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Resumen: Introducción. El consorcio europeo BIOMED-2 se creó para determinar si una población linfoide de difícil clasificación patológica es clonal. En Colombia, la implementación de estas pruebas comenzó en el 2015 en el Instituto Nacional de Cancerología E.S.E. (Bogotá). Objetivos. Determinar el comportamiento de las pruebas de reordenamiento clonal o clonalidad linfoide. y determinar las dificultades de su uso en nuestro medio verificando su adaptación local y los resultados en una serie retrospectiva de casos y consecutiva de proliferaciones linfoides sometidas a los protocolos BIOMED-2. Materiales y métodos. A partir de las historias clínicas, se recolectaron los datos clínicos e histológicos y los resultados de los análisis de los reordenamientos en todos los casos de proliferaciones linfoides sometidas a los protocolos BIOMED-2, entre febrero de 2015 y mayo de 2019. Resultados. Se hallaron 132 casos, de los cuales 47 se clasificaron mediante los protocolos de Biomed-2 como hiperplasias linfoides reactivas, 62 como linfomas T, 19 como linfomas B y 3 como neoplasias linfoides de linaje no establecido. Solo en un caso falló la extracción de ADN. Según estos resultados, la mayor dificultad diagnóstica para el patólogo fue el análisis de los infiltrados linfoides T, la mayoría (44) de los cuales correspondía a lesiones cutáneas. Conclusiones. Las pruebas de clonalidad pueden usarse en tejidos de diversa calidad en nuestro medio como ayuda en el diagnóstico de proliferaciones linfoides de difícil clasificación. Es importante hacerlas e interpretarlas de manera multidisciplinaria y considerar cada caso por separado. Abstract: Introduction: The European BIOMED-2 consortium was created to evaluate clonality in lymphoproliferations that are difficult to diagnose. In Colombia, the implementation of these tests began in 2015 at the Instituto Nacional de Cancerología E.S.E., Bogotá. Objectives: To determine the behavior of the rearrangement tests for lymphoid clonality and the difficulties of its implementation in our field through a series of retrospective and consecutive cases of lymphoid proliferation subjected to the BIOMED-2 protocols. Materials and methods: Clinical and histological data and the results of the rearrangement analysis of all cases of lymphoid proliferation subjected to the BIOMED-2 protocols between February 2015 and May 2019 were collected from clinical histories. Results: We recovered 132 samples from which 47 corresponded to reactive lymphoid hyperplasias, 62 to T lymphomas, 19 to B lymphomas, and three to lymphoid neoplasms of unestablished lineage. Only in one case did DNA extraction fail. According to these results, the greatest diagnostic difficulty for the pathologist was the analysis of T lymphoid infiltrates, most of which (44) were skin lesions. Conclusions: Clonality tests can be used in tissues of different quality to help in the diagnosis of lymphoid proliferations that are difficult to classify. It is important to implement and interpret them in an interdisciplinary way considering each case separately.

