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El presente trabajo experimental tuvo como objetivo determinar valores referenciales de reticulocitos y leucocitos en caninos aparentemente sanos en condiciones de altitud, en el cantón Cuenca, provincia del Azuay. Para obtener valores reales de la zona y poder mejorar diagnósticos y tratamientos en el área de Medicina Veterinaria. The objective of this experimental work was to determine reference values of reticulocytes and leukocytes in apparently healthy canines under high altitude conditions, in Cuenca canton, Azuay province. To obtain real values of the area and to be able to improve diagnoses and treatments in the area of Veterinary Medicine.

La presente investigación se enfocó en establecer valores de referencia en cerdos aparentemente sanos en condiciones de altitud, donde se analizó las formas leucocitarias, conteo de reticulocitos y niveles de hematocrito, se trabajó con 120 muestras, 60 hembras y 60 machos. Mediante el análisis estadístico se obtuvieron valores con diferencias significativas con respecto a los valores referenciales. The present investigation focused on establishing reference values in apparently healthy pigs in high altitude conditions, where leukocyte forms, reticulocyte count and hematocrit levels were analyzed, working with 120 samples, 60 females and 60 males. Through statistical analysis, values with significant differences were obtained with respect to the reference values.

Al determinar los valores de referencia de Serie Blanca y Reticulocitos en felinos de la ciudad de Cuenca, con su análisis respectivo, se obtuvo resultados favorables al factor altitud, por no presentar ninguna alteración en sus variables estudiadas comparándolo a altitudes similares, a diferencia de los Linfocitos que se alteró en referencia con una altitud menor. When determining the reference values of White Series and Reticulocytes in felines from the city of Cuenca, with their respective analysis, favorable results were obtained for the altitude factor, as they did not present any alteration in their studied variables when compared to similar altitudes, unlike the Lymphocytes that were altered in reference to a lower altitude.

El presente trabajo experimental tuvo como objetivo determinar valores referenciales de reticulocitos y leucocitos en equinos (Equus caballus) aparentemente sanos en condiciones de altitud, en el Cantón Cuenca, provincia del Azuay. Para obtener valores reales de la zona y poder mejorar diagnósticos y tratamientos en el área de Medicina Veterinaria. The objective of this experimental work was to determine the reference values of reticulocytes and leukocytes in apparently healthy equines (Equus caballus) in altitude conditions, at the city of Cuenca, located in the province of Azuay. To obtain real values of the area and to be able to improve diagnoses and treatments in Veterinary Medicine.

Plasmodium vivax is the most widely distributed malaria parasite affecting humans worldwide, causing ~5 million cases yearly. Despite the disease’s extensive burden, there are gaps in the knowledge of the pathophysiological mechanisms by which P. vivax invades reticulocytes. In contrast, this crucial step is better understood for P. falciparum, the less widely distributed but more often fatal malaria parasite. This discrepancy is due to the difficulty of studying P. vivax’s exclusive invasion of reticulocytes, which represent 1–2% of circulating cells. Its accurate targeting mechanism has not yet been clarified, hindering the establishment of long-term continuous in vitro culture systems. So far, only three reticulocyte invasion pathways have been characterised based on parasite interactions with DARC, TfR1 and CD98 host proteins. However, exposing the parasite’s alternative invasion mechanisms is currently being considered, opening up a large field for exploring the entry receptors used by P. vivax for invading host cells. New methods must be developed to ensure better understanding of the parasite to control malarial transmission and to eradicate the disease. Here, we review the current state of knowledge on cellular and molecular mechanisms of P. vivax’s merozoite invasion to contribute to a better understanding of the parasite’s biology, pathogenesis and epidemiology. Incluye referencias bibliográficas

