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Abstract Background A major outbreak of bloody diarrhea associated with Shiga toxin-producing Escherichia coli O104:H4 occurred early in 2011, to which an unusual number of hemolytic uremic syndrome cases were linked. Due to limited information regarding pathogenesis and/or virulence properties of this particular serotype, we investigated the contribution of the aerobactin iron transport system during in vitro and in vivo conditions. Results A bioluminescent reporter construct was used to perform real-time monitoring of E. coli O104:H4 in a mouse model of infection. We verified that our reporter strain maintained characteristics and growth kinetics that were similar to those of the wild-type E. coli strain. We found that the intestinal cecum of ICR (CD-1) mice was colonized by O104:H4, with bacteria persisting for up to 7 days after intragastric inoculation. MALDI-TOF analysis of heat-extracted proteins was performed to identify putative surface-exposed virulence determinants. A protein with a high similarity to the aerobactin iron receptor was identified and further demonstrated to be up-regulated in E. coli O104:H4 when grown on MacConkey agar or during iron-depleted conditions. Because the aerobactin iron acquisition system is a key virulence factor in Enterobacteriaceae, an isogenic aerobactin receptor (iutA) mutant was created and its intestinal fitness assessed in the murine model. We demonstrated that the aerobactin mutant was out-competed by the wild-type E. coli O104:H4 during in vivo competition experiments, and the mutant was unable to persist in the cecum. Conclusion Our findings demonstrate that bioluminescent imaging is a useful tool to monitor E. coli O104:H4 colonization properties, and the murine model can become a rapid way to evaluate bacterial factors associated with fitness and/or colonization during E. coli O104:H4 infections.

Background: Environmental tobacco smoke (ETS) is a known risk factor for severe respiratory syncytial virus (RSV) infections, yet the mechanisms of ETS/RSV comorbidity are largely unknown. Cystathionine γ-lyase regulates important physiological functions of the respiratory tract., Methods: We used mice genetically deficient in the cystathionine γ-lyase enzyme (CSE), the major H 2 S-generating enzyme in the lung to determine the contribution of H 2 S to airway disease in response to side-stream tobacco smoke (TS), and to TS/RSV co-exposure., Results: Following a 2-week period of exposure to TS, CSE-deficient mice (KO) showed a dramatic increase in airway hyperresponsiveness (AHR) to methacholine challenge, and greater airway cellular inflammation, compared with wild-type (WT) mice. TS-exposed CSE KO mice that were subsequently infected with RSV exhibited a more severe clinical disease, airway obstruction and AHR, enhanced viral replication, and lung inflammation, compared with TS-exposed RSV-infected WT mice. TS-exposed RSV-infected CSE KO mice had also a significant increase in the number of neutrophils in bronchoalveolar lavage fluid and increased levels of inflammatory cytokines and chemokines., Conclusion: This study demonstrates the critical contribution of the H 2 S-generating pathway to airway reactivity and disease following exposure to ETS alone or in combination with RSV infection.

The pathogenesis of respiratory syncytial virus (RSV) infections is characterized by lower airway obstruction driven at great extent by the exuberant production of inflammatory cytokines. We have previously shown that RSV infection in vitro and in vivo results in production of reactive oxygen species along with reduction in the expression of antioxidant enzymes (AOEs), which are involved in maintaining the cellular oxidant-antioxidant balance. These events were associated with the concomitant reduction in nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor that controls AOE expression. The objective of the current study was to establish the role of Nrf2 in shaping innate immune responses, clinical disease, airway inflammation, and viral replication in established experimental models of intranasal RSV and human metapneumovirus (hMPV) infections, by employing mice genetically deficient for the Nrf2 gene. Compared to control wild type (WT), mice genetically deficient in Nrf2 (Nrf2 KO) developed enhanced clinical disease, airway inflammation and pathology, and significantly greater lung viral titers following experimental infection with either RSV or hMPV. In particular, compared to control mice, RSV-infected Nrf2 KO mice lost more body weight and had increased airway obstruction at time points characterized by a remarkable increase in inflammatory cytokines and airway neutrophilia. Airway levels of AOEs and enzymes that regulate synthesis of the endogenous hydrogen sulfide (H 2 S) pathway, which we showed to play an important antiviral function, were also decreased in RSV-infected Nrf2 KO compared to WT. In conclusion, these results suggest that Nrf2 is a critical regulator of innate, inflammatory, and disease-associated responses in the airways of mice infected with viruses that are members of the Pneumoviridae family. Importantly, the results of this study suggest that Nrf2-dependent genes, including those controlling the cellular antioxidant and H 2 S-generating enzymes and cytokines can affect several aspects of the antiviral response, such as airway neutrophilia, clinical disease, airway obstruction, and viral replication.

Burkholderia pseudomallei , the etiologic agent of melioidosis, causes severe disease in humans and animals. Diagnosis and treatment of melioidosis can be challenging, and no licensed vaccines currently exist. Several studies have shown that this pathogen expresses a variety of structurally conserved protective antigens that include cell surface polysaccharides and cell-associated and cell-secreted proteins. Based on those findings, such antigens have become important components of the subunit vaccine candidates that we are currently developing. In the present study, the 6-deoxyheptan capsular polysaccharide (CPS) from B. pseudomallei was purified, chemically activated, and covalently linked to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce CPS-CRM197. Additionally, tandem nickel-cobalt affinity chromatography was used to prepare highly purified recombinant B. pseudomallei Hcp1 and TssM proteins. Immunization of C57BL/6 mice with CPS-CRM197 produced high-titer IgG and opsonizing antibody responses against the CPS component of the glycoconjugate, while immunization with Hcp1 and TssM produced high-titer IgG and robust gamma interferon-secreting T cell responses against the proteins. Extending upon these studies, we found that when mice were vaccinated with a combination of CPS-CRM197 and Hcp1, 100% of the mice survived a lethal inhalational challenge with B. pseudomallei Remarkably, 70% of the survivors had no culturable bacteria in their lungs, livers, or spleens, indicating that the vaccine formulation had generated sterilizing immune responses. Collectively, these studies help to better establish surrogates of antigen-induced immunity against B. pseudomallei as well as provide valuable insights toward the development of a safe, affordable, and effective melioidosis vaccine., (Copyright © 2017 Burtnick et al.)