Resumen Introducción. Las especies Rhodnius (Hemiptera: Reduviidae: Triatominae) están conformadas por insectos hematófagos vectores de Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, y T. rangeli, parásito infectivo pero no patógeno para el vertebrado. El estudio de la diversidad proteica de la saliva de estos insectos permite la obtención de perfiles electroforéticos unidimensionales característicos de algunas especies de triatominos. Sin embargo, el reporte de los patrones electroforéticos de proteínas salivales de las especies de Rhodnius ha sido escaso. Objetivo. Hacer un análisis comparativo de los perfiles electroforéticos unidimensionales de las proteínas salivales de R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus. Materiales y métodos. Se obtuvieron los perfiles electroforéticos de la saliva de las especies en estudio mediante electroforesis en gel de poliacrilamida con dodecilsulfato sódico (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) y se construyó un fenograma mediante el método UPGMA (Unweighted Pair Group Method Using Arithmetic Averages). Resultados. Los perfiles electroforéticos de las proteínas solubles de saliva presentaron bandas en un rango de masa aproximado de 15 a 45 kDa, los cuales permitieron diferenciar las cinco especies estudiadas. El fenograma reveló la existencia de dos grupos principales: uno conformado por los grupos cisandinos Pictipes y Prolixus y otro constituido por el grupo transandino Pallescens. Conclusiones. Existen diferencias en los perfiles electroforéticos de las proteínas salivales entre R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus, cuya variabilidad permitió construir un fenograma congruente con los grupos del género Rhodnius. Abstract Introduction: Rhodnius (Hemiptera: Reduviidae: Triatominae) species are made up of haematophagous insect vectors for Trypanosoma cruzi (Chagas’ disease aetiological agent) and T. rangeli, an infective parasite that is not pathogenic for vertebrate hosts. The study of their salivary protein diversity enables the obtention of characteristic one-dimensional electrophoretic profiles of some triatomine species; however, few reports have dealt with Rhodnius species salivary proteins electrophoretic patterns. Objective: To compare R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus’ salivary proteins one-dimensional electrophoretic profiles. Materials and methods: SDS-PAGE was used for obtaining electrophoretic profiles of saliva from the species under study. The unweighted pair group method with arithmetic mean (UPGMA) was used for constructing a phenogram. Results: Electrophoretic profiles of soluble saliva had protein bands ranging from 15 to 45 kDa, thereby enabling the five species studied to be differentiated. The phenogram revealed two main groups, one formed by the Pictipes and Prolixus cis-Andean groups and another consisting of the Pallescens trans-Andean group. Conclusion: Differences were revealed regarding R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus electrophoretic profiles of salivary proteins; their variability facilitated constructing a phenogram which was taxonomically congruent with the groups from the genus Rhodnius.

Paracoccidioidomycosis (PCM) is an infectious disease endemic to South America, caused by the thermally dimorphic fungi Paracoccidioides. Currently, there is no effective human vaccine that can be used in prophylactic or therapeutic regimes. We tested the hypothesis that the immunogenicity of the immunodominant CD4+ T-cell epitope (P10) of Paracoccidioides brasiliensis gp43 antigen might be significantly enhanced by using a hepatitis B virus-derived particle (VLP) as an antigen carrier. This chimera was administered to mice as a (His)6-purified protein (rPbT) or a replication-deficient human type 5 adenoviral vector (rAdPbT) in an immunoprophylaxis assay. The highly virulent Pb18 yeast strain was used to challenge our vaccine candidates. Fungal challenge evoked robust P10-specific memory CD4+ T cells secreting protective Th-1 cytokines in most groups of immunized mice. Furthermore, the highest level of fungal burden control was achieved when rAdPbT was inoculated in a homologous prime-boost regimen, with 10-fold less CFU recovering than in non-vaccinated mice. Systemic Pb18 spreading was only prevented when rAdPbT was previously inoculated. In summary, we present here VLP/P10 formulations as vaccine candidates against PCM, some of which have demonstrated for the first time their ability to prevent progression of this pernicious fungal disease, which represents a significant social burden in developing countries., Author summary Human paracoccidioidomycosis (PCM) represents a serious public health issue due to its disabling sequelae in working-age people and mortality rates (8th among chronic infectious parasitic diseases in endemic countries and first among mycoses in Brazil). Although antifungal drugs have been widely used and provide clinical improvement for patients, the long duration of treatments (commonly from 6 to 12 months) has contributed to non-compliance and worsening of the disease in many cases. Induction of protective immune responses (either prophylactic or immune-therapeutic) remains the most cost-effective approach against most infectious agents. Members of our group have previously reported the protective properties of P10, a peptide contained in Paracoccidioides brasiliensis gp43 antigen, against PCM. However, the magnitude of the CD4+ T-cell responses elicited lacked the capacity of completely protecting experimental animals. We demonstrate here that immunogenic virus-like particles (VLP) carrying multiple copies of P10 peptide substantially improve P10-related cellular immunity in mice. This was particularly true when an adenoviral immunization vector expressed the chimeric VLP. Moreover, VLP/P10 formulations were capable of controlling the host fungal burden and prevented fungal systemic dissemination. The efficacy of diverse VLP/P10 formulations in murine PCM showed at least one promising candidate vaccine against human PCM.

Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.

