Investigation of the electrostatic potential of a grain boundary in Y-substituted BaZrO3 using inline electron holography.

We apply inline electron holography to investigate the electrostatic potential across an individual BaZr0.9Y0.1O3 grain boundary. With holography, we measure a grain boundary potential of -1.3 V. Electron energy loss spectroscopy analyses indicate that barium vacancies at the grain boundary are the main contributors to the potential well in this sample. Furthermore, geometric phase analysis and density functional theory calculations suggest that reduced atomic density at the grain boundary also contributes to the experimentally measured potential well.

Evaluation of Combination Drug Therapy for Treatment of Antibiotic-Resistant Inhalation Anthrax in a Murine Model.

Bacillus anthracis is considered a likely agent to be used as a bioweapon, and the use of a strain resistant to the first-line antimicrobial treatments is a concern. We determined treatment efficacies against a ciprofloxacin-resistant strain of B. anthracis (Cipr Ames) in a murine inhalational anthrax model. Ten groups of 46 BALB/c mice were exposed by inhalation to 7 to 35 times the 50% lethal dose (LD50) of B. anthracis Cipr Ames spores. Commencing at 36 h postexposure, groups were administered intraperitoneal doses of sterile water for injections (SWI) and ciprofloxacin alone (control groups), or ciprofloxacin combined with two antimicrobials, including meropenem-linezolid, meropenem-clindamycin, meropenem-rifampin, meropenem-doxycycline, penicillin-linezolid, penicillin-doxycycline, rifampin-linezolid, and rifampin-clindamycin, at appropriate dosing intervals (6 or 12 h) for the respective antibiotics. Ten mice per group were treated for 14 days and observed until day 28. The remaining animals were euthanized every 6 to 12 h, and blood, lungs, and spleens were collected for lethal factor (LF) and/or bacterial load determinations. All combination groups showed significant survival over the SWI and ciprofloxacin controls: meropenem-linezolid (P = 0.004), meropenem-clindamycin (P = 0.005), meropenem-rifampin (P = 0.012), meropenem-doxycycline (P = 0.032), penicillin-doxycycline (P = 0.012), penicillin-linezolid (P = 0.026), rifampin-linezolid (P = 0.001), and rifampin-clindamycin (P = 0.032). In controls, blood, lung, and spleen bacterial counts increased to terminal endpoints. In combination treatment groups, blood and spleen bacterial counts showed low/no colonies after 24-h treatments. The LF fell below the detection limits for all combination groups yet remained elevated in control groups. Combinations with linezolid had the greatest inhibitory effect on mean LF levels.

Influences of environmental bacteria and their metabolites on allergies, asthma, and host microbiota.

The prevalence of allergic diseases and asthma has dramatically increased over the last decades, resulting in a high burden for patients and healthcare systems. Thus, there is an unmet need to develop preventative strategies for these diseases. Epidemiological studies show that reduced exposure to environmental bacteria in early life (eg, birth by cesarean section, being formula-fed, growing up in an urban environment or with less contact to various persons) is associated with an increased risk to develop allergies and asthma later in life. Conversely, a reduced risk for asthma is consistently found in children growing up on traditional farms, thereby being exposed to a wide spectrum of microbes. However, clinical studies with bacteria to prevent allergic diseases are still rare and to some extent contradicting. A detailed mechanistic understanding of how environmental microbes influence the development of the human microbiome and the immune system is important to enable the development of novel preventative approaches that are based on the early modulation of the host microbiota and immunity. In this mini-review, we summarize current knowledge and experimental evidence for the potential of bacteria and their metabolites to be used for the prevention of asthma and allergic diseases.

Dual-role FilmArray® diagnostics for high-impact viral diseases.

In this study, we explored the potential utility of the human-focused FilmArray® Respiratory Panel for the diagnosis of a broad range of influenza viruses of veterinary concern as compared with the standard portfolio of recommended TaqMan®-based diagnostic tests. In addition, we discuss some potential operational advantages associated with the use of such integrated sample extraction, amplification and analysis devices in the context of a future long-term, dual-role strategy for the detection of emergency diseases of both human and veterinary concern.

Interleukin-4 and interferon-γ orchestrate an epithelial polarization in the airways.

