with complete correlation in 17 of 33 (52%) specimens and partial correlation in 12 of 33 (36%) specimens. Of the preparation methods, touch preparation and smear preparation provided the most accurate results, with an overall diagnostic accuracy of 82% (36/44) for both methods. However, smear preparations appeared to be of greater diagnostic value, with fewer nondiagnostic specimens, compared with touch preparations. CONCLUSIONS AND CLINICAL RELEVANCE: Cytologic preparations provide a useful diagnostic tool for the intraoperative diagnosis of intracranial lesions. All stains examined yielded promising results, the most accurate of which appeared to be the modified Wright stain. The smear preparation appeared to be the preparation method of greatest diagnostic value.