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A new series of [Fe3-xLnx]O4 nanoparticles, with Ln = Gd; Dy; Lu and x = 0.05; 0.1; 0.15, was synthesized using the coprecipitation method. Analyses by X-ray diffraction (XRD), Rietveld refinement, and high-resolution transmission electron microscopy (HRTEM) indicate that all phases crystallized in space group Fd3¯m, characteristic of spinels. The XRD patterns, HRTEM, scanning electron microscopy analysis (SEM-EDS), and Raman spectra showed single phases. Transmission electron microscopy (TEM), Rietveld analysis, and Scherrer's calculations confirm that these materials are nanoparticles with sizes in the range of ~6 nm to ~13 nm. Magnetic measurements reveal that the saturation magnetization (Ms) of the as-prepared ferrites increases with lanthanide chemical substitution (x), while the coercivity (Hc) has low values. The Raman analysis confirms that the compounds are ferrites and the Ms behavior can be explained by the relationship between the areas of the signals. The magnetic measurements indicate superparamagnetic behavior. The blocking temperatures (TB) were estimated from ZFC-FC measurements, and the use of the Néel equation enabled the magnetic anisotropy to be estimated.

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Gastropod Molluscs rely exclusively on the innate immune system to protect from pathogens, defending their embryos through maternally transferred effectors. In this regard, Pomacea snail eggs, in addition to immune defenses, have evolved the perivitellin-2 or PV2 combining two immune proteins into a neurotoxin: a lectin and a pore-forming protein from the Membrane Attack Complex/Perforin (MACPF) family. This binary structure resembles AB-toxins, a group of toxins otherwise restricted to bacteria and plants. Many of these are enterotoxins, leading us to explore this activity in PV2. Enterotoxins found in bacteria and plants act mainly as pore-forming toxins and toxic lectins, respectively. In animals, although both pore-forming proteins and lectins are ubiquitous, no enterotoxins have been reported. Considering that Pomacea snail eggs ingestion induce morpho-physiological changes in the intestinal mucosa of rodents and is cytotoxic to intestinal cells in culture, we seek for the factor causing these effects and identified PmPV2 from Pomacea maculata eggs. We characterized the enterotoxic activity of PmPV2 through in vitro and in vivo assays. We determined that it withstands the gastrointestinal environment and resisted a wide pH range and enzymatic proteolysis. After binding to Caco-2 cells it promoted changes in surface morphology and an increase in membrane roughness. It was also cytotoxic to both epithelial and immune cells from the digestive system of mammals. It induced enterocyte death by a lytic mechanism and disrupted enterocyte monolayers in a dose-dependent manner. Further, after oral administration to mice PmPV2 attached to enterocytes and induced large dose-dependent morphological changes on their small intestine mucosa, reducing the absorptive surface. Additionally, PmPV2 was detected in the Peyer's patches where it activated lymphoid follicles and triggered apoptosis. We also provide evidence that the toxin can traverse the intestinal barrier and induce oral adaptive immunity with evidence of circulating antibody response. As a whole, these results indicate that PmPV2 is a true enterotoxin, a role that has never been reported to lectins or perforin in animals. This extends by convergent evolution the presence of plant- and bacteria-like enterotoxins to animals, thus expanding the diversity of functions of MACPF proteins in nature.

Assuntos

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Zinc oxide nanoparticles were successfully synthesized under precipitation processes, using ZnSO4·7H2O as a Zn2+ precursor and K2CO3 used as a basic source, and hydrozincite was obtained as an intermediary, which was treated under two procedures; first procedure involved multiple stages to get final precipitated with NaOH, and in the second procedure the hydrozincite was straightforwardly dried at 220 °C. By both processes ZnO structures were obtained, which were turned into nanoparticles by a solvothermal treatment, for four hours in ethylene glycol at 200 °C. The final products for the first procedure was conglomerate of spherical nanoparticles with sizes ranged between 5-10 nm and dispersed ellipsoidal nanoparticles for the second procedure. Apart off the two procedures mentioned above, another synthesis was carried out with the same Zn2+ precursor but now using NaOH, and the solvothermal treatment produced ZnO mixed micro-structures which under ultrasonic cavitation disaggregated on mesoporous ZnO nanoplates of hexagonal shapes with nanopore sizes of approximately 0.35 nm. All ZnOs synthesized were structurally characterized with XRD, TEM and FT-IR techniques, and electronically with UV-Vis absorption and diffuse reflectance spectroscopies.

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The reaction of 2-aminonicotinaldehyde with 2- or 4-methoxyacetophenone in basic media leads to the new ligands 2-(4-methoxyphenyl)-1,8-naphthyridine and 2-(2-methoxyphenyl)-1,8-naphthyridine, respectively, in high yield. The reaction of these naphthyridine derivatives with [RuCl2(CO)2]n leads to the respective complexes cis-dicarbonyldichloridobis[2-(4-methoxyphenyl)-1,8-naphthyridine-κN8]ruthenium(II) and cis-dicarbonyldichloridobis[2-(2-methoxyphenyl)-1,8-naphthyridine-κN8]ruthenium(II), both [RuCl2(C15H12N2O)2(CO)2], in good yield. Both ruthenium(II) complexes display a slightly distorted octahedron with two cis carbonyl, two cis chloride and two cis naphthyridine ligands, the latter coordinated in a monodentate fashion through the N atom in the 8-position. Both complexes exhibit a moderate catalytic activity in the hydrogen-transfer reaction from propan-2-ol to acetophenone in the presence of a base, with 100% selectivity.

