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

Targets and Patented Drugs for Chemotherapy of Chagas Disease in the Last 15 Years-Period

Fil: Duschak, Vilma G. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.The American trypanosomiasis, Chagas disease, is a parasitic infection typically spread by triatomine vectors affecting millions of people all over Latin America. Existing chemotherapy is centered on the nitroaromatic compounds benznidazole and nifurtimox that provide unsatisfactory results and substantial side effects. So, the finding and exploration of novel ways to challenge this neglected disease is a main priority

A decade of targets and patented drugs for chemotherapy of Chagas disease

Fil: Duschak, Vilma G. ANLIS Dr. C. G. Malbrán. Instituto Nacional de Parasitología Dr. Mario Fatala Chaben; Argentina.Chagas disease, a parasitic infection typically spread by triatomine bugs, affects millions of people throughout Latin America. Current chemotherapy based on the nitroaromatic compounds, benzonidazole and nifurtimox provides unsatisfactory results and suffers from considerable side effects. Therefore, there is still an urgent need for new drugs to treat this neglected disease. During the last decade, the advances and understanding in the biology and biochemistry of Trypanosoma cruzi have allowed the identification of multiple new targets for Chagas' disease chemotherapy. Among the most promising targets for antiparasitic drugs are: cruzipain, the main cysteine protease of T. cruzi, essential for parasite survival and proliferation in mammalian host; ergosterol biosynthesis pathway; trypanothione synthesis and thiol-dependant redox metabolism. Specific enzymes of the glycolytic, pentose phosphate, polyisoprenoid (farnesylpyrophosphate synthase) and other particular biosynthetic pathways as well as enzymes from purine salvage (hypoxanthine-guanine phosphoribosyl-transferase, dihydrofolate reductase) have also been intensively studied in T. cruzi. In particular, trypanocidal agents that target the validated biochemical pathways of the parasite including cysteine proteinase inhibitors and inhibitors capable to block ergosterol biosynthesis are currently in the pipeline. Among the latter, posaconazole and ravuconazole, are planned to enter in clinical trials for trypanocidal chemotherapy in the near future. This review will summarize advances on antichagasic agents directed to specific parasite targets such as metabolic pathways or specific enzymes. Related patents filed and issued from 2000 to 2010 claiming inhibitors for specific parasite targets will be also discussed. Among them, the most represented were those related with cysteine proteinase inhibitors

Cruzipain, the major cysteine protease of Trypanosoma cruzi: a sulfated glycoprotein antigen as relevant candidate for vaccine development and drug target. A review

This review aims to present different aspects related to cruzipain, one of the most important proteins of the etiological agent of Chagas disease that has been extensively studied in the last two decades, including all the particularities of the molecule as well as to highlight its participation in multiple relevant functions of the parasite to favour the cell invasion phenomena, to facilitate host tissues proteolytic degradation and to trigger the evasion mechanism from host immune response. Cruzipain has been related with parasite metabolism and identified as both an important candidate for vaccine development and for trypanocidal drug design. We have reported for the first time that this enzyme is a sulfated glycoprotein. Indeed, the sulfated oligosaccharides are main targets for immune responses and are involved in tissue damage in mice immunized in absence of infection contributing to get deeper into the knowledge of the molecule composition and helping to elucidate its role in the infection and/or pathogenesis of the disease. A whole view including all the aspects related to the major cysteine proteinase of Trypanosoma cruzi studied so far including recent advances as proteinase, antigen and glycoprotein will be discussed

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

Novel cysteine proteinase in Trypanosoma cruzi metacyclogenesis

Fil: Duschak, Vilma G. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Barboza, M. Universidad Nacional de General San Martín. Instituto de Investigaciones Biotecnológicas–INTECH; Argentina.Fil: Garcia, G. A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Lammel, Estela M. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología, Parasitología e Inmunología; Argentina.Fil: Couto, Alicia S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica. CIHIDECAR; Argentina.Fil: Isola, E. L. D. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología, Parasitología e Inmunología; Argentina.With the aim to study proteinases released to the culture medium during Trypanosoma cruzi metacyclogenesis, the presence of cysteine proteinases (CPs) was analysed in culture supernatants obtained throughout the differentiation induced by stimulation of epimastigotes with Triatoma infestans hindgut homogenate. In SDS-gelatin containing gels, an important endopeptidase activity with apparent molecular weight range between 97 and 116 kDa was encountered at pH 6, which was abolished by the specific cysteine proteinase inhibitor E-64 and TLCK, but not by pepstatin, 1,10 phenantroline or PMSF. This novel CP, named TcCPmet, showed affinity to cystatin-Sepharose, denoting its thiol-proteinase character as well as to ConA-Sepharose, indicating it contains N-linked oligosaccharides. However, it presented a different elution pattern on ConA-Sepharose than cruzipain and, in addition, it was not recognized by anti-cruzipain serum, facts that strongly suggest the different nature of both CPs. Moroever, evidence is presented indicating that TcCPmet was able to hydrolyse the same chromogenic peptides as cruzipain at optimal alkaline pH values, although with a different order of effectiveness. Our results indicate the presence of a novel CP secreted by metacyclic trypomastigotes and reinforces the important role of these enzymes in metacyclogenesis

Subcellular localization and post-secretory targeting of TgPI, a serine proteinase inhibitor from Toxoplasma gondii

Fil: Pszenny, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Ledesma, Bibiana A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Matrajt, Mariana. Department of Biology, University of Pennsylvania, Philadelphia; Estados Unidos.Fil: Duschak, Vilma G. Instituto de Investigaciones Biotecnológicas, UNSAM, Provincia de Buenos Aires; Argentina.Fil: Bontempi, Esteban J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina.Fil: Dubremetz, Jean-François. UMR5539 CNRS, Université de Montpellier II, Montpellier; Francia.Fil: Angel, Sergio O. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología; Argentina

Structural and immunological characterization of sulphatides: relevance of sulphate moieties in Trypanosoma cruzi glycoconjugates

Sulphoglycosphingolipids, present on the surface of diverse cells, participate in the regulation of various cellular events. However, little is known about the structure and the role of sulphoglycosphingolipids in trypanosomatids. Herein, sulphated dihexosylceramide structures - composed mainly of sphingosine as the long chain base acylated with stearic acid - have been determined for the first time in Trypanosoma cruzi epimastigotes by UV-MALDI-TOF-MS analysis. Interestingly, inhibition ELISA assays using cruzipain as antigen and polyclonal rabbit antibodies specific for cruzipain, the major cysteine proteinase of T. cruzi, or for its C-terminal domain, have demonstrated (i) that sulphate epitopes are shared between cruzipain and sulphatides of T. cruzi, (ii) that cross-reactivity maps to the C-terminal domain and (iii) the existence of other antigenic determinants in the glycolipidic structures. These features provide evidence that sulphate groups are antigenic in sulphate-containing parasite glycoconjugates. Furthermore, IgG2 antibody levels inversely correlate with disease severity in chronic Chagas disease patients, suggesting that IgG2 antibodies specific for sulphated epitopes might be associated with protective immunity and might be considered as potential surrogates of the course of chronic Chagas disease

Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii

Fil: Pszenny, Viviana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Angel, Sergio O. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Duschak, Vilma G. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina.Fil: Paulino, Margot. Universidad de la República. Facultad de Química. Cátedra de Química Cuántica, Montevideo; Uruguay.Fil: Ledesma, Bibiana A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Yabo, Miriam I. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Producción de Biológicos; Argentina.Fil: Guarnera, Eduardo. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Ruiz, Andrés M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.Fil: Bontempi, Esteban. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Parasitología; Argentina.A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host