Efficacy of subcutaneous immunotherapy achieved with a whole-body extract of Pseudomyrmex ant. A case reported in a young woman, in Argentina

The aim of this work is to report for the first time a patient case with anaphylaxis by Pseudomyrmex acanthobius /favidulus ant sting, prepare an extract with the wholeant-body, to study their biochemical and immunological properties and to validate the efficacy of the subcutaneous immunotherapy with this non-commercial extract. An argentine woman patient, 19 years old, with background of allergic rhinitis and bronchial asthma in the childhood, suffered repeated episodes of anaphylaxis by non-identified insect’s stings and previous immunotherapies treatment. The entomologic analysis revealed that the aggressor insect was an ant belonging to Pseudomyrmex genera and acanthobius or favidulus species. High serum total levels of IgE (202 UI/ml) were measured by ELISA and a positive 6-mm wheal and flare reaction (extract dilution 1/100000) was revealed by in vivo intradermal test. A sample of the extract proteins fraction was analysed by SDS⁄PAGE. The silver stained protein bands ranged since 20 to 220 kDa. The patient serum immune-recognized allergenic extract proteins at approximately 160, 90, and a double band at 42/46 kDa prior to desensitization treatment by electro-blotting. Interestingly, post-specific-subcutaneous-immunotherapy the mentioned double band almost disappeared. After a 6-years-treatment, the total IgE serum values strongly decreased and the specific intradermal test was negative up to 1/10 extract dilution. The tolerance to the treatment was good. Altogether, our findings revealed that the immunotherapy performed with the whole-body-Pseudomyrmex ant allergenic extract was highly effective and the IgE specific-double protein allergenic extract bands (42/46 kDa) that disappeared after immunotherapy were responsible for the anaphylactic shock.Fil: Rodríguez Rodríguez, Raquel Milagros. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Irañeta, Silvia G.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Olgiati, María Laura. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Soprano, Luciana Lía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mouchian, Krikor. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Alonso, Angel. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Duschak, Vilma Gladys. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Input of NAcGlc6SO3 epitopes (sulfotopes) present in Trypanosoma cruzi glycoproteins, and their specific antibodies, in the infection and immune pathogenesis of experimental Chagas disease

Background: Trypanosoma cruzi, the causative agent of Chagas disease contains a major antigen, cruzipain (Cz). The C-terminal domain (C-T) of this glycoprotein bears N-linked high mannose type sulfated oligosaccharide chains and is responsible for most antibodiesinnaturalandexperimentalinfections.Miceimmunization with C-T has shown that sulfate moieties of Cz molecule are targets for specific immune responses and responsible for cardiac ultrastructural abnormalities in absence of infection. Methods & Materials: After the molecular characterization of these sufotopes, BALB/c mice were immunized with Cz/C-T, prior and after desulfation treatment, and with NAcGlc6SO3-BSA, to be furthersublethallychallengedwithtrypomastigotestoinvestigate whether they are involved in immunepathogenesis and/or infection of experimental Chagas disease. Results: C-T-immunized mice showed low IL-4 levels and elevated IFN- concentration by capture ELISA using C-T as stimulus and a cytokines profile compatible with a mixed response showing: Th2 tendency with excessively high IFN and raised IL-17 levels. By contrast, dC-T-immunized-mice presented undetectable IL-4 levels, low IFN- level and a cytokines profile like that of control but with a significantly elevated IL-10 value. In addition, ultrastructuralcardiacalterationsandmainimmunorecognitionof fibrils and mitochondria were observed in C-T-immunized mice bothconfrontedwithpolyclonalanti-CzandmyosinadsorbedantiCz sera. After sublethal challenge, elevated parasitaemias were observed. Mortality was 20 and 80% in C-T and dC-T immunized mice, respectively and mice from dC-T group that survived presentedseveremusclealterations.BSA-NAcGlc6SO3-immunized mice mounted a predominant IgG1and IgG2b immune response followed by IgG2a, demonstrating the immunodominance of the sulfotope and a vigorous mice memory T cells response, similarly toC-T-immunizedmice.Aftersublethalinfection,miceimmunized with the sulfotope displayed excessively elevated parasitemias, similarIFN-levelsandsignificantlowermortalitypercentagethan those from BSA-NAcGlc control group. Furthermore, mice treated by passive transference of sulfate-specific IgGs purified from sera of BSA-NAcGlc6SO3-immunized mice, exhibited ultrastructural alterations in cardiac tissue. After challenge, those treated with sulfate-specific IgGs presented higher parasitemias than controls Conclusion:Altogether,thesefindingshavedemonstratedthat sulfotopes and their specific antibodies display a dual role, participating in the host-tissue immunopathogenicity of experimental Chagas disease and favoring the infection by T. cruziFil: Soprano, Luciana Lía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferrero, Maximiliano Ruben. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olgiati, María Laura. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Landoni, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Garcia, Gabriela Andrea. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Esteva, Mónica Inés. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Duschak, Vilma Gladys. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina18th International Congress of Infectious diseasesBuenos AiresArgentinaInternational Society of Infectious Disease