O sangue é um tipo especial de tecido conjuntivo, associando várias funções, como o transporte de nutrientes e gases respiratórios, assim como, atuando ao nível da defesa do organismo através de células bem específicas. O sangue é uma mistura de diversas células em suspensão num líquido denominado plasma, que representa cerca de 55% da sua composição e onde as células sanguíneas, que constituem cerca de 45%, são eritrócitos ou glóbulos vermelhos, leucócitos ou glóbulos brancos e plaquetas. A importância do sangue como elemento de vida, encontra-se igualmente no seu uso em aplicações clínicas, nomeadamente, nas transfusões, na recolha diferenciada de certos elementos sanguíneos para o tratamento específico de determinadas doenças do foro hematológico, ou ainda, na aplicação em imunoterapias. O nosso objetivo foi o de avaliar em vários buffy coats de dadores saudáveis, do CHUSJ, um conjunto de parâmetros celulares de referência, nomeadamente, a contagem de eritrócitos, de reticulócitos, associado ao valor de reticulócitos corrigido (VRC) e ao índice de produção de reticulócitos (IPR), bem como, a análise da morfologia das populações eritrocitária e leucocitária. Por outro lado, optou-se por uma análise quantitativa por citometria de fluxo de sub-populações linfocitárias, TCD4+, TCD8+ e linfócitos B, avaliando os parâmetros de perda da viabilidade celular ao longo de um timing de 7 dias e a sua concentração percentual, de forma a explorar o grau de variabilidade/integridade e degradação dos BC num determinado período de tempo. No nosso estudo e, no que respeita ao valor do IPR, valor mais representativo da atividade medular, verificou-se que a maioria dos dadores (84,6%) apresentou como resultado um valor de IPR =>3, o que denota uma resposta aumentada à solicitação do organismo à dádiva. Da análise das populações linfocitárias foi possível constatar uma redução da viabilidade celular e da percentagem de células, ao longo do período de tempo estudado.