Interferon-γ (IFN-γ) and interleukin-4 (IL-4) are key effector cytokines for the differentiation of T helper type 1 and 2 (Th1 and Th2) cells. Both cytokines induce fate-decisive transcription factors such as GATA3 and TBX21 that antagonize the polarized development of opposite phenotypes by direct regulation of each other's expression along with many other target genes. Although it is well established that mesenchymal cells directly respond to Th1 and Th2 cytokines, the nature of antagonistic differentiation programs in airway epithelial cells is only partially understood. In this study, primary normal human bronchial epithelial cells (NHBEs) were exposed to IL-4, IFN-γ, or both and genome-wide transcriptome analysis was performed. The study uncovers an antagonistic regulation pattern of IL-4 and IFN-γ in NHBEs, translating the Th1/Th2 antagonism directly in epithelial gene regulation. IL-4- and IFN-γ-induced transcription factor hubs form clusters, present in antagonistically and polarized gene regulation networks. Furthermore, the IL-4-dependent induction of IL-24 observed in rhinitis patients was downregulated by IFN-γ, and therefore IL-24 represents a potential biomarker of allergic inflammation and a Th2 polarized condition of the epithelium.

Farm-derived Gram-positive bacterium Staphylococcus sciuri W620 prevents asthma phenotype in HDM- and OVA-exposed mice.

BACKGROUND: Farm-derived dust samples have been screened for bacteria with potential allergo-protective properties. Among those was Staphylococcus sciuri W620 (S. sciuri W620), which we tested with regard to its protective capacities in murine models of allergic airway inflammation. METHODS: We employed two protocols of acute airway inflammation in mice administering either ovalbumin (OVA) or house dust mite extract (HDM) for sensitization. Mechanistic studies on the activation of innate immune responses to S. sciuri W620 were carried out using human primary monocytic dendritic cells (moDC) and co-culture with autologous T cells. RESULTS: The allergo-protective properties of S. sciuri W620 were proven in a T(H)2-driven OVA model as well as in a mixed T(H)1/T(H)2 phenotype HDM model as demonstrated by abrogation of eosinophils and neutrophils in the airways after intranasal treatment. In the HDM model, lymph node cell T(H)1/T(H)2 signature cytokines were decreased in parallel. Studies on human moDC revealed an activation of TLR2 and NOD2 receptors and initiation of DC maturation following incubation with S. sciuri W620. Cytokine expression analyses after exposure to S. sciuri W620 showed a lack of IL-12 production in moDC due to missing transcription of the IL-12p35 mRNA. However, such DC selectively supported T(H)1 cytokine release by co-cultured T cells. CONCLUSION AND CLINICAL RELEVANCE: Our proof-of-concept experiments verify the screening system of farm-derived dust samples as suitable to elucidate new candidates for allergo-protection. S. sciuri W620 was shown to possess preventive properties on airway inflammation providing the basis for further mechanistic studies and potential clinical implication.

Neonatal supplementation of processed supernatant from Lactobacillus rhamnosus GG improves allergic airway inflammation in mice later in life.

BACKGROUND: Oral supplementation with probiotic bacteria can protect against the development of allergic and inflammatory diseases. OBJECTIVE: The aim of this study was to investigate potential immunomodulatory and allergy-protective effects of processed Lactobacillus rhamnosus GG (LGG)-derived supernatants early in life in neonatal mice. METHODS: In vitro, RAW264.7 mouse macrophages were stimulated with viable LGG, LGG-derived supernatants, prepared from different growth phases, and different size fractions thereof, and pro- and anti-inflammatory cytokine production was analysed. Supernatant fractions were also treated with protease, DNAse or carbohydrate-digesting enzymes to define the nature of immunomodulatory components. In vivo, neonatal Balb/c mice were orally supplemented with differentially processed LGG supernatants. Starting at 4 weeks of age, a protocol of ovalbumin-induced acute allergic airway inflammation was applied and protective effects of processed LGG supernatants were assessed. RESULTS: Incubation of RAW264.7 cells with LGG-derived supernatants significantly increased TNFα and IL-10 production. These effects were not restricted to a particular molecular size fraction. Treatment with protease, but not with DNAse or carbohydrate-digesting enzymes, completely abolished the immunomodulatory activities. Incubation of TLR/NOD-transfected cells with LGG-derived supernatants revealed that recognition and signalling of bioactive components is mediated by TLR2 and NOD2. In vivo supplementation of newborn mice with processed LGG-derived supernatants resulted in pronounced protective effects on the allergic inflammatory response as reflected by reduced eosinophil numbers, modified T helper cell cytokine production, significantly less lung inflammation and reduced goblet cell numbers in comparison with sham-treated controls. CONCLUSION: LGG-derived supernatants exert immunomodulatory activities, and neonatal administration of specifically processed supernatants may provide an alternative to viable probiotics in reducing allergic inflammatory responses.

