Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus

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Glutathione transferases of Phanerochaete chrysosporium: S-glutathionyl-p-hydroquinone reductase belongs to a new structural class

The white rot fungus Phanerochaete chrysosporium, a saprophytic basidiomycete, possesses a large number of cytosolic glutathione transferases, eight of them showing similarity to the Omega class. PcGSTO1 (subclass I, the bacterial homologs of which were recently proposed, based on their enzymatic function, to constitute a new class of glutathione transferase named S-glutathionyl-(chloro)hydroquinone reductases) and PcGSTO3 (subclass II related to mammalian homologs) have been investigated in this study. Biochemical investigations demonstrate that both enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteinyl residue. This reaction leads to the formation of a disulfide bridge between the conserved cysteine and the removed glutathione from their substrate. The substrate specificity of each isoform differs. In particular PcGSTO1, in contrast to PcGSTO3, was found to catalyze deglutathionylation of S-glutathionyl-p-hydroquinone substrates. The three-dimensional structure of PcGSTO1 presented here confirms the hypothesis that it belongs not only to a new biological class but also to a new structural class that we propose to name GST xi. Indeed, it shows specific features, the most striking ones being a new dimerization mode and a catalytic site that is buried due to the presence of long loops and that contains the catalytic cysteine.     

New distributional records and conservation implications for the critically endangered greater bamboo lemur Prolemur simus

To improve our knowledge of the distribution of the critically endangered greater bamboo lemur Prolemur simus, we surveyed 6 sites in eastern Madagascar. We found its characteristic feeding signs at 5 sites and made a direct sighting at one of these. One site represents a northern extension of 45 km of the known extant range of the species. Two sites are located in a forest corridor approximately halfway between the previously known southern and northern populations, therefore suggesting a broadly continuous distribution of the species within its range rather than the previously suspected distribution of two distinct populations separated by a distance of over 200 km. Our results illustrate the benefit of species-focussed surveys in determining the true distribution of endangered species, a realistic measure which is necessary in order to assess their current status and to prioritise long-term conservation interventions.     

Development and validation of ultra high performance liquid chromatography-mass spectrometry method for LBH589 in mouse plasma and tissues

An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed and validated for the quantitation of LBH589, a novel histone deacetylase inhibitor (HDACi), in mouse plasma and tissues (liver, spleen, kidney and lung). Tobramycin was employed as the internal standard. Separation was performed on an Acquity UPLC? BEH column, with a mobile phase consisting of 10% water (with 0.1% of trifluoroacetic acid) and 90% methanol (with 0.1% trifluoroacetic acid). LBH589 and tobramycin were determined using an electrospray ionization (ESI) interface. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of LBH589 at m/z 349.42→157.95 and of tobramycin at 468.2→163. Calibration curves for the UHPLC method (0.0025-1 μg/mL for plasma and tissue homogenates, equivalent to 0.0357-14.2857 μg/g for tissue samples) showed a linear range of detector responses (r>0.998). Intra-batch and inter-batch precision expressed as coefficient of variation (CV) ranged from 0.92 to 8.40%. Accuracy expressed as bias, ranged from -2.41 to 2.62%. The lower limit of quantitation (LLOQ) was 0.0025 μg/mL for both plasma and tissue homogenate samples, equivalent to 0.0357 μg/g tissue. This method was successfully applied to quantify LBH589 in plasma and tissue samples obtained after the intraperitoneal administration of a single dose of 20 mg/kg of LBH589 in BALB/c mice.     

New strategies for echocardiographic evaluation of left ventricular function in a mouse model of long-term myocardial infarction

Background

The aim of this article is to present an optimized acquisition and analysis protocol for the echocardiographic evaluation of left ventricle (LV) remodeling in a mouse model of myocardial infarction (MI).

Methodology

13 female DBA/2J mice underwent permanent occlusion of the left anterior descending (LAD) coronary artery leading to MI. Mice echocardiography was performed using a Vevo 770 (Visualsonics, Canada) before infarction, and 7, 14, 30, 60, 90 and 120 days after LAD ligation. LV systolic function was evaluated using different parameters, including the fractional area change (FAC%) computed in four high-temporal resolution B-mode short axis images taken at different ventricular levels, and in one parasternal long axis. Pulsed wave and tissue Doppler modes were used to evaluate the diastolic function and Tei Index for global cardiac function. The echocardiographic measurements of infarct size were validated histologically using collagen deposition labeled by Sirius red staining. All data was analyzed using Shapiro-Wilk and Student''s t-tests.

Principal Findings

Our results reveal LV dilation resulting in marked remodeling an severe systolic dysfunction, starting seven days after MI (LV internal apical diameter, basal = 2.82±0.24, 7d = 3.49±0.42; p<0.001. End-diastolic area, basal = 18.98±1.81, 7d = 22.04±2.11; p<0.001). A strong statistically significant negative correlation exists between the infarct size and long-axis FAC% (r = −0.946; R₂ = 0.90; p<0.05). Moreover, the measured Tei Index values confirmed significant post-infarction impairment of the global cardiac function (basal = 0.46±0.07, 7d = 0.55±0.08, 14 d = 0.57±0.06, 30 d = 0.54±0.06, 60 d = 0.54±0.07, 90 d = 0.57±0.08; p<0.01).

Conclusions/Significance

In summary, we have performed a complete characterization of LV post-infarction remodeling in a DBA/2J mouse model of MI, using parameters adapted to the particular characteristics of the model In the future, this well characterized model will be used in both investigative and pharmacological studies that require accurate quantitative monitoring of cardiac recovery after myocardial infarction.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

Chimeras of labile toxin one and cholera toxin retain mucosal adjuvanticity and direct Th cell subsets via their B subunit

Native cholera toxin (nCT) and the heat-labile toxin 1 (nLT) of enterotoxigenic Escherichia coli are AB5-type enterotoxins. Both nCT and nLT are effective adjuvants that promote mucosal and systemic immunity to protein Ags given by either oral or nasal routes. Previous studies have shown that nCT as mucosal adjuvant requires IL-4 and induces CD4-positive (CD4+) Th2-type responses, while nLT up-regulates Th1 cell production of IFN-gamma and IL-4-independent Th2-type responses. To address the relative importance of the A or B subunits in CD4+ Th cell subset responses, chimeras of CT-A/LT-B and LT-A/CT-B were constructed. Mice nasally immunized with CT-A/LT-B or LT-A/CT-B and the weak immunogen OVA developed OVA-specific, plasma IgG Abs titers similar to those induced by either nCT or nLT. Both CT-A/LT-B and LT-A/CT-B promoted secretory IgA anti-OVA Ab, which established their retention of mucosal adjuvant activity. The CT-A/LT-B chimera, like nLT, induced OVA-specific mucosal and peripheral CD4+ T cells secreting IFN-gamma and IL-4-independent Th2-type responses, with plasma IgG2a anti-OVA Abs. Further, LT-A/CT-B, like nCT, promoted plasma IgG1 more than IgG2a and IgE Abs with OVA-specific CD4+ Th2 cells secreting high levels of IL-4, but not IFN-gamma. The LT-A/CT-B chimera and nCT, but not the CT-A/LT-B chimera or nLT, suppressed IL-12R expression and IFN-gamma production by activated T cells. Our results show that the B subunits of enterotoxin adjuvants regulate IL-12R expression and subsequent Th cell subset responses.