Hydrogen sulfide (H 2 S) is an endogenous gaseous transmitter whose role in the pathophysiology of several lung diseases has been increasingly appreciated. Our recent studies in vitro have shown, we believe for the first time, that H 2 S has an important antiviral and antiinflammatory activity in respiratory syncytial virus (RSV) infection, the leading cause of bronchiolitis and viral pneumonia in children. Our objective was to evaluate the therapeutic potential of GYY4137, a novel slow-releasing H 2 S donor, for the prevention and treatment of RSV-induced lung disease, as well as to investigate the role of endogenous H 2 S in a mouse model of RSV infection. Ten- to 12-week-old BALB/c mice treated with GYY4137, or C57BL/6J mice genetically deficient in the cystathionine γ-lyase enzyme, the major H 2 S-generating enzyme in the lung, were infected with RSV and assessed for viral replication, clinical disease, airway hyperresponsiveness, and inflammatory responses. Our results show that intranasal delivery of GYY4137 to RSV-infected mice significantly reduced viral replication and markedly improved clinical disease parameters and pulmonary dysfunction compared with the results in vehicle-treated control mice. The protective effect of the H 2 S donor was associated with a significant reduction of viral-induced proinflammatory mediators and lung cellular infiltrates. Furthermore, cystathionine γ-lyase-deficient mice showed significantly enhanced RSV-induced lung disease and viral replication compared with wild-type animals. Overall, our results indicate that H 2 S exerts a novel antiviral and antiinflammatory activity in the context of RSV infection and represent a potential novel pharmacological approach for ameliorating virus-induced lung disease.

Background: Neonatal respiratory distress syndrome in preterm infants is a leading cause of neonatal death. Pulmonary insufficiency-related infant mortality rates have improved with antenatal glucocorticoid treatment and neonatal surfactant replacement. However, the mechanism of glucocorticoid-promoted fetal lung maturation is not understood fully, despite decades of clinical use. We previously have shown that genetic deletion of Erk3 in mice results in growth restriction, cyanosis, and early neonatal lethality because of pulmonary immaturity and respiratory distress. Recently, we demonstrated that the addition of postnatal surfactant administration to antenatal dexamethasone treatment resulted in enhanced survival of neonatal Erk3-null mice., Objective: To better understand the molecular underpinnings of corticosteroid-mediated lung maturation, we used high-throughput transcriptomic and high-resolution morphologic analysis of the murine fetal lung. We sought to examine the alterations in fetal lung structure and function that are associated with neonatal respiratory distress and antenatal glucocorticoid treatment., Study Design: Dexamethasone (0.4 mg/kg) or saline solution was administered to pregnant dams on embryonic days 16.5 and 17.5. Fetal lungs were collected and analyzed by microCT and RNA-seq for differential gene expression and pathway interactions with genotype and treatment. Results from transcriptomic analysis guided further investigation of candidate genes with the use of immunostaining in murine and human fetal lung tissue., Results: Erk3(-/-) mice exhibited atelectasis with decreased overall porosity and saccular space relative to wild type, which was ameliorated by glucocorticoid treatment. Of 596 differentially expressed genes (q < 0.05) that were detected by RNA-seq, pathway analysis revealed 36 genes (q < 0.05) interacting with dexamethasone, several with roles in lung development, which included corticotropin-releasing hormone and surfactant protein B. Corticotropin-releasing hormone protein was detected in wild-type and Erk3(-/-) lungs at E14.5, with significantly temporally altered expression through embryonic day 18.5. Antenatal dexamethasone attenuated corticotropin-releasing hormone at embryonic day 18.5 in both wild-type and Erk3(-/-) lungs (0.56-fold and 0.67-fold; P < .001). Wild type mice responded to glucocorticoid administration with increased pulmonary surfactant protein B (P = .003). In contrast, dexamethasone treatment in Erk3(-/-) mice resulted in decreased surfactant protein B (P = .012). In human validation studies, we confirmed that corticotropin-releasing hormone protein is present in the fetal lung at 18 weeks of gestation and increases in expression with progression towards viability (22 weeks of gestation; P < .01)., Conclusion: Characterization of whole transcriptome gene expression revealed glucocorticoid-mediated regulation of corticotropin-releasing hormone and surfactant protein B via Erk3-independent and -dependent mechanisms, respectively. We demonstrated for the first time the expression and temporal regulation of corticotropin-releasing hormone protein in midtrimester human fetal lung. This unique model allows the effects of corticosteroids on fetal pulmonary morphologic condition to be distinguished from functional gene pathway regulation. These findings implicate Erk3 as a potentially important molecular mediator of antenatal glucocorticoid action in promoting surfactant protein production in the preterm neonatal lung and expanding our understanding of key mechanisms of clinical therapy to improve neonatal survival., Competing Interests: The authors report no conflict of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Burkholderia mallei is the causative agent of glanders, an incapacitating disease with high mortality rates in respiratory cases. Its endemicity and ineffective treatment options emphasize its public health threat and highlight the need for a vaccine. Live attenuated vaccines are considered the most viable vaccine strategy for Burkholderia, but single-gene-deletion mutants have not provided complete protection. In this study, we constructed the select-agent-excluded B. mallei ΔtonB Δhcp1 (CLH001) vaccine strain and investigated its ability to protect against acute respiratory glanders. Here we show that CLH001 is attenuated, safe, and effective at protecting against lethal B. mallei challenge. Intranasal administration of CLH001 to BALB/c and NOD SCID gamma (NSG) mice resulted in complete survival without detectable colonization or abnormal organ histopathology. Additionally, BALB/c mice intranasally immunized with CLH001 in a prime/boost regimen were fully protected against lethal challenge with the B. mallei lux (CSM001) wild-type strain., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objective: Uterine leiomyomas represent a common gynecologic problem with no satisfactory long-term medical treatment. The purpose of this study is to examine the effects of simvastatin on uterine leiomyoma, both in vitro and in vivo., Study Design: This is a laboratory-based experimental study. For in vitro studies, we used human and rat leiomyoma cells. For in vivo studies, we used immunodeficient mice supplemented with estrogen/progesterone pellets xenografted with human leiomyoma tissue explant., Results: For in vitro studies, cells were treated with different concentrations of simvastatin for 48 hours. Simvastatin induced dose-dependent apoptosis in leiomyoma cells as measured by a fluorometric caspase-3 activity assay, and inhibited proliferation as demonstrated by an (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (both were significant at 5 and 10 μM). In addition, simvastatin decreased Akt signaling pathway phosphorylation as examined using Western blot analysis. For in vivo studies, animals were treated for 28 days with simvastatin (20 μg/gm body weight/day) vs vehicle control. The treatment inhibited tumor growth as measured weekly using calipers and/ or ultrasound (P < .01). Finally, simvastatin decreased expression of the proliferation marker Ki67 in xenograft tumor tissue as examined by immunohistochemistry (P = .02)., Conclusion: Simvastatin can be a promising treatment for uterine leiomyoma. Further studies, including pharmacokinetic and drug delivery studies, are required., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis., Methodology/principal Findings: Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 10(4) CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001., Conclusions/significance: Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.

Adherent-invasive Escherichia coli (AIEC) pathogroup isolates are a group of isolates from the intestinal mucosa of Crohn's disease patients that can invade intestinal epithelial cells (IECs) or macrophages and survive and/or replicate within. We have identified the ibeA gene in the genome of AIEC strain NRG857c and report the contribution of IbeA to the interaction of AIEC with IECs and macrophages and colonization of the mouse intestine. An ibeA deletion mutant strain (NRG857cΔibeA) was constructed, and the in vitro effect on AIEC adhesion and invasion of nonpolarized and polarized Caco-2 cells, the adhesion and transcytosis of M-like cells, the intracellular survival in THP-1 macrophages, and the contribution to intestinal colonization of the CD-1 murine model of infection were evaluated. A significant reduction in invasion was observed with the ibeA mutant in Caco-2 and M-like cells, whereas adhesion was not affected. Complementation of the mutant reestablished Caco-2 invasive phenotype to wild-type levels. Reduction in invasion did not significantly affect transcytosis through M-like cells at early time points. The absence of ibeA significantly affected AIEC intramacrophage survival up to 24 h postinfection. No significant changes associated with IbeA were found in AIEC colonization across the murine gastrointestinal tract, but a slight reduction of gamma interferon was observed in the ceca of mice infected with the ibeA mutant. In addition, a decrease in the pathology scores was observed in the ilea and ceca of mice infected with the ibeA mutant. Our data support the function of IbeA in the AIEC invasion process, macrophage survival, and inflammatory response in the murine intestine., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The chemokine CXCL1 and its receptor CXCR2 play a crucial role in host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. Dysregulation in this process has been implicated in collateral tissue damage causing disease. CXCL1 reversibly exists as monomers and dimers, and it has been proposed that distinct monomer and dimer activities and the monomer-dimer equilibrium regulate the neutrophil function. However, the molecular mechanisms linking the CXCL1/CXCR2 axis and the neutrophil 'beneficial' and 'destructive' phenotypes are not known. In this study, we characterized neutrophil trafficking and its consequence in the mouse lung by the CXCL1 wild type (WT), which exists as monomers and dimers, and by a nondissociating dimer. Whereas the WT, compared to the dimer, was more active at low doses, both the WT and the dimer elicited a large neutrophil efflux at high doses. Importantly, robust neutrophil recruitment elicited by the WT or dimer was not detrimental to lung tissue integrity and, further, could not be correlated to surface CXCR2 levels. We conclude that the CXCL1 monomer-dimer distribution and receptor interactions are highly coupled and regulate neutrophil trafficking and that injury in the context of disease is a consequence of inappropriate CXCR2 activation at the target tissue and not due to mechanical forces exerted by neutrophils during recruitment., (© 2015 S. Karger AG, Basel.)

Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are leading causes of upper and lower respiratory tract infections in young children and among elderly and immunocompromised patients. The pathogenesis of hMPV-induced lung disease is poorly understood. The lung macrophage population consists of alveolar macrophages (AMs) residing at the luminal surface of alveoli and interstitial macrophages present within the parenchymal lung interstitium. The involvement of AMs in innate immune responses to virus infections remains elusive. In this study, BALB/c mice depleted of AMs by intranasal instillation of dichloromethylene bisphosphonate (L-CL2MBP) liposomes were examined for disease, lung inflammation, and viral replication after infection with hMPV or RSV. hMPV-infected mice lacking AMs exhibited improved disease in terms of body weight loss, lung inflammation, airway obstruction, and hyperresponsiveness compared with AM-competent mice. AM depletion was associated with significantly reduced hMPV titers in the lungs, suggesting that hMPV required AMs for early entry and replication in the lung. In contrast, AM depletion in the context of RSV infection was characterized by an increase in viral replication, worsened disease, and inflammation, with increased airway neutrophils and inflammatory dendritic cells. Overall, lack of AMs resulted in a broad-spectrum disruption in type I IFN and certain inflammatory cytokine production, including TNF and IL-6, while causing a virus-specific alteration in the profile of several immunomodulatory cytokines, chemokines, and growth factors. Our study demonstrates that AMs have distinct roles in the context of human infections caused by members of the Paramyxoviridae family.

Background: Respiratory distress syndrome (RDS) persists as a prevalent cause of infant morbidity and mortality. We have previously demonstrated that deletion of Erk3 results in pulmonary immaturity and neonatal lethality. Using RNA sequencing, we identified corticotrophin releasing hormone (CRH) and surfactant protein B (SFTPB) as potential molecular mediators of Erk3-dependent lung maturation. In this study, we characterized the impact of antenatal glucocorticoids and postnatal surfactant on neonatal survival of Erk3 null mice., Methods: In a double crossover design, we administered dexamethasone (dex) or saline to pregnant dams during the saccular stage of lung development, followed by postnatal surfactant or saline via inhalation intubation. Survival was recorded, and detailed lung histological analysis and staining for CRH and SFTPB protein expression were performed., Results: Without treatment, Erk3 null pups die within 6 h of birth with reduced aerated space, impaired thinning of the alveolar septa, and abundant glycogen stores, as described in human RDS. The administration of dex and surfactant improved RDS-associated lethality of Erk3(-/-) pups and partially restored functional fetal lung maturation by accelerating the downregulation of pulmonary CRH and partially rescuing the production of SFTPB., Conclusion: These findings emphasize that Erk3 is integral to terminal differentiation of type II cells, SFTPB production, and fetal pulmonary maturity.

Melioidosis is an endemic disease caused by the bacterium Burkholderia pseudomallei. Concerns exist regarding B. pseudomallei use as a potential bio-threat agent causing persistent infections and typically manifesting as severe pneumonia capable of causing fatal bacteremia. Development of suitable therapeutics against melioidosis is complicated due to high degree of genetic and phenotypic variability among B. pseudomallei isolates and lack of data establishing commonly accepted strains for comparative studies. Further, the impact of strain variation on virulence, disease presentation, and mortality is not well understood. Therefore, this study evaluate and compare the virulence and disease progression of B. pseudomallei strains K96243 and HBPUB10303a, following aerosol challenge in a standardized BALB/c mouse model of infection. The natural history analysis of disease progression monitored conditions such as weight, body temperature, appearance, activity, bacteremia, organ and tissue colonization (pathological and histological analysis) and immunological responses. This study provides a detailed, direct comparison of infection with different B. pseudomallei strains and set up the basis for a standardized model useful to test different medical countermeasures against Burkholderia species. Further, this protocol serves as a guideline to standardize other bacterial aerosol models of infection or to define biomarkers of infectious processes caused by other intracellular pathogens.

Objective: To compare placental lesions for stillbirth cases and live birth controls in a population-based study., Methods: Pathologic examinations were performed on placentas from singleton pregnancies using a standard protocol. Data were analyzed overall and within gestational age groups at delivery., Results: Placentas from 518 stillbirths and 1,200 live births were studied. Single umbilical artery was present in 7.7% of stillbirths and 1.7% of live births, velamentous cord insertion was present in 5% of stillbirths and 1.1% of live births, diffuse terminal villous immaturity was present in 10.3% of stillbirths and 2.3% of live births, inflammation (eg, acute chorioamnionitis of placental membranes) was present in 30.4% of stillbirths and 12% of live births, vascular degenerative changes in chorionic plate were present in 55.7% of stillbirths and 0.5% of live births, retroplacental hematoma was present in 23.8% of stillbirths and 4.2% of live births, intraparenchymal thrombi was present in 19.7% of stillbirths and 13.3% of live births, parenchymal infarction was present in 10.9% of stillbirths and 4.4% of live births, fibrin deposition was present in 9.2% of stillbirths and 1.5% of live births, fetal vascular thrombi was present in 23% of stillbirths and 7% of live births, avascular villi was present in 7.6% of stillbirths and 2.0% of live births, and hydrops was present in 6.4% of stillbirths and 1.0% of live births. Among stillbirths, inflammation and retroplacental hematoma were more common in placentas from early deliveries, whereas thrombotic lesions were more common in later gestation. Inflammatory lesions were especially common in early live births., Conclusions: Placental lesions were highly associated with stillbirth compared with live births. All lesions associated with stillbirth were found in live births but often with variations by gestational age at delivery. Knowledge of lesion prevalence within gestational age groups in both stillbirths and live birth controls contributes to an understanding of the association between placental abnormality and stillbirth., Level of Evidence: II.

