Hypochlorous Acid-Induced Heme Degradation from Lactoperoxidase as a Novel Mechanism of Free Iron Release and Tissue Injury in Inflammatory Diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H2O2) in the airways through its ability to oxidize thiocyanate (SCN−) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 µM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O2, Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN−. On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function

Semiclassical thermodynamics of scalar fields

We present a systematic semiclassical procedure to compute the partition function for scalar field theories at finite temperature. The central objects in our scheme are the solutions of the classical equations of motion in imaginary time, with spatially independent boundary conditions. Field fluctuations -- both field deviations around these classical solutions, and fluctuations of the boundary value of the fields -- are resummed in a Gaussian approximation. In our final expression for the partition function, this resummation is reduced to solving certain ordinary differential equations. Moreover, we show that it is renormalizable with the usual 1-loop counterterms.Comment: 24 pages, 5 postscript figure

Prophylactic properties of a Leishmania-specific hypothetical protein in a murine model of visceral leishmaniasis

In this work, the effect of vaccination of a newly described Leishmania infantum antigenic protein has been studied in BALB/c mice infected with this parasite species. The LiHyD protein was characterized after a proteomic screening performed with the sera from dogs suffering visceral leishmaniasis (VL). Its recombinant version was expressed, purified and administered to BALB/c mice in combination with saponin. As a result of vaccination and 10 weeks after challenge using an infective dose of L. infantum stationary promastigotes, vaccinated mice showed lower parasite burdens in different organs (liver, spleen, bone marrow and footpads' draining lymph nodes) than mice inoculated with the adjuvant alone or the vaccine diluent. Protected mice showed anti-Leishmania IgG2a antibodies and a predominant IL-12-driven IFN-γ production (mainly produced by CD4 T cells) against parasite proteins, whereas unprotected controls showed anti-Leishmania IgG1 antibodies and parasite-mediated IL-4 and IL-10 responses. Vaccinated mice showed an anti-LiHyD IgG2a humoral response, and their spleen cells were able to secrete LiHyD-specific IFN-γ, IL-12 and GM-CSF cytokines before and after infection. The protection was correlated with the Leishmania-specific production on nitric oxide. Altogether, the results indicate that the new LiHyD protein could be considered in vaccine formulations against VL.Instituto Nacional de Ci^encia e Tecnologia em Nano-biofarmac^eutica (INCT-NanoBiofar), FAPEMIG (CBB-APQ-00819-12 and CBB-APQ-01778-2014) and CNPq (APQ-482976/2012-8, APQ-488237/2013-0 and APQ-467640/2014-9). In addition, this study was partially funded by the Spanish grant from Ministerio de Economía y Competitividad-FEDER (FIS PI14/00366 from the Instituto de Salud Carlos III)Peer Reviewe

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs

A Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis

Background: LiHyV is an antigenic hypothetical protein present in both promastigote and amastigote stages of Leishmania infantum, which was recently identified by an immunoproteomic approach. A recombinant version of this protein (rLiHyV) was evaluated as a diagnostic marker for canine VL (CVL). In addition, the prophylactic efficacy of the rLiHyV protein, and two of its CD8+ T cell epitopes, has been analyzed in a murine model of visceral leishmaniasis (VL). Methods: Initially, the rLiHyV protein was evaluated by an ELISA technique for the serodiagnosis of CVL. Secondly, vaccines composed of the recombinant protein and both chemically synthesized peptides, combined with saponin as an adjuvant; were administered subcutaneously into BALB/c mice. The cellular and humoral responses generated by vaccination were evaluated. In addition, the parasite burden and immune response were studied 10 weeks after L. infantum infection. Results: The rLiHyV protein was recognized by antibodies of VL dogs. No cross-reactivity was obtained with sera from dogs vaccinated with a Brazilian commercial vaccine, with sera from animals infected with Trypanosoma cruzi, Babesia canis and Ehrlichia canis, or those from non-infected animals living in an endemic area for leishmaniasis. After challenge with L. infantum, spleen cells of BALB/c mice vaccinated with rLiHyV/saponin stimulated with parasite antigens showed a higher production of IFN-γ, IL-12 and GM-CSF, than the same cells obtained from mice vaccinated with the individual peptides, or mice from control (inoculated with saline or saponin) groups. This Th1-type cellular response observed in rLiHyV/saponin vaccinated mice was accompanied by the induction of parasite-specific IgG2a isotype antibodies. Animals immunized with rLiHyV/saponin showed significant reductions in the parasite burden in the liver, spleen, bone marrow and in the lymph nodes draining the paws relative to control mice. Conclusions: The present study showed for the first time that the L. infantum LiHyV protein could be considered as a vaccine candidate against L. infantum infection, as well as a diagnostic marker for CVL.This work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, RHAE-456287/2012-4, APQ-482976/2012-8, and APQ-488237/2013-0). MACF is a grant recipient of FAPEMIG/CAPES. EAFC and APF are grant recipient of CNPq.Peer Reviewe

A Leishmania-specific hypothetical protein expressed in both promastigote and amastigote stages of Leishmania infantum employed for the serodiagnosis of, and as a vaccine candidate against, visceral leishmaniasis

