Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

Background

The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein.

Methods

We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation.

Results

The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections.

Conclusion

These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.     

Repertoire Sharing and Song Similarity between Great Tit Males Decline with Distance between Forest Fragments

Birdsong can play a critical role in establishing a territory and finding a mate among individuals from local and foreign populations. Variation in birdsong among populations can be influenced by habitat fragmentation and might affect successful dispersal among habitat fragments. We studied variation in great tit song in a long‐term study population distributed over nine forest fragments. All individual males recorded had a known dispersal history within the fragmented forest habitat. We found spatial structure of declining song‐type sharing with distance, with a marked drop from an individual’s own forest fragment to another across a habitat gap. We also found decreasing song similarity among increasingly distant fragments in terms of temporal and spectral characteristics of shared song types. The change in acoustic structure was more gradual and seemed less affected by habitat discontinuity but also showed a tight correlation with dispersal index among forest fragments. Immigrant birds shared fewer song types with neighbouring birds that were born within the same forest fragment, but not less compared to birds born in another forest fragment within the study area. Our data provide detailed insight into the relationship between song differentiation and male dispersal and contribute to our understanding of the potential role of song in reproductive exchange and avian speciation. The fact that birds in small forest fragments shared more songs than birds in larger forest fragments confirms that song analysis has potential as a tool for conservation in rare species.     

The importance of forest patch networks for the conservation of the Thorn-tailed Rayaditos in central Chile

Conservation of forest birds in fragmented landscapes requires not only determining the critical patch characteristics influencing local population persistence but also identifying patch networks providing connectivity and suitable habitat conditions necessary to ensure regional persistence. In this study, we assessed the importance of patch attributes, patch connectivity, and network components (i.e., groups of interconnected patches) in explaining the occupancy pattern of the Thorn-tailed Rayadito (Aphrastura spinicauda), a forest bird species of central Chile. Using a daily movement threshold distance, we identified a total of 16 network components of sclerophyllous forest within the study area. Among those components, patch area and vegetation structure-composition were important predictors of patch occupancy. However, the inclusion of patch connectivity and component size (i.e., the area of a network component) into the models greatly increases the models’ accuracy and parsimony. Using the best-fitted model, a total of 33 patches were predicted to be occupied by rayaditos within the study area, but such occupied patches were distributed in only six network components. These results suggest that persistence of rayaditos in central Chile requires the maintenance of large single patches and patch networks providing habitat and connectivity.     

Multistability in a Model for CTL Response to HTLV-I Infection and Its Implications to HAM/TSP Development and Prevention

Human T-cell leukaemia/lymphoma virus type I (HTLV-I) is a retrovirus that has been identified as the causative agent of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and other illnesses. HTLV-I infects primarily CD4⁺ T cells and the transmission occurs through direct cell-to-cell contact. HAM/TSP patients harbor higher proviral loads in peripheral blood lymphocytes than asymptomatic carriers. Also, HAM/TSP patients exhibit a remarkably high number of circulating HTLV-I-specific CD8⁺ cytotoxic T lymphocytes (CTLs) in the peripheral blood. While CTLs have a protective role by killing the infected cells and lowering the proviral load, a high level of CTLs and their cytotoxicity are believed to be a main cause of the development of HAM/TSP. A mathematical model for HTLV-I infection of CD4⁺ T cells that incorporates the CD8⁺ cytotoxic T-cell (CTL) response is investigated. Our mathematical analysis reveals that the system can stabilize at a carrier steady-state with persistent viral infection but no CTL response, or at a HAM/TSP steady-state at which both the viral infection and CTL response are persistent. We also establish two threshold parameters R ₀ and R ₁, the basic reproduction numbers for viral persistence and for CTL response, respectively. We show that the parameter R ₁ can be used to distinguish asymptomatic carriers from HAM/TSP patients, and as an important control parameter for preventing the development of HAM/TSP.     

Optimization of the Water-Insoluble Procedures for <Emphasis Type="Italic">USP</Emphasis> General Chapter <Emphasis Type="Italic">Residual Solvents</Emphasis> <467>

The water-insoluble procedures in US Pharmacopeia (USP) General Chapter Residual Solvents <467>, which are based on European Pharmacopoeia procedures, were optimized and modified before their inclusion in the chapter to improve their scope, performance, and ruggedness. The optimized procedures use a static headspace introduction system with a gas chromatograph equipped with a flame ionization detector. This article describes some of the key changes made to the USP published procedures, including use of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) as the solvent, addition of 5 mL of water and 1 mL of sample (dissolved in DMSO or DMF) to the headspace vial, use of a 3:1 GC split ratio, and use of new matrix-matched system suitability solutions. These procedures were verified with two different active pharmaceutical ingredients—hydroxyzine pamoate and prednisone. In the investigation, the more polar material (hydroxyzine pamoate) showed greater recoveries for the optimized procedures when prepared in DMSO. The less polar material (prednisone) typically had greater recoveries in DMF for the optimized procedures. During experimentation, insights into sample preparation, additional types of headspace instrumentation, solvent purity, and other parameters were also gained.     

