Non-isoplethic measurement on the solid-liquid-vapor equilibrium of binary mixtures at cryogenic temperatures.

We measured the solid-liquid-vapor (SLV) equilibrium of binary mixtures during experiments that alternated between cooling the mixture and injecting the more-volatile component into the sample chamber; thus, the composition of the mixture changed (non-isoplethic) throughout the experiment. Four binary mixtures were used in the experiments to represent mixtures with miscible solid phases (N2/CO) and barely miscible solid solutions (N2/C2H6), as well as mixtures with intermediate solid miscibility (N2/CH4 and CO/CH4). We measured new SLV pressure data for the binary mixtures, except for N2/CH4, which are also available in the literature for verification in this work. While these mixtures are of great interest in planetary science and cryogenics, the resulting pressure data are also needed for modeling purposes. We found the results for N2/CH4 to be consistent with the literature. The resulting new SLV curve for CO/CH4 shows similarities to N2/CH4. Both have two density inversion points (bracketing the temperature range where the solid floats). This result is important for places such as Pluto, Triton, and Titan, where these mixtures exist in vapor, liquid, and solid phases. Based on our experiments, the presence of a eutectic is unlikely for the N2/CH4 and CO/CH4 systems. An azeotrope with or without a peritectic is likely, but further investigations are needed to confirm. The N2/CO system does not have a density inversion point, as the ice always sinks in its liquid. For N2/C2H6, new SLV pressure data were measured near each triple point of the pure components.

Complex Brines and Their Implications for Habitability.

There is evidence that life on Earth originated in cold saline waters around scorching hydrothermal vents, and that similar conditions might exist or have existed on Mars, Europa, Ganymede, Enceladus, and other worlds. Could potentially habitable complex brines with extremely low freezing temperatures exist in the shallow subsurface of these frigid worlds? Earth, Mars, and carbonaceous chondrites have similar bulk elemental abundances, but while the Earth is depleted in the most volatile elements, the Icy Worlds of the outer solar system are expected to be rich in them. The cooling of ionic solutions containing substances that likely exist in the Icy Worlds could form complex brines with the lowest eutectic temperature possible for the compounds available in them. Indeed, here, we show observational and theoretical evidence that even elements present in trace amounts in nature are concentrated by freeze-thaw cycles, and therefore contribute significantly to the formation of brine reservoirs that remain liquid throughout the year in some of the coldest places on Earth. This is interesting because the eutectic temperature of water-ammonia solutions can be as low as ~160 K, and significant fractions of the mass of the Icy Worlds are estimated to be water substance and ammonia. Thus, briny solutions with eutectic temperature of at least ~160 K could have formed where, historically, temperature have oscillated above and below ~160 K. We conclude that complex brines must exist in the shallow subsurface of Mars and the Icy Worlds, and that liquid saline water should be present where ice has existed, the temperature is above ~160 K, and evaporation and sublimation have been inhibited.

Stratification Dynamics of Titan's Lakes via Methane Evaporation.

Saturn's moon Titan is the only extraterrestrial body known to host stable lakes and a hydrological cycle. Titan's lakes predominantly contain liquid methane, ethane, and nitrogen, with methane evaporation driving its hydrological cycle. Molecular interactions between these three species lead to non-ideal behavior that causes Titan's lakes to behave differently than Earth's lakes. Here, we numerically investigate how methane evaporation and non-ideal interactions affect the physical properties, structure, dynamics, and evolution of shallow lakes on Titan. We find that, under certain temperature regimes, methane-rich mixtures are denser than relatively ethane-rich mixtures. This allows methane evaporation to stratify Titan's lakes into ethane-rich upper layers and methane-rich lower layers, separated by a strong compositional gradient. At temperatures above 86K, lakes remain well-mixed and unstratified. Between 84 and 86K, lakes can stratify episodically. Below 84K, lakes permanently stratify, and develop very methane-depleted epilimnia. Despite small seasonal and diurnal deviations (<5K) from typical surface temperatures, Titan's rain-filled ephemeral lakes and "phantom lakes" may nevertheless experience significantly larger temperature fluctuations, resulting in polymictic or even meromictic stratification, which may trigger ethane ice precipitation.

A Synthetic Nanoparticle Based Vaccine Approach Targeting MSP4/5 Is Immunogenic and Induces Moderate Protection Against Murine Blood-Stage Malaria.

Malaria remains a significant health problem in many tropical and sub-tropical regions. The development of vaccines against the clinically active blood-stage of infection needs to consider variability and polymorphism in target antigens, and an adjuvant system able to induce broad spectrum immunity comprising both antibodies and helper T cells. Moreover, recent studies have shown some conventional pro-inflammatory adjuvants can also promote expansion of immunosuppressive regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), both of which could negatively impact malaria disease progression. Herein, we explore the ability of a model nanoparticle delivery system (polystyrene nanoparticles; PSNPs), previously proven to not induce conventional inflammation, Treg or MDSC, to induce immunity to MSP4/5 from Plasmodium yoelii, a member of the MSP4 and MSP5 family of proteins which are highly conserved across diverse malaria species including P. falciparum. The results show PSNPs-MSP4/5 conjugates are highly immunogenic, inducing immune responses comprising both T helper 1 (Th1) and Th2 cellular immunity, and a spectrum of antibody subclasses including IgG1, IgG2a, and IgG2b. Benchmarked against Alum and Complete Freund's Adjuvant (CFA), the immune responses that were induced were of comparable or higher magnitude, for both T cell frequencies by ELISpot and antibody responses in terms of ELISA end titer. Importantly, immunization with PSNPs-MSP4/5 induced partial protection against malaria blood-stage infection (50-80%) shown to be mechanistically dependent on interferon gamma (IFN-γ) production. These results expand the scope of adjuvants considered for malaria blood-stage vaccine development to those that do not use conventional adjuvant pathways and emphasizes the critical role of cellular immunity and specifically IFN-γ producing cells in providing moderate protection against blood-stage malaria comparable to Freunds adjuvant.

Nanoparticles modify dendritic cell homeostasis and induce non-specific effects on immunity to malaria.

BACKGROUND: Many current vaccines to a specific pathogen influence responses to other pathogens in a process called heterologous immunity. We propose that their particulate nature contributes to non-specific effects. Herein, we demonstrate polystyrene nanoparticles modulate dendritic cell (DC) homeostasis, thereby promoting a persistent enhanced state of immune readiness to a subsequent infectious challenge. METHODS: Particles (approximately 40 nm and 500 nm carboxylated polystyrene nanoparticles; PSNPs) alone or conjugated to a model antigen were injected in mice, and DCs in draining lymph nodes (dLNs) and bone-marrow (BM) quantified by flow cytometry. BM cells were tested for capacity to generate DCs upon culture with granulocyte and macrophage colony stimulating factor. Mice were challenged with Plasmodium yoelli. Blood parasitaemias were monitored by GIEMSA. Sera was analyzed for antibodies by ELISA. RESULTS: Intradermal administration of 40 nm PSNPs induced anti-inflammatory cytokines, chemokines and growth factors, increased numbers and proportions of DCs in the dLN, and increased the capacity of BM to generate DCs. Consistent with these unexpected changes, 40 nm PSNPs pre-injected mice had enhanced ability to generate immunity to a subsequent malarial infection. CONCLUSIONS: Intradermal administration of 40 nm PSNPs modifies DC homeostasis, which may at least in part explain the observed beneficial heterologous effects of current particulate vaccines. Further nanotechnological developments may exploit such strategies to promote beneficial non-specific effects.

Antibodies to Plasmodium falciparum and Plasmodium vivax merozoite surface protein 5 in Indonesia: species-specific and cross-reactive responses.

BACKGROUND: Merozoite surface protein (MSP) 5 is a candidate antigen for a malaria vaccine. In cross-sectional and longitudinal studies, we measured MSP5 antibody responses in Papuans with acute Plasmodium falciparum malaria, Plasmodium vivax malaria, and mixed P. falciparum and P. vivax malaria and in those with past exposure. METHODS: Enzyme-linked immunosorbant assay (ELISA) was used to quantitate antibody responses to P. falciparum MSP5 (PfMSP5) and P. vivax MSP5 (PvMSP5) in 82 subjects with P. falciparum infection, 86 subjects with P. vivax infection, 85 subjects with mixed infection, and 87 asymptomatic individuals. Longitudinal responses through day 28 were tested in 20 persons. Cross-reactivity was tested by competition ELISA. RESULTS: PfMSP5 or PvMSP5 immunoglobulin (Ig)Gwas detected in 39%-52% of subjects, and IgM was detected in 44%-72%. IgG responses were distributed equally between IgG3 and IgG1 for PfMSP5 but were predominantly IgG3 for PvMSP5. Although IgG responses were generally specific for PfMSP5 or PvMSP5, cross-species reactivity was found in 7 of 107 dual-positive responders. No significant difference was seen in the magnitude, frequency, or subclass of PfMSP5 or PvMSP5 IgG antibodies between groups. There was no significant association between antibody responses and therapeutic response. CONCLUSION: PfMSP5 and PvMSP5 were frequently recognized by short-lived, species-specific antibodies. Although infrequent, the cross-reactive MSP5 antibodies indicate that an appropriately formulated vaccine may elicit and/or enhance cross-species recognition, which may be very useful in areas where both parasites are endemic.

Poly-L-lysine-coated nanoparticles: a potent delivery system to enhance DNA vaccine efficacy.

DNA formulations provide the basis for safe and cost efficient vaccines. However, naked plasmid DNA is only poorly immunogenic and new effective delivery strategies are needed to enhance the potency of DNA vaccines. In this study, we present a novel approach for the delivery of DNA vaccines using inert poly-L-lysine (PLL) coated polystyrene particles, which greatly enhance DNA immunogenicity. Intradermal injection of plasmid DNA encoding for chicken egg ovalbumin (OVA) complexed with PLL-coated polystyrene nanoparticles induced high levels of CD8 T cells as well as OVA-specific antibodies in C57BL/6 mice and furthermore inhibited tumour growth after challenge with the OVA expressing EG7 tumour cell line. Importantly, vaccine efficacy depended critically on the size of the particles used as well as on the presence of the PLL linker. Our data show that PLL-coated polystyrene nanoparticles of 0.05 microm but not 0.02 microm or 1.0 microm in diameter are highly effective for the delivery of DNA vaccines.

Short peptide sequences containing MHC class I and/or class II epitopes linked to nano-beads induce strong immunity and inhibition of growth of antigen-specific tumour challenge in mice.

Peptide based vaccines offer practical advantages, but unmodified peptides usually require an adjuvant or delivery vehicle to promote immunogenicity. When peptides containing ovalbumin (OVA) derived CD4 and CD8 T cell epitopes were conjugated to 0.05 microm nano-beads, they gave strong immune responses and inhibition of growth of tumour cells expressing the CD8 T cell epitope with MHC class I. These responses were inducible with both high (50 microg) and low (5 microg) peptide doses after a single immunisation. The helper CD4 T cell epitope was unnecessary for induction of CD8 T cell or tumour challenge responses. However, the CD4 T cell epitope contained a B cell epitope and triggered strong antibody responses. This simple approach offers a convenient experimental tool and a potentially useful clinical method for peptide immunisation.

Vaccines that facilitate antigen entry into dendritic cells.

Although vaccines have been highly successful in preventing and treating many infectious diseases (including smallpox, polio and diphtheria) diseases prevalent in the developing world such as malaria and HIV, that suppress the host immune system, require new, multiple strategies that will be defined by our growing understanding of specific immune activation. The definition of adjuvants, previously thought of as any substance that enhanced the immunogenicity of antigen, could now include soluble mediators and antigenic carriers that interact with surface molecules present on DC (e.g. LPS, Flt3L, heat shock protein) particulate antigens which are taken up by mechanisms available to APC but not other cell types (e.g. immunostimulatory complexes, latex, polystyrene particles) and viral/bacterial vectors that infect antigen presenting cells (e.g. vaccinia, lentivirus, adenovirus). These approaches, summarized herein, have shown potential in vaccinating against disease in animal models, and in some cases in humans. Of these, particle-antigen conjugates provide rapid formulation of the vaccine, easy storage and wide application, with both carrier and adjuvant functions that activate DC. Combined vaccines of the future could use adjuvants such as virus-like particles and particles targeted towards a predominant cellular type or immune response, with target cell activation enhanced by growth factors or maturation signals prior to, or during immunization. Collectively, these new additions to adjuvant technology provide opportunities for more specific immune regulation than previously available.