Background: LiHyV is an antigenic hypothetical protein present in both promastigote and amastigote stages of Leishmania infantum, which was recently identified by an immunoproteomic approach. A recombinant version of this protein (rLiHyV) was evaluated as a diagnostic marker for canine VL (CVL). In addition, the prophylactic efficacy of the rLiHyV protein, and two of its CD8+ T cell epitopes, has been analyzed in a murine model of visceral leishmaniasis (VL). Methods: Initially, the rLiHyV protein was evaluated by an ELISA technique for the serodiagnosis of CVL. Secondly, vaccines composed of the recombinant protein and both chemically synthesized peptides, combined with saponin as an adjuvant; were administered subcutaneously into BALB/c mice. The cellular and humoral responses generated by vaccination were evaluated. In addition, the parasite burden and immune response were studied 10 weeks after L. infantum infection. Results: The rLiHyV protein was recognized by antibodies of VL dogs. No cross-reactivity was obtained with sera from dogs vaccinated with a Brazilian commercial vaccine, with sera from animals infected with Trypanosoma cruzi, Babesia canis and Ehrlichia canis, or those from non-infected animals living in an endemic area for leishmaniasis. After challenge with L. infantum, spleen cells of BALB/c mice vaccinated with rLiHyV/saponin stimulated with parasite antigens showed a higher production of IFN-γ, IL-12 and GM-CSF, than the same cells obtained from mice vaccinated with the individual peptides, or mice from control (inoculated with saline or saponin) groups. This Th1-type cellular response observed in rLiHyV/saponin vaccinated mice was accompanied by the induction of parasite-specific IgG2a isotype antibodies. Animals immunized with rLiHyV/saponin showed significant reductions in the parasite burden in the liver, spleen, bone marrow and in the lymph nodes draining the paws relative to control mice. Conclusions: The present study showed for the first time that the L. infantum LiHyV protein could be considered as a vaccine candidate against L. infantum infection, as well as a diagnostic marker for CVLThis work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, RHAE-456287/2012-4, APQ-482976/2012-8, and APQ-488237/2013-0). MACF is a grant recipient of FAPEMIG/CAPES. EAFC and APF are grant recipient of CNP

USE OF COMBINATION OF FLUORESCENT PROBES TO IDENTIFY SPERM SUBPOPULATIONS FOR QUALITY ASSESSMENT OF FRESH AND CRYOPRESERVED CANINE SEMEN. PRELIMINARY RESULTS

The use of fluorescent markers in the evaluation of sperm morphophysiology allows a better accuracy, compared to the subjective nature of some routine tests in semen qualification. In this study was used the combination of fluorescence probes: propionate iodide, Hoechst 33342 and FITC-PSA in fresh and thawed dog semen, to the identification of the following morphological subpopulations: II (intact plasma and acrosomal membranes), IL (intact plasma membrane and lesioned acrosomal membrane), LI (lesioned plasma membrane and intact acrosomal membrane) and LL (both membranes lesioned). When comparing the results obtained with the results of the tests used conventionally in semen evaluation (sperm motility and vigor, hypoosmotic test and morphological alterations), little correlation was observed. The II population declined from fresh semen to thawed, while LL population increased (p <0.05). The IL population was composed of extremely small numbers of cells but increased (p <0.05) from fresh semen to thawed semen. In the thawed semen the major defects had a positive correlation with the LL population (p <0.01). For the thawed semen, the results of the hypoosmotic test (number of cells that reacted to the medium) correlated positively with population II (p <0.025), that is, different from that observed in fresh semen. Although all tests were able to detect decrease in sperm quality post-thawing (p <0.05). The use of this fluorescent probe association allowed qualification and more accurately quantification of plasma membrane and acrosomal insults mediated by cryopreservation. El uso de marcadores fluorescentes en la evaluación de la morfofisiología espermática permite una mayor precisión, comparada con la naturaleza subjetiva de algunas pruebas de rutina en la valoración del semen. En este estudio se usó la combinación de pruebas fluorescentes: yoduro de propidio; Hoechst 33342 y FITC-PSA en semen fresco y descongelado de perro, para la identificación de las siguientes subpoblaciones morfológicas: II (membranas plasmática y acrosomal intactas), IL (membrana plasmática intacta y membrana acrosomal dañada), LI (membrana plasmática dañada y membrana acrosomal intacta) y LL (ambas membranas dañadas). Cuando se comparan los resultados obtenidos con los resultados de las pruebas usadas convencionalmente en la evaluación seminal (motilidad y vigor espermáticos, prueba hipoosmótica y alteraciones morfológicas), se observó poca correlación. La población II disminuyó desde el semen fresco al descongelado, mientras que la población LL se incrementó (p<0.05). la población IL estuvo compuesta por un número extremadamente pequeño de células, pero incremento (p<0.05) desde el semen fresco al descongelado. En el semen descongelado los defectos mayores tuvieron una correlación positiva con la población LL (p<0.01). En el semen descongelado, los resultados de la prueba hipoosmótica (número de células que reaccionan al medio) se correlacionaron positivamente con la población II (p<0.05). El uso de esta asociación de pruebas fluorescentes permitió la valoración y la cuantificación más precisa de los daños a la membrana plasmática y acrosomal mediados por la criopreservación.

Characterization of Soybean Cultivars for Biodiesel Production

Due to environmental issues involving the polluting gasesemission, Brazil has adopted the policy of using oil and biodiesel. For biodiesel production, the main raw material used in Brazil is soybean oil. The development of the numerous genotypes of this culture has always considered quantitative aspects. The objective was to qualitatively characterize 12 soybean cultivars for biodiesel production. The experimental design was randomized blocks with three replicates. The cultivars were sown in December 2016, in no-tillage system, in Ponta Grossa, Paraná, Brazil (-25.093056, -50.063327 UTM). The analyzed variables were: oil and protein contents, acidity index and specific mass. It was concluded that there were no significant differences among the cultivars for oil and protein contents. For the variables acidity index and specific mass, there were significant differences among the cultivars, being below the limits established by the Brazilian legislation for vegetable oil, but with potential for biodiesel production