Diagnosis of equine influenza virus infections in quarantine stations in Australia, 2007.

In August 2007, several horses showed pyrexia and respiratory signs while in post-arrival quarantine in Australia. Subsequent investigations diagnosed equine influenza by serology and PCR in two quarantine stations. A common origin in a shipment of horses from Japan was indicated.

Use of an in vitro pharmacodynamic model to derive a moxifloxacin regimen that optimizes kill of Yersinia pestis and prevents emergence of resistance.

Yersinia pestis, the causative agent of bubonic, septicemic, and pneumonic plague, is classified as a CDC category A bioterrorism pathogen. Streptomycin and doxycycline are the "gold standards" for the treatment of plague. However, streptomycin is not available in many countries, and Y. pestis isolates resistant to streptomycin and doxycycline occur naturally and have been generated in laboratories. Moxifloxacin is a fluoroquinolone antibiotic that demonstrates potent activity against Y. pestis in in vitro and animal infection models. However, the dose and frequency of administration of moxifloxacin that would be predicted to optimize treatment efficacy in humans while preventing the emergence of resistance are unknown. Therefore, dose range and dose fractionation studies for moxifloxacin were conducted for Y. pestis in an in vitro pharmacodynamic model in which the half-lives of moxifloxacin in human serum were simulated so as to identify the lowest drug exposure and the schedule of administration that are linked with killing of Y. pestis and with the suppression of resistance. In the dose range studies, simulated moxifloxacin regimens of ≥175 mg/day killed drug-susceptible bacteria without resistance amplification. Dose fractionation studies demonstrated that the AUC (area under the concentration-time curve)/MIC ratio predicted kill of drug-susceptible Y. pestis, while the C(max) (maximum concentration of the drug in serum)/MIC ratio was linked to resistance prevention. Monte Carlo simulations predicted that moxifloxacin at 400 mg/day would successfully treat human infection due to Y. pestis in 99.8% of subjects and would prevent resistance amplification. We conclude that in an in vitro pharmacodynamic model, the clinically prescribed moxifloxacin regimen of 400 mg/day is predicted to be highly effective for the treatment of Y. pestis infections in humans. Studies of moxifloxacin in animal models of plague are warranted.

The allergy-protective properties of Acinetobacter lwoffii F78 are imparted by its lipopolysaccharide.

BACKGROUND: An increasing number of epidemiological studies show that exposure to farming environment during early childhood strongly influences the development of allergic reactions later in life ('hygiene hypothesis'). Also, it had been shown that certain bacteria from this environment may have allergy-protective properties. In the present study, we further characterized one of these bacteria, namely Acinetobacter lwoffii F78, with regard to the bacteria-induced signaling and possible mechanisms of allergy protection. METHODS: The impact of A. lwoffii F78 on human monocyte-derived dendritic cells especially with respect to their T(Helper) cell polarization capacity was investigated by ELISA and real-time PCR experiments as well as confocal microscopy. The responsible molecule for these effects was further characterized and identified using blocking experiments. RESULTS: It was shown that A. lwoffii F78 induced a T(H)1-polarizing program in human dendritic cells which led to T(H)1 differentiation. In addition, a positive influence on the TBet/GATA3 level could be detected. Blocking experiments revealed that the lipopolysaccharide (LPS) of A. lwoffii F78 was the responsible molecule promoting these effects. CONCLUSION: We found evidence that the allergy-protecting effects of A. lwoffii F78 are because of the activation of a T(H)1-polarizing program in human dendritic cells, and that the LPS of A. lwoffii F78 is responsible for these beneficial effects.

Impact of spore biology on the rate of kill and suppression of resistance in Bacillus anthracis.