Molecular phylogeny of mangrove oysters (Crassostrea) from Brazil

As a result of phenotypic plasticity, the cupped oysters (Crassostrea)are difficult to identify by means of their morphology. However,molecular DNA markers are a useful means of discriminating amongthese species. Cupped oysters are one of the most widely culturedmarine invertebrates and correct species identification is importantin aquaculture. Moreover, the molecular phylogeny of the genusCrassostrea and the subfamily Crassostreinae is still not clear.In order to identify the Brazilian cupped oysters and to clarifythe phylogenetic relationships of these species, we sequenceda fragment of mitochondrial DNA (16S rRNA gene) from 120 specimenscollected at nine different sites distributed along the Braziliancoast. The results identified two native species of oyster:Crassostrea gasar, from the Amazon to the Parnaíba delta;and Crassostrea rhizophorae, from the northeast (Fortim) tothe south of Brazil. An exotic Crassostrea species, closelyrelated to Indo-Pacific Crassostrea, was found in one locationin the north of Brazil. Crassostrea showed monophyly and theAtlantic oysters are clearly separated from the Indo-Pacificcluster. (Received 30 May 2006; accepted 12 April 2007)     

Chitons (Mollusca: Polyplacophora) from El Salvador, Central America

Collections of 11 species of shallow water Polyplacophora from El Salvador were made in July 2002. Previously only five species had been documented in El Salvador: Chaetopleura lurida (Sowerby, 1832); Ischnochiton guatemalensis (Thiele, 1910); Ceratozona angusta (Thiele, 1909); Chiton stokesii (Broderip, 1832) and Acantochitona exquisita (Pilsbry, 1893). Of these, L. guatemalensis and A. exquisita were not collected in this census. Seven other species are reported here for El Salvador for the first time: Lepidochitona beanii (Carpenter, 1857); Ischnochiton dispar (Sowerby, 1832); Stenoplax limaciformis (Sowerby, 1832); Callistochiton expressus (Carpenter, 1865); Acanthochitona arragonites (Carpenter, 1867); A. ferreirai (Lyons, 1988) and A. hirudiniformis (Sowerby, 1832). The known geographic distribution of 1. dispar is extended to the north. An un-named species of Lepidochitona is briefly described.     

Excretion and conservation of glycerol, and expression of aquaporins and glyceroporins, during cold acclimation in Cope's gray tree frog Hyla chrysoscelis

Cope's gray tree frog Hyla chrysoscelis accumulates glycerol during cold acclimation. We hypothesized that, during this process, gray tree frogs adjust renal filtration and/or reabsorption rates to retain accumulated glycerol. During cold acclimation, plasma concentrations of glycerol rose >200-fold, to 51 mmol/l. Although fractional water reabsorption decreased, glomerular filtration rate (GFR) and, consequently, urine flow were <5% of warm levels, and fractional glycerol reabsorption increased. In contrast, dehydrated frogs increased fractional water reabsorption, decreased GFR, and did not accumulate glycerol. We hypothesized that expression of proteins from the aquaporin (AQP)/glyceroporin (GLP) family was associated with changing patterns of water and glycerol movement. We cloned the cDNA for three such proteins, quantified mRNA expression in nine tissues using real-time quantitative PCR, and functionally characterized them using a Xenopus oocyte expression system. HC-1, an AQP1-like water channel conferring low glycerol permeability, is expressed ubiquitously in warm- and cold-acclimated tissues. HC-2, a water channel most similar to AQP2, is primarily expressed in organs of osmoregulation. HC-3, which is most similar to AQP3, is functionally characterized as a GLP, with low permeability to water but high permeability to glycerol. Aspects of expression levels and functional characteristics varied between cold and warm conditions for each of the three AQPs, suggesting a complex pattern of involvement in osmoregulation related to thermal acclimation.     

Resveratrol increases vascular oxidative stress resistance

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its vasculoprotective effects are not completely understood. Because oxidative stress and endothelial cell injury play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits oxidative stress-induced endothelial apoptosis. We found that oxidized LDL and TNF-alpha elicited significant increases in caspase-3/7 activity in endothelial cells and cultured rat aortas, which were prevented by resveratrol pretreatment (10(-6)-10(-4) mol/l). The protective effect of resveratrol was attenuated by inhibition of glutathione peroxidase and heme oxygenase-1, suggesting a role for antioxidant systems in the antiapoptotic action of resveratrol. Indeed, resveratrol treatment protected cultured aortic segments and/or endothelial cells against increases in intracellular H(2)O(2) levels and H(2)O(2)-mediated apoptotic cell death induced by oxidative stressors (exogenous H(2)O(2), paraquat, and UV light). Resveratrol treatment also attenuated UV-induced DNA damage (comet assay). Resveratrol treatment upregulated the expression of glutathione peroxidase, catalase, and heme oxygenase-1 in cultured arteries, whereas it had no significant effect on the expression of SOD isoforms. Resveratrol also effectively scavenged H(2)O(2) in vitro. Thus resveratrol seems to increase vascular oxidative stress resistance by scavenging H(2)O(2) and preventing oxidative stress-induced endothelial cell death. We propose that the antioxidant and antiapoptotic effects of resveratrol, together with its previously described anti-inflammatory actions, are responsible, at least in part, for its cardioprotective effects.