Age-related sensitivity and pathological differences in infections by 2009 pandemic influenza A (H1N1) virus

<p>Abstract</p> <p>Background</p> <p>The highly pandemic 2009 influenza A H1N1 virus infection showed distinguished skewed age distribution with majority of infection and death in children and young adults. Although previous exposure to related antigen has been proposed as an explanation, the mechanism of age protection is still unknown.</p> <p>Methods</p> <p>In this study, murine model of different ages were inoculated intranasally with H1N1 (A/Beijing/501/09) virus and the susceptibility and pathological response to 2009 H1N1 infection were investigated.</p> <p>Results</p> <p>Our results showed that the younger mice had higher mortality rate when infected with the same dose of virus and the lethal dose increased with age. Immunohistochemical staining of H1N1 antigens in mice lung indicated infection was in the lower respiratory tract. Most bronchial and bronchiolar epithelial cells in 4-week mice were infected while only a minor percentage of those cells in 6-month and 1-year old mice did. The young mice developed much more severe lung lesions and had higher virus load in lung than the two older groups of mice while older mice formed more inducible bronchus-associated lymphoid tissue in their lungs and more severe damage in spleen.</p> <p>Conclusions</p> <p>These results suggest that young individuals are more sensitive to H1N1 infection and have less protective immune responses than older adults. The age factor should be considered when studying the pathogenesis and transmission of influenza virus and formulating strategies on vaccination and treatment.</p

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Latex Agglutination Test for Monitoring Antibodies to Avian Influenza Virus Subtype H5N1

A latex agglutination test (LAT) based on polystyrene beads sensitized with inactivated avian influenza virus H5N1 particles was developed. Compared with the hemagglutination inhibition test, the sensitivity and specificity of the LAT were 88.8 and 97.6%, respectively, in detecting 830 serum samples from vaccinated chickens. The test has application potential in field practice

Extensins: Self-Assembly, Crosslinking, and the Role of Peroxidases

The extensin (EXT) network is elaborated by the covalent intermolecular crosslinking of EXT glycoprotein monomers, and its proper assembly is important for numerous aspects of basic wall architecture and cellular defense. In this review, we discuss new advances in the secretion of EXT monomers and the molecular drivers of EXT network self-assembly. Many of the functions of EXTs are conferred through covalent crosslinking into the wall, so we also discuss the different types of known intermolecular crosslinks, the enzymes that are involved, as well as the potential for additional crosslinks that are yet to be identified. EXTs also function in wall architecture independent of crosslinking status, and therefore, we explore the role of non-crosslinking EXTs. As EXT crosslinking is upregulated in response to wounding and pathogen infection, we discuss a potential regulatory mechanism to control covalent crosslinking and its relationship to the subcellular localization of the crosslinking enzymes

Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Vaccination ▿

The lethal toxin (LeTx) of Bacillus anthracis plays a key role in the pathogenesis of anthrax. The protective antigen (PA) is a primary part of the anthrax toxin and forms LeTx by combination with lethal factor (LF). Phenylalanine-427 (F427) is crucial for PA function. This study was designed to discover potential novel therapeutic agents and vaccines for anthrax. This was done by screening PA mutants that were mutated at the F427 residue for a dominant-negative inhibitory (DNI) phenotype which was nontoxic but inhibited the toxicity of the wild-type LeTx. For this, PA residue F427 was first mutated to each of the other 19 naturally occurring amino acids. The cytotoxicity and DNI phenotypes of the mutated PA proteins were tested in the presence of 1 μg/ml LF in RAW264.7 cells and were shown to be dependent on the individual amino acid replacements. A total of 16 nontoxic mutants with various levels of DNI activity were identified in vitro. Among them, F427D and F427N mutants had the highest DNI activities in RAW264.7 cells. Both mutants inhibited LeTx intoxication in mice in a dose-dependent way. Furthermore, they induced a Th2-predominant immune response and protected mice against a challenge with five 50% lethal doses of LeTx. The protection was correlated mainly with a low level of interleukin-1β (IL-1β) and with high levels of PA-specific immunoglobulin G1, IL-6, and tumor necrosis factor alpha. Thus, PA DNI mutants, such as F427D and F427N mutants, may serve in the development of novel therapeutic agents and vaccines to fight B. anthracis infections

A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine▿

Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PAF427D. In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax