Synthesis and Detection of Oxygen-18 Labeled Phosphate

Phosphorus (P) has only one stable isotope and therefore tracking P dynamics in ecosystems and inferring sources of P loading to water bodies have been difficult. Researchers have recently employed the natural abundance of the ratio of 18O/16O of phosphate to elucidate P dynamics. In addition, phosphate highly enriched in oxygen-18 also has potential to be an effective tool for tracking specific sources of P in the environment, but has so far been used sparingly, possibly due to unavailability of oxygen-18 labeled phosphate (OLP) and uncertainty in synthesis and detection. One objective of this research was to develop a simple procedure to synthesize highly enriched OLP. Synthesized OLP is made up of a collection of species that contain between zero and four oxygen-18 atoms and, as a result, the second objective of this research was to develop a method to detect and quantify each OLP species. OLP was synthesized by reacting either PCl5 or POCl3 with water enriched with 97 atom % oxygen-18 in ambient atmosphere under a fume hood. Unlike previous reports, we observed no loss of oxygen-18 enrichment during synthesis. Electrospray ionization mass spectrometertry (ESI-MS) was used to detect and quantify each species present in OLP. OLP synthesized from POCl3 contained 1.2% P18O16O3, 18.2% P18O216O2, 67.7% P18O316O, and 12.9% P18O4, and OLP synthesized from PCl5 contained 0.7% P16O4, 9.3% P18O316O, and 90.0% P18O4. We found that OLP can be synthesized using a simple procedure in ambient atmosphere without the loss of oxygen-18 enrichment and ESI-MS is an effective tool to detect and quantify OLP that sheds light on the dynamics of synthesis in ways that standard detection methods cannot

Ears of the Armadillo: Global Health Research and Neglected Diseases in Texas

Neglected tropical diseases (NTDs) have\ud been recently identified as significant public\ud health problems in Texas and elsewhere in\ud the American South. A one-day forum on the\ud landscape of research and development and\ud the hidden burden of NTDs in Texas\ud explored the next steps to coordinate advocacy,\ud public health, and research into a\ud cogent health policy framework for the\ud American NTDs. It also highlighted how\ud U.S.-funded global health research can serve\ud to combat these health disparities in the\ud United States, in addition to benefiting\ud communities abroad

Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasisThe study was supported in Spain by grants from Ministerio de Ciencia e Innovación FIS PI11/00095 and FISPI14/00366 from the Instituto de Salud Carlos III within the Network of TropicalDiseases Research (VI P I+D+I 2008-2011, ISCIII -Subdirección General de Redes y Centros de Investigación Cooperativa (RD12/0018/0009)). This work was also supported in Brazil by a grant from CNPq (Ciencia sem Fronteiras-PVE 300174/2014-4). A CBMSO institutional grant from Fundación Ramón Areces is also acknowledged. EAFC is a grant recipient of CNPq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Impact of the HIV-1 env Genetic Context outside HR1–HR2 on Resistance to the Fusion Inhibitor Enfuvirtide and Viral Infectivity in Clinical Isolates

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1–HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1–HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1–HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1–HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy

Study of Leishmania pathogenesis in mice : experimental considerations

Although leishmaniases are endemic in 98 countries, they are still considered neglected tropical diseases. Leishmaniases are characterized by the emergence of new virulent and asymptomatic strains of Leishmania spp. and, as a consequence, by a very diverse clinical spectrum. To fight more efficiently these parasites, the mechanisms of host defense and of parasite virulence need to be thoroughly investigated. To this aim, animal models are widely used. However, the results obtained with these models are influenced by several experimental parameters, such as the mouse genetic background, parasite genotype, inoculation route/infection site, parasite dose and phlebotome saliva. In this review, we propose an update on their influence in the two main clinical forms of the disease: cutaneous and visceral leishmaniases

Evaluation of Leishmania donovani Protein Disulfide Isomerase as a Potential Immunogenic Protein/Vaccine Candidate against Visceral Leishmaniasis