Bacillus anthracis is complex because of its spore form. The spore is invulnerable to antibiotic action. It also has an impact on the emergence of resistance. We employed the hollow-fiber infection model to study the impacts of different doses and schedules of moxifloxacin on the total-organism population, the spore population, and the subpopulations of vegetative- and spore-phase organisms that were resistant to moxifloxacin. We then generated a mathematical model of the impact of moxifloxacin, administered by continuous infusion or once daily, on vegetative- and spore-phase organisms. The ratio of the rate constant for vegetative-phase cells going to spore phase (K(vs)) to the rate constant for spore-phase cells going to vegetative phase (K(sv)) determines the rate of organism clearance. The continuous-infusion drug profile is more easily sensed as a threat; the K(vs)/K(sv) ratio increases at lower drug exposures (possibly related to quorum sensing). This movement to spore phase protects the organism but makes the emergence of resistance less likely. Suppression of resistance requires a higher level of drug exposure with once-daily administration than with a continuous infusion, a difference that is related to vegetative-to-spore (and back) transitioning. Spore biology has a major impact on drug therapy and resistance suppression. These findings explain why all drugs of different classes have approximately the same rate of organism clearance for Bacillus anthracis.

Design and evaluation of consensus PCR assays for henipaviruses.

Henipaviruses were first discovered in the 1990s, and their potential threat to public health is of increasing concern with increasing knowledge. Old-world fruit bats are the reservoir hosts for these viruses, and spill-over events cause lethal infections in a wide range of mammalian species, including humans. In anticipation of these spill-over events, and to investigate further the geographical range of these genetically diverse viruses, assays for detection of known and potentially novel strains of henipaviruses are required. The development of multiple consensus PCR assays for the detection of henipaviruses, including both SYBR Green and TaqMan real-time PCRs and a conventional heminested PCR is described. The assays are highly sensitive and have defined specificity. In addition to being useful tools for detection of known and novel henipaviruses, evaluation of assay efficiency and sensitivity across both biological and synthetic templates has provided valuable insight into consensus PCR design and use.

Efficacy of oritavancin in a murine model of Bacillus anthracis spore inhalation anthrax.

The inhaled form of Bacillus anthracis infection may be fatal to humans. The current standard of care for inhalational anthrax postexposure prophylaxis is ciprofloxacin therapy twice daily for 60 days. The potent in vitro activity of oritavancin, a semisynthetic lipoglycopeptide, against B. anthracis (MIC against Ames strain, 0.015 microg/ml) prompted us to test its efficacy in a mouse aerosol-anthrax model. In postexposure prophylaxis dose-ranging studies, a single intravenous (i.v.) dose of oritavancin of 5, 15, or 50 mg/kg 24 h after a challenge with 50 to 75 times the median lethal dose of Ames strain spores provided 40, 70, and 100% proportional survival, respectively, at 30 days postchallenge. Untreated animals died within 4 days of challenge, whereas 90% of control animals receiving ciprofloxacin at 30 mg/kg intraperitoneally twice daily for 14 days starting 24 h after challenge survived. Oritavancin demonstrated significant activity post symptom development; a single i.v. dose of 50 mg/kg administered 42 h after challenge provided 56% proportional survival at 30 days. In a preexposure prophylaxis study, a single i.v. oritavancin dose of 50 mg/kg administered 1, 7, 14, or 28 days before lethal challenge protected 90, 100, 100, and 20% of mice at 30 days; mice treated with ciprofloxacin 24 h or 24 and 12 h before challenge all died within 5 days. Efficacy in pre- and postexposure models of inhalation anthrax, together with a demonstrated low propensity to engender resistance, promotes further study of oritavancin pharmacokinetics and efficacy in nonhuman primate models.

Application of carbohydrate microarray technology for the detection of Burkholderia pseudomallei, Bacillus anthracis and Francisella tularensis antibodies.

We developed a microarray platform by immobilizing bacterial 'signature' carbohydrates onto epoxide modified glass slides. The carbohydrate microarray platform was probed with sera from non-melioidosis and melioidosis (Burkholderia pseudomallei) individuals. The platform was also probed with sera from rabbits vaccinated with Bacillus anthracis spores and Francisella tularensis bacteria. By employing this microarray platform, we were able to detect and differentiate B. pseudomallei, B. anthracis and F. tularensis antibodies in infected patients, and infected or vaccinated animals. These antibodies were absent in the sera of naïve test subjects. The advantages of the carbohydrate microarray technology over the traditional indirect hemagglutination and microagglutination tests for the serodiagnosis of melioidosis and tularemia are discussed. Furthermore, this array is a multiplex carbohydrate microarray for the detection of all three biothreat bacterial infections including melioidosis, anthrax and tularemia with one, multivalent device. The implication is that this technology could be expanded to include a wide array of infectious and biothreat agents.

Use of an in vitro pharmacodynamic model to derive a linezolid regimen that optimizes bacterial kill and prevents emergence of resistance in Bacillus anthracis.