In order to determine the effects of pravastatin (Pra) on angiogenic and placental hypoxic imbalance in a model of preeclampsia induced by overexpression of soluble fms-like tyrosine kinase 1 (sFlt-1), we randomly allocated pregnant CD1 mice to injection with adenovirus-carrying sFlt-1 or mFc (control). The sFlt-1 group received either Pra (sFlt-1 + Pra) or water (sFlt-1). Mice were sacrificed at day 18, and serum levels of sFlt-1 and soluble endoglin (sEng) were measured. Placental expression of placental (PLGF) and vascular endothelial (VEGF) growth factors and other markers of angiogenesis and hypoxia were assayed. We observed that Pra treatment in sFlt-1 mice reduced sFlt-1 and sEng concentrations at day 18 to levels similar to control group. Placental PLGF and VEGF expression were upregulated, and markers of hypoxia downregulated to levels similar to control group. Hence, Pra prevents the rise in circulating antiangiogenic factors in a mouse model of preeclampsia. Statins may represent a novel approach to prevention of preeclampsia.

Background: Successful development of topical rectal microbicides requires preclinical evaluation in suitable large animal models. Our previous studies have demonstrated the benefits of high-resolution optical coherence tomography (OCT) to visualize subclinical microbicide toxicity in the sheep vagina. In the current study, we evaluated the potential application of colonoscopy and OCT to visualize and quantify the effects of topical products on sheep colorectal tissue, as assessed by advanced imaging techniques., Methods: Yearling virginal female sheep were treated rectally with a single 8-mL dose of 0.2% benzalkonium chloride (BZK) solution or phosphate-buffered saline control. Imaging was performed before and 30 minutes after treatment. Colonoscopy findings were evaluated based on mucosal disruption. Optical coherence tomography images were graded based on the integrity of the mucosal layer. Biopsies collected after treatment were evaluated by histology for validation of OCT scoring., Results: Mucosal disruption was observed by colonoscopy in BZK-treated animals, whereas none was present in controls. In contrast to colonoscopy, high-resolution in-depth OCT imaging provided visualization of the morphology of the mucosal layer and underlying muscularis, thus enabling detection of microscopic abnormalities. Noninvasive quantification of drug-induced injury after validation of the scoring system (categories 1, 2, 3) showed increased scores after treatment with BZK (P < 0.001), indicating mucosal injury., Conclusions: High-resolution OCT can be used as highly sensitive tool to evaluate rectal microbicide effects. Because the sheep rectum has both gross and microscopic similarities to the human, this model is a useful addition to current methods of rectal product toxicity.

Burkholderia mallei , the etiologic agent of glanders, are Category B select agents with biothreat potential, and yet effective therapeutic treatments are lacking. In this study, we showed that CpG administration increased survival, demonstrating protection in the murine glanders model. Bacterial recovery from infected lungs, liver and spleen was significantly reduced in CpG-treated animals as compared with non-treated mice. Reciprocally, lungs of CpG-treated infected animals were infiltrated with higher levels of neutrophils and inflammatory monocytes, as compared to control animals. Employing the B. mallei bioluminescent strain CSM001 and the Neutrophil-Specific Fluorescent Imaging Agent, bacterial dissemination and neutrophil trafficking were monitored in real-time using multimodal in vivo whole body imaging techniques. CpG-treatment increased recruitment of neutrophils to the lungs and reduced bioluminescent bacteria, correlating with decreased bacterial burden and increased protection against acute murine glanders. Our results indicate that protection of CpG-treated animals was associated with recruitment of neutrophils prior to infection and demonstrated, for the first time, simultaneous real time in vivo imaging of neutrophils and bacteria. This study provides experimental evidence supporting the importance of incorporating optimized in vivo imaging methods to monitor disease progression and to evaluate the efficacy of therapeutic treatment during bacterial infections.

The safety and immunogenicity of two authentic recombinant (ar) Rift Valley fever (RVF) viruses, one with a deletion in the NSs region of the S RNA segment (arMP-12ΔNSs16/198) and the other with a large deletion of the NSm gene in the pre Gn region of the M RNA segment (arMP-12ΔNSm21/384) of the RVF MP-12 vaccine virus were tested in crossbred ewes at 30-50 days of gestation. First, we evaluated the neutralizing antibody response, measured by plaque reduction neutralization (PRNT(80)), and clinical response of the two viruses in groups of four ewes each. The virus dose was 1×10(5)plaque forming units (PFU). Control groups of four ewes each were also inoculated with a similar dose of RVF MP-12 or the parent recombinant virus (arMP-12). Neutralizing antibody was first detected in 3 of 4 animals inoculated with arMP-12ΔNSm21/384 on Day 5 post inoculation and all four animals had PRNT(80) titers of ≥1:20 on Day 6. Neutralizing antibody was first detected in 2 of 4 ewes inoculated with arMP-12ΔNSs16/198 on Day 7 and all had PRNT(80) titers of ≥1:20 on Day 10. We found the mean PRNT(80) response to arMP-12ΔNSs16/198 to be 16- to 25-fold lower than that of ewes inoculated with arMP-12ΔNSm21/384, arMP-12 or RVF MP-12. No abortions occurred though a single fetal death in each of the arMP-12 and RVF MP-12 groups was found at necropsy. The poor PRNT(80) response to arMP-12ΔNSs16/198 caused us to discontinue further testing of this candidate and focus on arMP-12ΔNSm21/384. A dose escalation study of arMP-12ΔNSm21/384 showed that 1×10(3)plaque forming units (PFU) stimulate a PRNT(80) response comparable to doses of up to 1×10(5)PFU of this virus. With further study, the arMP-12ΔNSm21/384 virus may prove to be a safe and efficacious candidate for a livestock vaccine. The large deletion in the NSm gene may also provide a negative marker that will allow serologic differentiation of naturally infected animals from vaccinated animals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To estimate the effects of prostaglandin E1 (PGE1) and E2 (PGE2) on myometrial contractility and structure in vitro., Study Design: Myometrial strips from 18 women were incubated with PGE1 (10-5 mol/L), PGE2 (10-5 mol/L), or solvent (CTR) for up to 360 minutes in organ chambers for isometric tension recording. The area under the contraction curve, total collagen content, and percentage of the area covered by connective tissue were calculated at various time periods., Results: PGE1 significantly increased in vitro myometrial contractility up to 90 minutes when compared with PGE2 and CTR (p < 0.01) and up to 180 minutes as compared with PGE2 (p < 0.05). After 360 minutes, CTR and PGE1 samples had lower total collagen content and area covered by connective tissue than PGE2 (p < 0.01)., Conclusion: The effects of prostaglandins on the uterus cannot be solely explained by contractility. Treatment with PGE1 significantly increased myometrial contractions and decreased both total collagen content and the area covered by connective tissue. Such findings may explain the higher rates of vaginal delivery, tachysystole, and uterine rupture associated with PGE1 use., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathologic-type lung disease., Design: Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated i.m. on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathology., Results: All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil prominence., Conclusions: These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.