Fil: Cardoso, María de las Mercedes Elisa. Universidad Nacional del Litoral. Facultad de Ingeniería Química; Argentina. Las bacterias ácido lácticas, que gozan del status de “Generalmente Reconocidas Como Seguras”, otorgado por la Organización Mundial de la Salud, pueden producir sustancias antimicrobianas de naturaleza proteica, denominadas bacteriocinas, que actúan como bioconservantes alimentarios. Se investigó la capacidad bacteriocinogénica de la cepa de Enterococcus faecalis DBFIQ E24, aislada de leche cruda producida en la región santafesina. Esta cepa fue sensible a vancomicina y carente de capacidad hemolítica. El sobrenadante libre de células de sus cultivos presentó actividad inhibitoria contra bacterias Gram positivas y Gram negativas, hongos filamentosos y levaduras. Los ensayos enzimáticos demostraron la naturaleza proteica de la sustancia activa, que además resultó ser resistente a altas temperaturas, estable en un extenso rango de pH y a distintos tiempos y temperaturas de almacenamiento. El Peso Molecular aparente del sobrenadante, determinado por ultrafiltración y diálisis, estuvo comprendido entre 1000 y 2000 Dalton, y su título fue de 266 Unidades Arbitrarias por mililitro.El modo de acción de la sustancia inhibitoria fue bactericida y bacteriolítico. Mediante electroforesis en geles de poliacrilamida se determinó el Peso Molecular aproximado, el que resultó estar entre 3.5 y 1.06 kilo Dalton. Se realizó la purificación parcial de la sustancia antimicrobiana aplicando dos protocolos de purificación: 1) Cromatografías de Intercambio catiónico, Fase reversa y Líquida de alta resolución en fase reversa; 2) Precipitación salina con sulfato de amonio, seguida de cromatografías de filtración por geles, intercambio catiónico, fase reversa y líquida de alta resolución en fase reversa. Ambos protocolos presentaron perfiles cromatográficos similares. Lactic acid bacteria, whose present-day status, given by the World Health Organization, is that of “Normally Regarded as Safe”, can produce antimicrobian substances derived from protein, called bacteriocins, which act as food biopreservers. The bacteriocinogenic potential of an Enterococcus faecalis DBFIQ E24 strain was surveyed which was isolated from raw milk yielded in the province of Santa Fe. The strain was sensitive to vancomycin and presented no hemolitic capacity. The culture cell-free supernatant showed inhibitory activity against Gram-positive and Gram-negative bacteria, filamentous fungi and yeast. Enzyme assays confirmed the proteinic nature of the active substance, which also proved to be resistant to high temperature, stable in a wide range of pH and to various storage times and temperatures. The supernatant apparent Molecular Weight, measured through ultrafiltration and dialysis, was somewhere between 1000 and 2000 Dalton, its titer was 266 Arbitrary Units per mililiter. The inhibitory substance presented bactericide and bacteriolytic actions. The approximate Molecular Weight, which was obtained through polyacrylamide gel electrophoresis, was between 3.5 and 1.06 kilo Dalton. An antimicrobial substance partial purification was performed by applying two purification protocols: 1) Cation-exchange, Reversed-phase and Reverse-phase High-performance Liquid chromatographies; 2) Ammonium sulfate saline precipitation followed by gel permeation, Cation-exchange, Reversed-phase and Reverse-phase High-performance Liquid chromatographies. In both protocols, chromatography profiles were similar. Agencia Nacional para la Promoción Científica y Tecnológica Universidad Nacional del Litoral