Sulfatación y sulfotopes en Trypanosoma cruzi, agente causal de la Enfermedad de Chagas

Trypanosoma cruzi, agente causal de la enfermedad de Chagas- (ChD), contiene un antígeno principal, cruzipain-(Cz), que retiene el dominio C-terminal-(C-T) en su forma madura y contiene varias modificaciones-post-traduccionales. La presencia de oligosacáridos sulfatados se demostró en la Cz, en una glicoproteína con actividad serina-carboxipeptidasa-(SCP) y en los glicoesfingolípidos o sulfátidos parasitarios. Los glicoconjugados sulfatados en los tripanosomátidos son blanco de respuestas inmunes específicas. Los sujetos con infección crónica por T. cruzi desarrollan respuestas inmunes humorales específicas a la Cz sulfatada. En ausencia de infección, los ratones inmunizados con C-T pero no con C-T desulfatado, mostraron efectos ultraestructurales patológicos cardíacos sorprendentes. Además, se demostró la composición química e inmunológica del epitope sulfatado, que el mismo imita la respuesta humoral del sulfotope presente en la Cz nativa, la participación de los sulfotopes en la inmunomodulación por interacción huésped-parásito a través de la unión de lectinas específicas de tipo Ig-ácido siálico- (Siglec) a glicoproteínas sulfosialiladas, así como en el proceso de infección del parásito. Sorprendentemente, evidencias recientes involucraron a los anticuerpos específicos para los sulfotopes de la Cz en los procesos de inmunopatogénesis e infección en la ChD experimental. Curiosamente, los sueros de individuos crónicos infectados por T. cruzi con enfermedad leve mostraron niveles más altos de anticuerpos-IgG2-específicos para glicoproteínas sulfatadas y sulfátidos en comparación con aquellos en formas más graves de la enfermedad, demostrando que los sulfotopes de T. cruzi son antigénicos independientemente del tipo de glicoconjugado, y que los anticuerpos-IgG2-específicos podrían considerarse bio-marcadores de progresión cardíaca de la ChD. Trypanosoma cruzi, the causative agent of Chagas disease-(ChD), contains a major antigen, cruzipain-(Cz), which retains the C-terminal-(C-T) domain in its mature form and contains several post-translational-modifications. The presence of sulfated oligosaccharides in the C-T of Cz, in a minor antigen with serine-carboxypeptidase activity and in parasitic glycosphingolipids or sulfatides was demonstrated. Sulfate-bearing glycoproteins in trypanosomatids are targets of specific immune responses. Subjects with chronic T. cruzi infection develop specific humoral immune responses to sulfated Cz. In the absence of infection, mice immunized with C-T but not sulfate-depleted C-T showed striking ultrastructural cardiac pathologic effects. Furthermore, the structural and immunological characterization of the synthetic sulfated epitope GlcNAc6SO3 was demonstrated and that it mimics the humoral response of the present in native Cz. In addition, the participation of sulfotopes in immunomodulation by host-parasite interaction through the binding of specific lectins of the Ig-sialic acid type (Siglec) to sulfosialylated glycoproteins, as well as in the parasite infection process, has been reported. Surprisingly, recent evidence implicates Cz-sulfotopes-specific antibodies in the processes of immunopathogenesis and infection of experimental ChD. Interestingly, sera from chronically T. cruzi-infected individuals with mild disease showed higher levels of IgG2-antibodies-specific for sulfated and sulfatide glycoproteins compared to those with more severe forms of the disease, demonstrating that T. cruzi sulfotopes are antigenic regardless of the type of glycoconjugate. Ongoing trials indicate that sulfotope-specific antibodies could play a role as predictors of stability from early stages of chronic coronary disease and could be considered bio-markers of human heart disease progression.Fil: Duschak, Vilma Gladys. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Sulfatación y sulfotopes en Trypanosoma cruzi, agente causal de la Enfermedad de Chagas