Simulating the average non-protein-bound (free) human serum drug concentration-time profiles for linezolid in an in vitro pharmacodynamic model, we characterized the pharmacodynamic parameter(s) of linezolid predictive of kill and for prevention of resistance in Bacillus anthracis. In 10-day dose-ranging studies, the average exposure for > or =700 mg of linezolid given once daily (QD) resulted in >3-log CFU/ml declines in B. anthracis without resistance selection. Linezolid at < or =600 mg QD amplified for resistance. With twice-daily (q12h) dosing, linezolid at > or =500 mg q12 h was required for resistance prevention. In dose fractionation studies, killing of B. anthracis was predicted by the area under the time-concentration curve (AUC)/MIC ratio. However, resistance prevention was linked to the maximum serum drug concentration (C(max))/MIC ratio. Monte Carlo simulations predicted that linezolid at 1,100 mg QD would produce in 96.7% of human subjects a free 24-h AUC that would match or exceed the average 24-h AUC of 78.5 mg x h/liter generated by linezolid at 700 mg QD while reproducing the shape of the concentration-time profile for this pharmacodynamically optimized regimen. However, linezolid at 700 mg q12h (cumulative daily dose of 1,400 mg) would produce an exposure that would equal or exceed the average free 24-h AUC of 90 mg x h/liter generated by linezolid at 500 mg q12h in 93.8% of human subjects. In conclusion, in our in vitro studies, the QD-administered, pharmacodynamically optimized regimen for linezolid killed drug-susceptible B. anthracis and prevented resistance emergence at lower dosages than q12h regimens. The lower dosage for the pharmacodynamically optimized regimen may decrease drug toxicity. Also, the QD administration schedule may improve patient compliance.

A serological and virological survey for evidence of infection with Newcastle disease virus in Australian chicken farms.

OBJECTIVE: To determine the prevalence and distribution of antibodies to Newcastle disease virus on Australian chicken farms and to determine the pathotype and relationships of the Newcastle disease viruses present on those farms. DESIGN: A cross-sectional survey of 753 commercial chicken farms. PROCEDURE: The survey comprised a detailed questionnaire and collection of venous blood samples. The titre of antibodies to Newcastle disease virus was determined by haemagglutination inhibition. Virus isolation was conducted from cloacal and tracheal swabs taken from chickens in serologically positive flocks. Virus isolates were pathotyped on the basis of the deduced Fusion protein cleavage site determined by nucleotide sequencing of a 265 bp region of the genome in the region of the cleavage site. RESULTS: Antibody evidence of Newcastle disease virus infection was found on 300 of the 753 surveyed farms throughout all 11 geographic regions of the survey. The highest prevalence occurred in the Sydney basin, New South Wales and Victoria east regions. Antibody titres were also highest in the regions where serologically positive flocks were most prevalent. The 259 virus isolates revealed nine different RNA sequences. Of the nine virus groups isolated, the most common group W was identical in sequence to the V4 vaccine strain. Five of the other groups had novel RNA sequences in the region of the F protein cleavage site. CONCLUSIONS: Antibodies to Newcastle disease virus are highly prevalent in the Australian chicken flock but all identified strains were avirulent in nature.

Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses.

Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haematopoietic necrosis virus (EHNV) and Bohle iridovirus (BIV) in Australia, and European sheatfish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OIE (Office International des Epizooties) notifiable systemic necrotizing iridovirus syndrome and are currently identified by protein-based assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop TaqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.

Rapid detection of highly pathogenic avian influenza H5N1 virus by TaqMan reverse transcriptase-polymerase chain reaction.

Highly pathogenic avian influenza (AI) H5N1 viruses have been spreading from Asia since late 2003. Early detection and classification are paramount for control of the disease because these viruses are lethal to birds and have caused fatalities in humans. Here, we described TaqMan reverse transcriptase-polymerase chain reaction assays for rapid detection of all AI viruses (influenza type A) and for identification of H5N1 of the Eurasian lineage. The assays were sensitive and quantitative over a 10(5)-10(6) linear range, detected all of the tested AI viruses, and enabled differentiation between H5 and H7 subtypes. These tests allow definitive confirmation of an AI virus as H5 within hours, which is crucial for rapid implementation of control measures in the event of an outbreak.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

OBJECTIVE: To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. DESIGN: A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. RESULTS: The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. CONCLUSION: The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.