A retrospective review of cases of tuberculosis examined by our Autopsy Division was undertaken to determine the most common associated co-morbidities. Forty-six cases of tuberculosis were examined between 2000 and 2010. The subpopulation of decedents studied included a large number of incarcerated individuals and showed an age distribution from 30 to 78 years. Thirty-five of the cases reviewed showed one or more co-morbidities, primarily viral hepatitis C, cancer, human immunodeficiency virus (HIV), cardiovascular diseases, and chronic obstructive pulmonary diseases. Almost 30% of the cases showed evidence of extrapulmonary disease, including one case of tuberculous meningitis. In approximately 20% of the cases, rapid progressive or disseminated tuberculosis was identified as immediate cause of death. Tuberculosis was the immediate cause of death in 20% of the hepatitis C-infected group and in 14% of the decedents diagnosed with cancer, compared to over 45% of the HIV-infected decedents. This observation is consistent with previous studies reporting an enhanced mortality from tuberculosis in HIV-infected subjects. Interestingly, rapid progressive tuberculosis was identified as immediate cause of death in two cases with no associated co-morbidities; both decedents were young immunocompetent adults, suggesting an increasing susceptibility of this subpopulation to tuberculosis exposure and to severe disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We investigated the effect of the angiotensin-converting enzyme (ACE) inhibitor captopril in a clinically relevant ovine model of smoke and burn injury, with special reference to oxidative stress and activation of poly(ADP-ribose) polymerase, in the lung and in circulating leukocytes. Female, adult sheep (28-40 kg) were divided into three groups. After tracheostomy and under deep anesthesia, both vehicle-control-treated (n = 5) and captopril-treated (20 mg/kg per day, i.v., starting 0.5 h before the injury) (n = 5) groups were subjected to 2 × 20%, third-degree burn injury and were insufflated with 48 breaths of cotton smoke. A sham group not receiving burn/smoke was also studied (n = 5). Animals were mechanically ventilated and fluid resuscitated for 24 h in the awake state. Burn and smoke injury resulted in an upregulation of ACE in the lung, evidenced by immunohistochemical determination and Western blotting. Burn and smoke injury resulted in pulmonary dysfunction, as well as systemic hemodynamic alterations. Captopril treatment of burn and smoke animals improved PaO2/FiO2 ratio and pulmonary shunt fraction and reduced the degree of lung edema. There was a marked increase in PAR levels in circulating leukocytes after burn/smoke injury, which was significantly decreased by captopril. The pulmonary level of ACE and the elevated pulmonary levels of transforming growth factor β in response to burn and smoke injury were significantly decreased by captopril treatment. Our results suggest that the ACE inhibitor captopril exerts beneficial effects on the pulmonary function in burn/smoke injury. The effects of the ACE inhibitor may be related to the prevention of reactive oxygen species-induced poly(ADP-ribose)polymerase overactivation. Angiotensin-converting enzyme inhibition may also exert additional beneficial effects by inhibiting the expression of the profibrotic mediator transforming growth factor β.