Trypanosoma cruzi, agente causal de la enfermedad de Chagas- (ChD), contiene un antígeno principal, cruzipain-(Cz), que retiene el dominio C-terminal-(C-T) en su forma madura y contiene varias modificaciones-post-traduccionales. La presencia de oligosacáridos sulfatados se demostró en la Cz, en una glicoproteína con actividad serina-carboxipeptidasa-(SCP) y en los glicoesfingolípidos o sulfátidos parasitarios. Los glicoconjugados sulfatados en los tripanosomátidos son blanco de respuestas inmunes específicas. Los sujetos con infección crónica por T. cruzi desarrollan respuestas inmunes humorales específicas a la Cz sulfatada. En ausencia de infección, los ratones inmunizados con C-T pero no con C-T desulfatado, mostraron efectos ultraestructurales patológicos cardíacos sorprendentes. Además, se demostró la composición química e inmunológica del epitope sulfatado, que el mismo imita la respuesta humoral del sulfotope presente en la Cz nativa, la participación de los sulfotopes en la inmunomodulación por interacción huésped-parásito a través de la unión de lectinas específicas de tipo Ig-ácido siálico- (Siglec) a glicoproteínas sulfosialiladas, así como en el proceso de infección del parásito. Sorprendentemente, evidencias recientes involucraron a los anticuerpos específicos para los sulfotopes de la Cz en los procesos de inmunopatogénesis e infección en la ChD experimental. Curiosamente, los sueros de individuos crónicos infectados por T. cruzi con enfermedad leve mostraron niveles más altos de anticuerpos-IgG2-específicos para glicoproteínas sulfatadas y sulfátidos en comparación con aquellos en formas más graves de la enfermedad, demostrando que los sulfotopes de T. cruzi son antigénicos independientemente del tipo de glicoconjugado, y que los anticuerpos-IgG2-específicos podrían considerarse bio-marcadores de progresión cardíaca de la ChD. Trypanosoma cruzi, the causative agent of Chagas disease-(ChD), contains a major antigen, cruzipain-(Cz), which retains the C-terminal-(C-T) domain in its mature form and contains several post-translational-modifications. The presence of sulfated oligosaccharides in the C-T of Cz, in a minor antigen with serine-carboxypeptidase activity and in parasitic glycosphingolipids or sulfatides was demonstrated. Sulfate-bearing glycoproteins in trypanosomatids are targets of specific immune responses. Subjects with chronic T. cruzi infection develop specific humoral immune responses to sulfated Cz. In the absence of infection, mice immunized with C-T but not sulfate-depleted C-T showed striking ultrastructural cardiac pathologic effects. Furthermore, the structural and immunological characterization of the synthetic sulfated epitope GlcNAc6SO3 was demonstrated and that it mimics the humoral response of the present in native Cz. In addition, the participation of sulfotopes in immunomodulation by host-parasite interaction through the binding of specific lectins of the Ig-sialic acid type (Siglec) to sulfosialylated glycoproteins, as well as in the parasite infection process, has been reported. Surprisingly, recent evidence implicates Cz-sulfotopes-specific antibodies in the processes of immunopathogenesis and infection of experimental ChD. Interestingly, sera from chronically T. cruzi-infected individuals with mild disease showed higher levels of IgG2-antibodies-specific for sulfated and sulfatide glycoproteins compared to those with more severe forms of the disease, demonstrating that T. cruzi sulfotopes are antigenic regardless of the type of glycoconjugate. Ongoing trials indicate that sulfotope-specific antibodies could play a role as predictors of stability from early stages of chronic coronary disease and could be considered bio-markers of human heart disease progression.Fil: Duschak, Vilma Gladys. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Major kinds of drug targets in chagas disease or american trypanosomiasis

