Recombinant lipidated dengue-4 envelope protein domain III elicits protective immunity

AbstractThe combination of recombinant protein antigens with an immunostimulator has the potential to greatly increase the immunogenicity of recombinant protein antigens. In the present study, we selected the dengue-4 envelope protein domain III as a dengue vaccine candidate and expressed the protein in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-4 envelope protein domain III folded into the proper conformation and competed with the dengue-4 virus for cellular binding sites. Mice immunized with lipidated dengue-4 envelope protein domain III without exogenous adjuvant had higher frequencies of dengue-4 envelope protein domain III-specific B cells secreting antibodies than mice immunized with the nonlipidated form. Importantly, lipidated dengue-4 envelope protein domain III-immunized mice demonstrated a durable neutralizing antibody response and had reduced viremia levels after challenge. The study demonstrates that lipidated dengue-4 envelope protein domain III is immunogenic and may be a potential dengue vaccine candidate. Furthermore, the lipidation strategy can be applied to other serotypes of dengue virus

A Novel Single-Dose Dengue Subunit Vaccine Induces Memory Immune Responses

To protect against dengue viral infection, a novel lipidated dengue subunit vaccine was rationally designed to contain the consensus amino acid sequences derived from four serotypes of dengue viruses. We found that the lipidated consensus dengue virus envelope protein domain III (LcED III) is capable of activating antigen-presenting cells and enhancing cellular and humoral immune responses. A single-dose of LcED III immunization in mice without extra adjuvant formulation is sufficient to elicit neutralizing antibodies against all four serotypes of dengue viruses. In addition, strong memory responses were elicited in mice immunized with a single-dose of LcED III. Quick, anamnestic neutralizing antibody responses to a live dengue virus challenge were elicited at week 28 post-immunization. These results demonstrate the promising possibility of a future successful tetravalent vaccine against dengue viral infections that utilizes one-dose vaccination with LcED III

Dengue-1 Envelope Protein Domain III along with PELC and CpG Oligodeoxynucleotides Synergistically Enhances Immune Responses

The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus

CD74 deficiency ameliorates Pseudomonas aeruginosa-induced ocular infection

Eye trauma and contact lens wear are the main factors that predispose to the development of infectious keratitis. The existing therapies fail to control the inflammation-driven tissue damage that occurs during Pseudomonas aeruginosa infection. Antibiotic treatment reduces bacterial burdens, but better interventions are needed to alleviate tissue damage resulting from local inflammation. We have previously documented that inhibition of macrophage migration inhibitory factor (MIF) reduces the bacterial levels and the inflammatory damage during keratitis. Here, we report that mice deficient for CD74, the putative MIF receptor, developed milder Pseudomonas aeruginosa-induced disease, characterized by decreased proinflammatory mediators and reduced bacterial presence in the cornea. However, topical inhibition of MIF using antibodies applied to the cornea further promoted recovery from disease, suggesting that in addition to MIF-dependent signaling events, MIF-triggered CD74-independent signaling pathways regulate sensitization to P. aeruginosa-induced infection

Crack formation and prevention in colloidal drops

Crack formation is a frequent result of residual stress release from colloidal films made by the evaporation of colloidal droplets containing nanoparticles. Crack prevention is a significant task in industrial applications such as painting and inkjet printing with colloidal nanoparticles. Here, we illustrate how colloidal drops evaporate and how crack generation is dependent on the particle size and initial volume fraction, through direct visualization of the individual colloids with confocal laser microscopy. To prevent crack formation, we suggest use of a versatile method to control the colloid-polymer interactions by mixing a nonadsorbing polymer with the colloidal suspension, which is known to drive gelation of the particles with short-range attraction. Gelation-driven crack prevention is a feasible and simple method to obtain crack-free, uniform coatings through drying-mediated assembly of colloidal nanoparticlesopen0

Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors

TRPM2 channel deficiency prevents delayed cytosolic Zn²⁺ accumulation and CA1 pyramidal neuronal death after transient global ischemia

Transient ischemia is a leading cause of cognitive dysfunction. Postischemic ROS generation and an increase in the cytosolic Zn²⁺ level ([Zn²⁺]c) are critical in delayed CA1 pyramidal neuronal death, but the underlying mechanisms are not fully understood. Here we investigated the role of ROS-sensitive TRPM2 (transient receptor potential melastatin-related 2) channel. Using in vivo and in vitro models of ischemia-reperfusion, we showed that genetic knockout of TRPM2 strongly prohibited the delayed increase in the [Zn²⁺]c, ROS generation, CA1 pyramidal neuronal death and postischemic memory impairment. Time-lapse imaging revealed that TRPM2 deficiency had no effect on the ischemia-induced increase in the [Zn²⁺]c but abolished the cytosolic Zn²⁺ accumulation during reperfusion as well as ROS-elicited increases in the [Zn²⁺]c. These results provide the first evidence to show a critical role for TRPM2 channel activation during reperfusion in the delayed increase in the [Zn²⁺]c and CA1 pyramidal neuronal death and identify TRPM2 as a key molecule signaling ROS generation to postischemic brain injury

Co-localization and regulation of basic fibroblast growth factor and arginine vasopressin in neuroendocrine cells of the rat and human brain

<p>Abstract</p> <p>Background</p> <p>Adult rat hypothalamo-pituitary axis and choroid plexus are rich in basic fibroblast growth factor (FGF2) which likely has a role in fluid homeostasis. Towards this end, we characterized the distribution and modulation of FGF2 in the human and rat central nervous system. To ascertain a functional link between arginine vasopressin (AVP) and FGF2, a rat model of chronic dehydration was used to test the hypothesis that FGF2 expression, like that of AVP, is altered by perturbed fluid balance.</p> <p>Methods</p> <p>Immunohistochemistry and confocal microscopy were used to examine the distribution of FGF2 and AVP neuropeptides in the normal human brain. In order to assess effects of chronic dehydration, Sprague-Dawley rats were water deprived for 3 days. AVP neuropeptide expression and changes in FGF2 distribution in the brain, neural lobe of the pituitary and kidney were assessed by immunohistochemistry, and western blotting (FGF2 isoforms).</p> <p>Results</p> <p>In human hypothalamus, FGF2 and AVP were co-localized in the cytoplasm of supraoptic and paraventricular magnocellular neurons and axonal processes. Immunoreactive FGF2 was associated with small granular structures distributed throughout neuronal cytoplasm. Neurohypophysial FGF2 immunostaining was found in axonal processes, pituicytes and Herring bodies. Following chronic dehydration in rats, there was substantially-enhanced FGF2 staining in basement membranes underlying blood vessels, pituicytes and other glia. This accompanied remodeling of extracellular matrix. Western blot data revealed that dehydration increased expression of the hypothalamic FGF2 isoforms of ca. 18, 23 and 24 kDa. In lateral ventricle choroid plexus of dehydrated rats, FGF2 expression was augmented in the epithelium (Ab773 as immunomarker) but reduced interstitially (Ab106 immunostaining).</p> <p>Conclusions</p> <p>Dehydration altered FGF2 expression patterns in AVP-containing magnocellular neurons and neurohypophysis, as well as in choroid plexus epithelium. This supports the involvement of centrally-synthesized FGF2, putatively coupled to that of AVP, in homeostatic mechanisms that regulate fluid balance.</p