Objective: We sought to extend our prior observations and histopathologically characterize key metabolic enzymes (CYP1A1) with markers of oxidative damage in the placental sections from smokers., Study Design: Placental specimens were collected from term singleton deliveries from smokers (n = 10) and nonsmokers (n = 10) and subjected to a detailed histopathological examination. To quantify the extent of oxidative damage, masked score-graded (0-6) histopathology against 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxydeoxyguanisine (8-OHdG) was performed. Minimal significance (P < .05) was determined with a Fisher's exact and a 2-tailed Student t test as appropriate., Results: We observed a significant increase in the presence of syncytial knots in placentas from smokers (70% vs 10%, P = .02). These gross observations were accompanied by a significant aberrant placental aromatic hydrocarbon metabolism (increased CYP1A1, 4.4 vs 2.1, P = .002) in addition to evidence of oxidative damage (4-HNE 3.4 vs 1.1, P = .00005; 8-OHdG 4.9 vs 3.1, P = .0038)., Conclusion: We observed a strong association between maternal tobacco use and aberrant placental metabolism, syncytial knot formation, and multiple markers of oxidative damage., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) has been shown to promote cellular energetic collapse and cellular necrosis in various forms of critical illness. Most of the evidence implicating the PARP pathway in disease processes is derived from preclinical studies. With respect to PARP and burns, studies in rodent and large animal models of burn injury have demonstrated the activation of PARP in various tissues and the beneficial effect of its pharmacological inhibition. The aims of the current study were to measure the activation of PARP in human skeletal muscle biopsies at various stages of severe pediatric burn injury and to identify the cell types where this activation may occur. Another aim of the study was to test the effect of propranolol (an effective treatment of patients with burns) on the activation of PARP in skeletal muscle biopsies. Poly(ADP-ribose) polymerase activation was measured by Western blotting for its product, poly(ADP-ribose) (PAR). The localization of PARP activation was determined by PAR immunohistochemistry. The results showed that PARP becomes activated in the skeletal muscle tissue after burns, with the peak of the activation occurring in the middle stage of the disease (13-18 days after burns). Even at the late stage of the disease (69-369 days after burn), an elevated degree of PARP activation persisted in some of the patients. Immunohistochemical studies localized the staining of PAR primarily to vascular endothelial cells and occasionally to resident mononuclear cells. There was a marked suppression of PARP activation in the skeletal muscle biopsies of patients who received propranolol treatment. We conclude that human burn injury is associated with the activation of PARP. We hypothesize that this response may contribute to the inflammatory responses and cell dysfunction in burns. Some of the clinical benefit of propranolol in burns may be related to its inhibitory effect on PARP activation.

Mice infected with Dhori virus (DHOV) develop a fulminant, systemic, and uniformly fatal illness that has many of the clinical and pathologic findings seen in H5N1 influenza A virus infection. However, the role of host's immune response in DHOV infection remains unclear. In this study, the concentrations of 23 inflammatory cytokines and chemokines were measured in the liver, lungs, and sera of mice during the course of DHOV infection. Liver function, level of viremia, and hematologic response were also monitored. Infected animals exhibited significant leucopenia and lymphopenia, which directly correlated with the disease progression. High yields of infectious virus along with strikingly elevated expression of various inflammatory mediators, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, IL-10, macrophage inflammatory protein (MIP)-1alpha, manocyte chemoattractant protein (MCP)-1, and interferon (IFN)-alpha, indicate that these responses play an important role in the observed disease and pathology. The overall clinical, pathologic, and immunologic responses of ICR mice to DHOV infection closely resemble those described for highly virulent influenza A virus infection in humans, thereby offering a realistic, safe, and alternative animal model for studying the pathogenesis and treatment of highly pathogenic avian influenza virus.

Objective: The objective of the study was to investigate the effect of fetal programming on the development of atherosclerosis in the offspring in a mouse model., Study Design: Male and female mice of the wild type and the knockout for the apoprotein E (apoE) gene were cross-bred to obtain all 4 possible genetic offspring types. The offspring were kept on regular chow and killed at 8 months of age. Levels of total cholesterol and triglycerides were determined. The aortic arch was examined for the presence and severity of atherosclerosis. Kidney and liver sections were analyzed for pathologic changes., Results: We found increased total cholesterol levels and incidence of atherosclerosis in offspring born to hypercholesterolemic mothers as compared with genomically similar animals born to wild-type mothers. These animals also showed kidney and liver lesions consistent with chronic hypercholesterolemia., Conclusions: There is a strong effect of fetal programming on the development of atherosclerosis in the apoE mouse model.

Efficacy of the new antipoxvirus compound ST-246 was evaluated as treatment of monkeypox (MPX) virus infection in a ground squirrel model of the disease. Ground squirrels were given a lethal dose of MPX virus and were then treated orally at various times post-inoculation (pi) with 100 mg/kg/day of ST-246. Morbidity and mortality, clinical laboratory results, viral load, and pathology of placebo and treatment groups were compared. All animals that started treatment with ST-246 on days 0, 1, 2, and 3 pi survived lethal challenge with MPX virus; 67% of animals treated on day 4 pi also survived. In contrast, 100% of the placebo group died. Most of the ST-246-treated animals showed no evidence of clinical disease or alteration of baseline clinical laboratory values and had minimal histopathologic changes. These results suggest that ST-246 is a promising candidate for early treatment of severe orthopoxvirus infection.

The first human cases of monkeypox (MPX) were recognized in central Africa in 1970. Since then, sporadic outbreaks of the disease have occurred in central and west Africa. In 2003, an outbreak of human MPX occurred in the United States after importation of infected rodents from west Africa. Clinical features of the 2003 outbreak were less severe than accounts of the disease among people in central Africa. The reasons for this observed difference are unknown. In this study, the clinical and pathologic characteristics of experimental infection with representative central African and North American MPX virus strains were compared in a ground squirrel model of the disease. The results indicate that the US 2003 virus, which phylogenetically is a member of the west African MPX virus clade, was less virulent than central African MPX virus strains.

Using a recently described hamster model of yellow fever (YF), we compared the hematologic and clinical chemistry changes that occur in blood with the histopathologic alternations observed in liver and other organs. Inflammatory foci and necroapoptotic hepatocytes were first observed in the liver three days after YF infection. This was accompanied by a rapid increase in serum transaminase and bilirubin values, elevation of prothrombin times, thrombocytopenia, and leukocytosis. Maximum liver pathology was observed on the sixth and seventh days post-infection; this corresponded to the peak alternations in clinical chemistry and hematologic values. In surviving hamsters, regenerating hepatocytes began to appear on the eighth day post-infection; this was accompanied by a corresponding return to baseline levels of most of the aforementioned clinical laboratory values. The histopathologic and clinical laboratory findings in the hamster model were very similar to those observed in severe human cases of YF. These results provide further validation of the utility of the hamster model for studying the pathogenesis and treatment of YF.

The clinical laboratory, virologic, and pathologic changes occurring in hamsters after infection with Pirital virus (Arenaviridae) are described. Pirital virus infection in the hamsters was characterized by high titered viremia, leukocytosis, coagulopathy, pulmonary hemorrhage and edema, hepatocellular and splenic necrosis, and marked elevation of serum transaminase levels. All of the animals died within 9 days. The clinical and histopathological findings in the Pirital virus-infected hamsters were very similar to those reported in severe human cases of Lassa fever, suggesting that this new animal model could serve as a low-cost and relatively safe alternative for studying the pathogenesis and therapy of Lassa fever.

Studies of experimental infection of prairie dogs (Cynomys ludovicianus) with monkeypox virus are described. After intraperitoneal infection, all of the animals died within 11 days. Virus was cultured from their blood and oropharynx several days before death; at necropsy, most of the organs tested contained monkeypox virus. Marked hepatic and splenic necrosis were observed, along with mild inflammatory changes in the lungs. After intranasal (IN) infection, the primary pathologic changes were in the lungs and pleural cavity. Some of the IN infected animals (40%) survived, and monkeypox virus could be cultured from their nasal discharge and oropharynx for <22 days. Ulcerative lesions also developed on the lips, tongue, and buccal mucosa of the surviving animals. Our findings support an earlier report, which suggested that infected prairie dogs can transmit monkeypox virus by respiratory and mucocutaneous contact with susceptible animals and persons.

The results of experiments comparing the pathogenesis of West Nile virus (WNV) following infection by mosquito bite, needle inoculation, and ingestion are reported. Adult hamsters were readily infected by all three routes. The level and duration of viremia, clinical manifestations, pathology, and antibody response in the hamsters following mosquito infection and needle inoculation were similar; after oral infection, the onset of viremia was delayed and the mortality was lower, but the level and duration of viremia, histopathology, and antibody response were similar to the other routes. The results from this and previously published studies indicate that a wide variety of animal species are susceptible to oral infection with WNV and that orally infected animals develop a viremia and illness similar to that following the bite of infected mosquitoes. Oral infection appears to be an alternative transmission mechanism used by a number of different flaviviruses; its potential role in the natural history of WNV is discussed.

Ribavirin was evaluated as a potential treatment of yellow fever (YF) in a hamster model of the disease. Ribavirin treatment during the first five days after YF virus infection improved survival, reduced tissue damage in target organs (liver and spleen), prevented hepatocellular steatosis, and normalized alanine aminotransferase levels. The results of this study suggest that ribavirin may be effective in the early treatment of YF, and that its mechanism of action in reducing liver pathology in YF virus infection may be similar to that observed with ribavirin in the treatment of chronic hepatitis C virus infection.

A proposed new small-animal (rodent) model for studying the pathogenesis and treatment of severe orthopoxvirus infections is described. Thirteen-lined ground squirrels (Spermophilus tridecemlineatus) were infected intraperitoneally and intranasally with monkeypox virus (MPXV). A fulminant illness developed in all animals, and they died 6-9 days after infection. Virus was cultured from the blood and oropharynx several days before death; at necropsy, all of the organs tested contained relatively high titers of MPXV. The major pathologic findings were in the liver, which showed centrilobular necrosis, steatosis, and basophilic inclusion bodies in hepatocytes. Splenic necrosis was also observed, as well as interstitial inflammation in the lungs. The pathologic features of MPXV in ground squirrels are similar to that described with MPXV in macaques and severe variola (smallpox) virus infection in humans.

S-Adenosyl-L-homocysteine hydrolase (SAHh) inhibitors have long been used as broad-range antivirals and have been recently evaluated as an experimental therapy of filovirus infections. In response to the need for a rapid laboratory testing method that could assess antiviral potency in vivo, our group developed a capillary electrophoresis (CE) method for the determination of the S-adenosyl-L-homocysteine (SAH) to S-adenosyl-L-methionine (SAM) ratio. After chloroacetaldehyde derivatization, SAH and SAM were detected using laser-induced fluorescence detection with a HeCd laser. Separation and quantitation of both SAH and SAM in human plasma were achieved in less than 1 min. The proposed method is rapid and reliable, and could be easily applied to routine monitoring of clinical and preclinical trials subjects.

Plasma viral load (VL) values and CD4(+) cell count are employed clinically for initiation of therapy in the treatment of patients infected with human immunodeficiency virus (HIV), as previous clinical studies have shown a marked prevalence of acquired immunodeficiency syndrome (AIDS) development in seropositive individuals with VL values over 30 000 copies/mL. Many studies have shown that reduced glutathione (GSH) and cysteine (Cys) deficiency play an important role in the infection. We have developed capillary zone electrophoresis (CZE)-based assays and have used them to investigate the relationship between plasma and intracellular thiol levels and HIV-1 viremia in plasma. Blood samples from healthy volunteers and seropositive patients undergoing different antiretroviral regimes were analyzed in the study. The VL assay was based on CZE-UV detection of viral RNA at 260 nm. Determination of endogenous reduced Cys and GSH was achieved by CZE-UV detection of their mercurial complexes at 200 nm. We found that a decrease in GSH and Cys levels may be associated with disease progress. In fact, reduced GSH and Cys levels appear progressively reduced with increasing VL.