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Most pathogens infect through mucosal surfaces, and parenteral immunization typically fails to induce effective immune responses at these sites. Development of oral-administered vaccines capable of inducing mucosal as well as systemic immunity while bypassing the issues of antigen degradation and immune tolerance could be crucial for the control of enteropathogens. This study demonstrates that U-Omp19, a bacterial protease inhibitor with immunostimulatory features, coadministered with Salmonella antigens by the oral route, enhances mucosal and systemic immune responses in mice. U-Omp19 was able to increase antigen-specific production of IFN-γ and IL-17 and mucosal (IgA) antibody response. Finally, oral vaccination with U-Omp19 plus Salmonella antigens conferred protection against virulent challenge with Salmonella Typhimurium, with a significant reduction in bacterial loads. These findings prove the efficacy of this novel adjuvant in the Salmonella infection model and support the potential of U-Omp19 as a suitable adjuvant in oral vaccine formulations against mucosal pathogens requiring T helper (Th)1-Th17 protective immune responses.

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The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

Assuntos

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We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.

Assuntos

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Food allergies are increasingly common disorders and no therapeutic strategies are yet approved. The unlipidated Omp16 (U-Omp16) is the outer membrane protein of 16 kDa from B. abortus and possesses a mucosal adjuvant property. In this study, we aimed to examine the U-Omp16 capacity to abrogate an allergen-specific Th2 immune response when it is administered as an oral adjuvant in a mouse model of food allergy.   Balb/c mice were sensitized with cholera toxin and cow's milk proteins (CMP) by gavage and simultaneously treated with U-Omp16 and CMP. Oral challenge with CMP was performed to evaluate the allergic status of mice. Symptoms, local (small bowel cytokine and transcription factor gene expression) and systemic (specific isotypes and spleen cell-secreted cytokines) parameters, and skin tests were done to evaluate the immune response. We found that the oral administration of U-Omp16 with CMP during sensitization dampened the allergic symptoms, with negativization of immediate skin test and increased skin DTH response. Serum specific IgE and IL-5 were inhibited and a Th1 response was promoted (specific IgG2a antibodies and CMP-induced IFN-γ secretion). We found at the mucosal site an inhibition of the gene expression corresponding to IL-13 and Gata-3, with an induction of IFN-γ and T-bet. These results indicated that the oral administration of U-Omp16 significantly controlled the allergic response in sensitized mice with a shift of the balance of Th1- and Th2-T cells toward Th1 predominance. These findings suggest that U-Omp16 may be useful as a Th1-directing adjuvant in an oral vaccine.

Assuntos

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Canine brucellosis is an infectious disease caused by the Gram-negative bacterium Brucella canis. Unlike conventional control programs for other species of the genus Brucella, currently there is no vaccine available against canine brucellosis, and preventive measures are simply diagnosis and isolation of infected dogs. New approaches are therefore needed to develop an effective and safe immunization strategy against this zoonotic pathogen. In this study, BALB/c mice were subcutaneously immunized with the following: (i) the recombinant Brucella Omp31 antigen formulated in different adjuvants (incomplete Freund adjuvant, aluminum hydroxide, Quil A, and Montanide IMS 3012 VGPR), (ii) plasmid pCIOmp31, or (iii) pCIOmp31 plasmid followed by boosting with recombinant Omp31 (rOmp31). The immune response and the protective efficacy against B. canis infection were characterized. The different strategies induced a strong immunoglobulin G (IgG) response. Furthermore, spleen cells from rOmp31-immunized mice produced gamma interferon and interleukin-4 (IL-4) after in vitro stimulation with rOmp31, indicating the induction of a mixed Th1-Th2 response. Recombinant Omp31 administered with different adjuvants as well as the prime-boost strategy conferred protection against B. canis. In conclusion, our results suggest that Omp31 could be a useful candidate for the development of a subcellular vaccine against B. canis infection.

Assuntos

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Brucella, the etiological agent of animal and human brucellosis, is a bacterium with the capacity to modulate the inflammatory response. Cyclic ß-1,2-glucan (CßG) is a virulence factor key for the pathogenesis of Brucella as it is involved in the intracellular life cycle of the bacteria. Using comparative studies with different CßG mutants of Brucella, cgs (CßG synthase), cgt (CßG transporter) and cgm (CßG modifier), we have identified different roles for this polysaccharide in Brucella. While anionic CßG is required for bacterial growth in low osmolarity conditions, the sole requirement for a successful Brucella interaction with mammalian host is its transport to periplasmic space. Our results uncover a new role for CßG in promoting splenomegaly in mice. We showed that CßG-dependent spleen inflammation is the consequence of massive cell recruitment (monocytes, dendritics cells and neutrophils) due to the induction of pro-inflammatory cytokines such as IL-12 and TNF-α and also that the reduced splenomegaly response observed with the cgs mutant is not the consequence of changes in expression levels of the characterized Brucella PAMPs LPS, flagellin or OMP16/19. Complementation of cgs mutant with purified CßG increased significantly spleen inflammation response suggesting a direct role for this polysaccharide.

Assuntos