American Trypanosomiasis, a parasitic infection commonly named Chagas disease, affects millions of people all over Latin American countries. Presently, the World Health Organization (WHO) predicts that the number of international infected individuals extends to 7 to 8 million, assuming that more than 10,000 deaths occur annually. The transmission of the etiologic agent, Trypanosoma cruzi, through people migrating to non-endemic world nations makes it an emergent disease. The best promising targets for trypanocidal drugs may be classified into three main groups: Group I includes the main molecular targets that are considered among specific enzymes involved in the essential processes for parasite survival, principally Cruzipain, the major antigenic parasite cysteine proteinase. Group II involves biological pathways and their key specific enzymes, such as Sterol biosynthesis pathway, among others, specific antioxidant defense mechanisms, and bioenergetics ones. Group III includes the atypical organelles /structures present in the parasite relevant clinical forms, which are absent or considerably different from those present in mammals and biological processes related to them. These can be considered potential targets to develop drugs with extra effectiveness and fewer secondary effects than the currently used therapeutics. An improved distinction between the host and the parasite targets will help fight against this neglected disease.Fil: Duschak, Vilma Gladys. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Synthetic Biology: Computational Modeling Bridging the Gap between In Vitro and In Vivo Reactions

The synthetic biology firstly refers to the design and fabrication of biological components and systems that do not already exist in the natural world and to the redesign and fabrication of existing biological systems. The link of computational tools to cell-free systems, converts to synthetic biology is an emerging field expert to build artificial biological systems through the combination of molecular biology and engineering approaches. Herein, most findings describing the differences between in vivo and in vitro reactions and systems have been extensively described. The specific applications of computational tools to the design of an in vitro gene expression platform known as the artificial cell, its components and the strategies developed to predict activities of processor modules and to control the expression of genes have been discussed in detail. Potential applications of artificial cells in drug delivery, in biosynthesis, among others, have been described. Two sources of models for the possible developing of the computational toolbox for cell-free synthetic biology include i) Physical models of single cellular components able to be created from original principles, guiding to focus on tools to predict structure and dynamics of particular components; ii) A wide-range of mathematical models for predicting system dynamics of natural cells. Regarding modeling algorithms, there is a broad kind of models available for synthetic biologists and some areas of potential growth identified for researchers interested in developing tools for cell-free systems. Among them, deterministic, exploratory, molecular dynamic, stochastic, all atom models, among others, have been described and discussed. By using computational models to set up quantitative differences between in vitro reactions and in vivo systems, could identify specific mechanisms in living organisms to be further used in in vitro reactions in order to facilitate their processes. Thus, computational modeling would bridge the gap between in vitro and in vivo reactions.Fil: Duschak, Vilma Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentin

Importancia de los antígenos cítricos en la patología alérgica humana

ResúmenLa importancia del estudio de la inmuno-reactividad cruzada entre los extractos alergénicos de polen del naranjo (OTPE) y de la naranja (OFE) radica en la alta incidencia de alergias que relacionan al polen con ciertos alimentos en todo el mundo. El propósito del presente estudio es determinar la relación antigénica entre los extractos de OTPE y de OFE. Sueros de conejos anti-OTPE y anti-OFE, tanto como sueros de pacientes alérgicos a OTPE y a OFE se aplicaron comparativamente en ensayos específicos para IgG y/o IgE (inhibición de ELISA, cross-over e inhibición de immunoblot) usando extractos alergénicos de OTPE y de OFE como fase sólida. Los tratamientos con periodato y con proteinasa K se usaron para ensayos de depleción de carbohidratos y proteínas, respectivamente. La antigenicidad de OTPE y la presencia de estructuras comunes entre los extractos de OTPE y de OFE se demostraron mediante ensayos de inhibición de ELISA e inmunoblot cruzado usando antisueros de conejo específicos para cada uno de estos extractos. Una proteína de 30 kDa. blanco común de la respuesta IgE para los extractos de OTPE, de OFE y de mandarina (MFE), pero ausente en el extracto de limón (LFE) se identificó por ensayos de ELISA e inhibición de inmunoblot en pacientes con sensibilización primaria a OTPE en el contexto de exposición ocupacional. Además, por tratamientos bioquímicos se mostró que los epitopes antigénicos presentes en la proteína de 30 kDa contienen una estructura peptídica libre de carbohidratos. En conclusión, este trabajo describe la antigenicidad del polen del naranjo, la presencia de determinantes antigénicos comunes entre el polen y las frutas cítricas, y la presencia de una banda proteica de 30 kDa con reacción cruzada IgE específica, la cual comparte epitopes libres de carbohidratos. Este antígeno común, después de su aislamiento y purificación podría ser de utilidad para la inmunoterapia con un alérgeno específico en los pacientes alérgicos al polen y a las frutas cítricas.It is important to study the cross-reactivity between orange tree pollen (OTPE) and orange fruit (OFE) due to the high incidence of pollen/food-related allergies worldwide. The aim of the present study was to determine the antigenic relationship between OTPE and OFE. OTPE and OFE rabbit antisera as well as sera from patients allergic to OTPE and OFE were comparatively applied in IgG- and/or IgE-specific ELISA inhibition, crossover or inhibition immunoblotting assays using OTPE and OFE allergenic extracts as solid phase. Periodate and proteinase K treatments were used for carbohydrate and protein depletion, respectively. The antigenicity of OTPE and the presence of common structures between OTPE and OFE extracts were demonstrated by rabbit IgG-specific ELISA inhibition and crossover immunoblotting assays. A 30-kDa protein. common target of the IgE response on OTPE, OFE and mandarín extract, but absent in lemon extract, was identifíed by ELISA and immunoblot inhibition assays in patients suffering from primary sensitization to OTPE in the context of occupational exposure. Moreover, biochemical treatments showed that antigenic epitopes on the 30-kDa protein contain polypeptidic but no carbohydrate moieties. We can conclude that the antigenicity of OTPE, the presence of common antigenic determinants between pollen and citrus fruits and an IgE-specific cross-reactive protein band of 30 kDa sharing carbohydrate-free epitopes were described. After isolation and purification, this common antigen might be useful for allergen immunotherapy in pollen/fruit-related allergic patients.Fil: Irañeta, Silvia G.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Mouchian, Krikor. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Alonso, Angel. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Duschak, Vilma Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología ; Argentin

UV-MALDI Mass Spectrometry Analysis of NBD-Glycosphingolipids Without an External Matrix

Each day, advances in the instrumentation and operating protocols bring new applications and insights into the molecular processes of ultra violet-matrix assisted laser desorption/ionization-mass spectrometry (UV-MALDI MS), increasing its potential use. We report here an approach in which mass spectrometry analysis of sphingolipids has been performed using a fluorescent tag (nitrobenz-2-oxa-1, 3-diazole, NBD) covalently linked to the sphingoid base as matrix. Thus, different labeled-sphingolipids were analyzed: ceramide, dihydroceramide, acetylceramide, glucosylceramide, galactosylceramide, galactosyldihydroceramide. In addition an extract of glycosphingolipids obtained from epimastigote forms of Trypanosoma cruzi metabolically labeled with NBD-ceramide was analyzed. The goal of this work is to show that no matrix needs to be added for the mass spectrometry analysis as the same tag used to label the lipids may generate efficiently analyte ions to obtain high quality signals.Fil: Landoni, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Duschak, Vilma Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Erra Balsells, Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentin

Effect of tamoxifen on the sphingolipid biosynthetic pathway in the different intraerythrocytic stages of the apicomplexa Plasmodium falciparum

Parasites of the genus Plasmodium responsible for Malaria are obligate intracellular pathogens residing in mammalian red blood cells, hepatocytes, or mosquito midgut epithelial cells. Regarding that detailed knowledge on the sphingolipid biosynthetic pathway of the apicomplexan protozoan parasites is scarce, different stages of Plasmodium falciparum were treated with tamoxifen in order to evaluate the effects of this drug on the glycosphingolipid biosynthesis. Thin layer chromatography, High performance reverse phase chromatography and UV-MALDI-TOF mass spectrometry were the tools used for the analysis. In the ring forms, the increase of NBD-phosphatidyl inositol biosynthesis was notorious but differences at NBD-GlcCer levels were undetectable. In trophozoite forms, an abrupt decrease of NBD-acylated GlcDHCer and NBD-GlcDHCer in addition to an increase of NBD-PC biosynthesis was observed. On the contrary, in schizonts, tamoxifen seems not to be producing substantial changes in lipid biosynthesis. Our findings indicate that in this parasite, tamoxifen is exerting an inhibitory action on Glucosylceramidesynthase and sphingomyelin synthase levels. Moreover, regarding that Plasmodium does not biosynthesize inositolphosphoceramides, the accumulation of phosphatidylinositol should indicate an inhibitory action on glycosylinositol phospholipid synthesis.Fil: Piñero, Tamara Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Landoni, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Duschak, Vilma Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; ArgentinaFil: Katzin, Alejandro M.. Universidade de Sao Paulo; BrasilFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentin

Hallmarks of the relationship between host and Trypanosoma cruzi sulfated glycoconjugates along the course of Chagas disease

American Trypanosomiasis or Chagas disease (ChD), a major problem that is still endemic in large areas of Latin America, is caused by Trypanosoma cruzi. This agent holds a major antigen, cruzipain (Cz). Its C-terminal domain (C-T) is retained in the glycoprotein mature form and bears several post-translational modifications. Glycoproteins containing sulfated N-linked oligosaccharides have been mostly implicated in numerous specific procedures of molecular recognition. The presence of sulfated oligosaccharides was demonstrated in Cz, also in a minor abundant antigen with serine-carboxypeptidase (SCP) activity, as well as in parasite sulfatides. Sulfate-bearing glycoproteins in Trypanosomatids are targets of specific immune responses. T. cruzi chronically infected subjects mount specific humoral immune responses to sulfated Cz. Unexpectedly, in the absence of infection, mice immunized with C-T, but not with sulfate-depleted C-T, showed ultrastructural heart anomalous pathological effects. Moreover, the synthetic anionic sugar conjugate GlcNAc6SO3-BSA showed to mimic the N-glycan-linked sulfated epitope (sulfotope) humoral responses that natural Cz elicits. Furthermore, it has been reported that sulfotopes participate via the binding of sialic acid Ig-like-specific lectins (Siglecs) to sulfosialylated glycoproteins in the immunomodulation by host–parasite interaction as well as in the parasite infection process. Strikingly, recent evidence involved Cz-sulfotope-specific antibodies in the immunopathogenesis and infection processes during the experimental ChD. Remarkably, sera from chronically T. cruzi-infected individuals with mild disease displayed higher levels of IgG2 antibodies specific for sulfated glycoproteins and sulfatides than those with more severe forms of the disease, evidencing that T. cruzi sulfotopes are antigenic independently of the sulfated glycoconjugate type. Ongoing assays indicate that antibodies specific for sulfotopes might be considered biomarkers of human cardiac ChD progression, playing a role as predictors of stability from the early mild stages of chronic ChD.Fil: Soprano, Luciana Lía. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferrero, Maximiliano Ruben. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Jacobs, Thomas. Bernhard Nocht Institute For Tropical Medicine.; AlemaniaFil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; ArgentinaFil: Duschak, Vilma Gladys. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”. Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben”; Argentin