The mycobacterial cell envelope has been implicated in the pathogenicity of tuberculosis and therefore has been a prime target for the identification and characterization of surface proteins with potential application in drug and vaccine development. In this study, the genome of Mycobacterium tuberculosis H37Rv was screened using Machine Learning tools that included feature-based predictors, general localizers and transmembrane topology predictors to identify proteins that are potentially secreted to the surface of M. tuberculosis, or to the extracellular milieu through different secretory pathways. The subcellular localization of a set of 8 hypothetically secreted/surface candidate proteins was experimentally assessed by cellular fractionation and immunoelectron microscopy (IEM) to determine the reliability of the computational methodology proposed here, using 4 secreted/surface proteins with experimental confirmation as positive controls and 2 cytoplasmic proteins as negative controls. Subcellular fractionation and IEM studies provided evidence that the candidate proteins Rv0403c, Rv3630, Rv1022, Rv0835, Rv0361 and Rv0178 are secreted either to the mycobacterial surface or to the extracellular milieu. Surface localization was also confirmed for the positive controls, whereas negative controls were located on the cytoplasm. Based on statistical learning methods, we obtained computational subcellular localization predictions that were experimentally assessed and allowed us to construct a computational protocol with experimental support that allowed us to identify a new set of secreted/surface proteins as potential vaccine candidates., Author Summary Since the publication of the Mycobacterium tuberculosis genome in 1998, great expectations have emerged regarding speeding up the process of developing vaccines against tuberculosis. Our group has been focused on identifying molecules localized on the mycobacterial surface that could act as ligands facilitating this pathogen's entry into host cells. Immune responses exerted against these proteins might block receptor-ligand interactions, thus hampering mycobacterial invasion. Since protein fragments involved in these interactions might serve as vaccine candidates and, taking into account that a relatively small number of mycobacterial surface proteins have been experimentally identified to date due to the inherent difficulty of proteomics studies for characterizing surface proteins, in this study, we used Machine Learning-based tools available on the World Wide Web to obtain accurate predictions of surface and secreted proteins from this pathogen and found experimental support of such predictions for a group of candidate proteins selected based on novel criteria.

Objetivo: Caracterizar las proteínas solubles que se encuentran en la raíz del Lepidium peruvianum G. Chacón, maca, mediante electroforesis unidimensional y electroforesis bidimensional. Diseño: Estudio de tipo observacional y transversal. Lugar: Centro de Investigación de Bioquímica y Nutrición Alberto Guzmán Barrón. Facultad de Medicina, Universidad Nacional Mayor de San Marcos. Lima, Perú. Materiales: Raíces de Lepidium peruvianum G. Chacón ‘maca’ de los ecotipos blanco, amarillo y morado, procedentes de Junín que fueron obtenidas a través de la Universidad Nacional del Centro del Perú. Métodos: La extracción de las proteínas totales solubles se realizó con una solución antioxidante, seguida de electroforesis unidimensional y bidimensional para su caracterización. Principales medidas de resultados: Número de proteínas solubles, peso molecular de las proteínas y puntos isoelectricos de las proteínas más abundantes. Resultados: El análisis electroforético unidimensional mostró predominio de 2 proteínas (72% de las proteínas solubles totales). Una de 22,5 kDa, denominada en el presente trabajo ‘macatina’ (51% de la proteína total); la otra de 17,0 kDa (21% de la proteína soluble total). El mapa electroforético bidimensional mostró que tanto la ‘macatina’ como la proteína de 17,0 kDa son básicas y presentan 3 isómeros de carga que se distribuyen en un rango de punto isoeléctrico (pI) de 7,1 a 8,2. Conclusiones: Las proteínas solubles mostraron un patrón electroforético complejo, siendo la macatina la proteína más abundante., Objective: To characterize soluble proteins present in Lepidium peruvianum G. Chacon ‘maca’ root by both unidimensional and bidimensional electrophoresis. Design: Observational and cross-sectional type study. Setting: Alberto Guzman Barron Biochemistry and Nutrition Research Center, Faculty of Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru. Materials: Roots of Lepidium peruvianum G. Chacon ‘maca’ white, yellow and purple ecotypes, collected in Junin and obtained from Universidad Nacional del Centro del Peru. Methods: Soluble total proteins extraction with an antioxidant solution and characterization by both unidimensional and bidimensional electrophoresis were done. Main outcome measures: Soluble protein number, molecular weight and most abundant proteins’ isoelectrical points. Results: The unidimensional electrophoretic analysis showed predominance of two proteins (72% of total soluble proteins): one with 22,5 kDa denominated in the present work as “macatina” (51% of the total protein) and another with 17,0 kDa (21% of total soluble protein). The bidimensional electrophoretic map sample showed that both ‘macatina’ and the 17,0 kDa protein are basic and display three charge isomers distributed in an isoelectric point (pI) ranging from 7,1 to 8,2. Conclusions: The soluble proteins ecotypes studied showed a complex electrophoretical pattern, being macatina the most abundant protein.

Journal Article; Research Support, Non-U.S. Gov't; BACKGROUND Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. OBJECTIVE To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma MATERIALS AND METHODS An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. RESULTS Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA-), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p