In Leishmania species, Protein disulfide isomerase (PDI) - a redox chaperone, is reported to be involved in its virulence and survival. This protein has also been identified, through proteomics, as a Th1 stimulatory protein in the soluble lysate of a clinical isolate of Leishmania donovani (LdPDI). In the present study, the molecular characterization of LdPDI was carried out and the immunogenicity of recombinant LdPDI (rLdPDI) was assessed by lymphocyte proliferation assay (LTT), nitric oxide (NO) production, estimation of Th1 cytokines (IFN-γ and IL-12) as well as IL-10 in PBMCs of cured/endemic/infected Leishmania patients and cured L. donovani infected hamsters. A significantly higher proliferative response against rLdPDI as well as elevated levels of IFN-γ and IL-12 were observed. The level of IL-10 was found to be highly down regulated in response to rLdPDI. A significant increase in the level of NO production in stimulated hamster macrophages as well as IgG2 antibody and a low level of IgG1 in cured patient's serum was observed. Higher level of IgG2 antibody indicated its Th1 stimulatory potential. The efficacy of pcDNA-LdPDI construct was further evaluated for its prophylactic potential. Vaccination with this construct conferred remarkably good prophylactic efficacy (∼90%) and generated a robust cellular immune response with significant increases in the levels of iNOS transcript as well as TNF-α, IFN-γ and IL-12 cytokines. This was further supported by the high level of IgG2 antibody in vaccinated animals. The in vitro as well as in vivo results thus indicate that LdPDI may be exploited as a potential vaccine candidate against visceral Leishmaniasis (VL)

Critical Role of IRF-5 in the Development of T helper 1 responses to Leishmania donovani infection

The transcription factor Interferon Regulatory Factor 5 (IRF-5) has been shown to be involved in the induction of proinflammatory cytokines in response to viral infections and TLR activation and to play an essential role in the innate inflammatory response. In this study, we used the experimental model of visceral leishmaniasis to investigate the role of IRF-5 in the generation of Th1 responses and in the formation of Th1-type liver granulomas in Leishmania donovani infected mice. We show that TLR7-mediated activation of IRF-5 is essential for the development of Th1 responses to L. donovani in the spleen during chronic infection. We also demonstrate that IRF-5 deficiency leads to the incapacity to control L. donovani infection in the liver and to the formation of smaller granulomas. Granulomas in Irf5-/- mice are characterized by an increased IL-4 and IL-10 response and concomitant low iNOS expression. Collectively, these results identify IRF-5 as a critical molecular switch for the development of Th1 immune responses following L. donovani infections and reveal an indirect role of IRF-5 in the regulation of iNOS expression

Critical Role of IRF-5 in the Development of T helper 1 responses to Leishmania donovani infection

The transcription factor Interferon Regulatory Factor 5 (IRF-5) has been shown to be involved in the induction of proinflammatory cytokines in response to viral infections and TLR activation and to play an essential role in the innate inflammatory response. In this study, we used the experimental model of visceral leishmaniasis to investigate the role of IRF-5 in the generation of Th1 responses and in the formation of Th1-type liver granulomas in Leishmania donovani infected mice. We show that TLR7-mediated activation of IRF-5 is essential for the development of Th1 responses to L. donovani in the spleen during chronic infection. We also demonstrate that IRF-5 deficiency leads to the incapacity to control L. donovani infection in the liver and to the formation of smaller granulomas. Granulomas in Irf5-/- mice are characterized by an increased IL-4 and IL-10 response and concomitant low iNOS expression. Collectively, these results identify IRF-5 as a critical molecular switch for the development of Th1 immune responses following L. donovani infections and reveal an indirect role of IRF-5 in the regulation of iNOS expression

HIV-1 Residual Viremia Correlates with Persistent T-Cell Activation in Poor Immunological Responders to Combination Antiretroviral Therapy

BACKGROUND:The clinical significance and cellular sources of residual human immunodeficiency virus type 1 (HIV-1) production despite suppressive combination antiretroviral therapy (cART) remain unclear and the effect of low-level viremia on T-cell homeostasis is still debated. METHODOLOGY/PRINCIPAL FINDINGS:We characterized the recently produced residual viruses in the plasma and short-lived blood monocytes of 23 patients with various immunological responses to sustained suppressive cART. We quantified the residual HIV-1 in the plasma below 50 copies/ml, and in the CD14(high) CD16(-) and CD16+ monocyte subsets sorted by flow cytometry, and predicted coreceptor usage by genotyping V3 env sequences. We detected residual viremia in the plasma of 8 of 10 patients with poor CD4+ T-cell reconstitution in response to cART and in only 5 of 13 patients with good CD4+ T-cell reconstitution. CXCR4-using viruses were frequent among the recently produced viruses in the plasma and in the main CD14(high) CD16(-) monocyte subset. Finally, the residual viremia was correlated with persistent CD4+ and CD8+ T-cell activation in patients with poor immune reconstitution. CONCLUSIONS:Low-level viremia could result from the release of archived viruses from cellular reservoirs and/or from ongoing virus replication in some patients. The compartmentalization of the viruses between the plasma and the blood monocytes suggests at least two origins of residual virus production during effective cART. CXCR4-using viruses might be produced preferentially in patients on cART. Our results also suggest that low-level HIV-1 production in some patients may contribute to persistent immune dysfunction despite cART