Introduction: Anemia is a complication with impact on morbidity and mortality in chronic kidney disease (CKD) patients. Current markers for the diagnosis and monitoring of anemia in CKD are limited by the interrelation between erythropoiesis, iron stores, inflammation, and the resistance to treatment with erythropoiesis stimulant agents (ESA). Objective: The aim of this study was to analyze the role of immature reticulocyte fraction (IRF) and hemoglobin concentration in reticulocytes (RET-He) by the hematology analyser Sysmex XN-5000 in the monitoring of CKD anemia in peritoneal dialysis patients. Methods: This was a prospective, observational multicenter study which compared IRF and RET-He with parameters recommended by the guidelines. Inflammatory biomarkers were analyzed by the Luminex® Multiplexing Instruments system. Thirty-five patients (59 ± 13 years old; 51% men) were included in the analysis. Results: Hemoglobin was 12.2 ± 2 g/dl; 87% had resistance to ESA. Patients with erythropoietin resistance index (ERI) in the upper quartile presented a significantly higher of IRF and a lower percentage of iron deficiency (12%) compared to ferritin (82%) and transferrin saturation index (STI) (51%). Interleukin-6 (IL-6) levels correlated with the percentage of medium fluorescence reticulocyte (MFR) (r = 0.45, p < 0.03). Hemoglobin values after 60 and 180 days were consistently higher in the group of patients with a IRF% lower than 10.5. Conclusion: IRF and RET-He may add value in the iron deficiency investigation, as well as in the identification of patients with ERI. Due to the restricted number of patients analyzed in this study, future studies should be encouraged in larger populations and with prospective follow-up, to validate our findings. RESUMEN Introducción: La anemia es una complicación con impacto en la morbimortalidad en pacientes con enfermedad renal crónica (ERC). Los biomarcadores usados para el diagnóstico y seguimiento de la anemia en la ERC están limitados por la interrelación entre eritropoyesis, depósitos de hierro, inflamación y resistencia al tratamiento con agentes estimulantes de la eritropoyesis (AEE). Objetivo: El objetivo de este estudio fue analizar el papel de la fracción de reticulocitos inmaduros (IRF) y el equivalente de hemoglobina en reticulocitos (RET-He) mediante el analizador hematológico Sysmex XN-5000 en el seguimiento de la anemia por ERC en pacientes en diálisis peritoneal. Métodos: Estudio prospectivo, observacional multicéntrico que comparó IRF y RET-He con los parámetros recomendados por las guías. Los biomarcadores inflamatorios fueron analizados por el sistema Luminex® Multiplexing Instruments. Este estudio incluyó a 35 pacientes (59 ± 13 años; 51% hombres). Resultados: La hemoglobina fue de 12,2 ± 2 g/dl; el 87% tenía resistencia a AEE. Los pacientes con índice de resistencia a la eritropoyetina (IRE) en el cuartil superior tenían un IRF significativamente más alto y un porcentaje más bajo de deficiencia de hierro (12%) en comparación con la ferritina (82%) y las ITS (51%). Los niveles de interleucina-6 (IL-6) se correlacionaron con el porcentaje de reticulocitos de fluorescencia media (MFR) (r = 0,45, p < 0,03). Los valores de hemoglobina después de 60 y 180 días, fueron consistentemente más altos en el grupo de pacientes con IRF% inferior a 10,5. Conclusión: IRF y RET-He pueden agregar valor en la investigación de ferropenia, así como en la identificación de pacientes con ERI. Debido al número limitado de pacientes analizados en este estudio, se deben impulsar estudios futuros en poblaciones más grandes y con seguimiento prospectivo, para validar nuestros hallazgos. RESUMO Introdução: Anemia é uma complicação com impacto na morbidade e na mortalidade de pacientes com doença renal crônica (DRC). Os biomarcadores utilizados no diagnóstico e no monitoramento de anemia na DRC são limitados devido à inter-relação entre eritropoiese, estoque de ferro, inflamação e resistência à terapêutica com agentes estimuladores da eritropoiese (AEE). Objetivo: O objetivo deste estudo foi analisar o papel dos marcadores fração de reticulócitos imaturos (IRF) e concentração de hemoglobina nos reticulócitos (RET-He) do analisador hematológico Sysmex XN 5000 no monitoramento da anemia em pacientes em diálise peritoneal. Métodos: Estudo prospectivo, observacional e multicêntrico que comparou IRF e RET-He com parâmetros laboratoriais recomendados pelos guidelines. Biomarcadores inflamatórios foram analisados pelo sistema Luminex® Multiplexing Instruments. Este estudo incluiu 35 pacientes (59 ± 13 anos; 51% homens). Resultados: Os valores de hemoglobina foram 12,2 ± 2 g/dl; 87% apresentaram resistência a AEE. Pacientes com índice de resistência à eritropoietina (IRE) no quartil superior apresentaram valores significativamente maiores de IRF e menor porcentagem de deficiência de ferro (12%) em comparação com pacientes com ferritina (82%) e índice de saturação de transferrina (IST) (51%). Os níveis de interleucina 6 (IL-6) correlacionaram-se com a porcentagem de reticulócitos de fluorescência média (MFR) (r = 0,45, p < 0,03). Valores de hemoglobina após 60 e 180 dias foram consistentemente mais altos no grupo de pacientes com IRF% menor que 10,5. Conclusão: IRF e RET-He podem agregar valor na investigação da deficiência de ferro, bem como na identificação do índice de existência à eritropoietina (ERI). Devido ao número restrito de pacientes analisados neste trabalho, estudos futuros devem ser estimulados em populações maiores e com acompanhamento prospectivo, para validação dos nossos achados.

El trabajo experimental consiste en establecer valores de referencia de reticulocitos y leucograma (granulocitos, monocitos y linfocitos) en pollos de engorde cobb 500 a una altitud de 2260 m.s.n.m., se trabajó con 200 muestras sanguíneas, 100 de machos y 100 de hembras clínicamente evaluados y criados dentro de un sistema de producción controlado. Experimental work is to establish reticulocyte and leukogram reference values (granulocytes, monocytes and lymphocytes) in broiler chickens cobb 500 at an altitude of 2260 m.s.n.m., 200 blood samples, 100 male samples and 100 females clinically evaluated and bred within a controlled production system.

Resumen Introducción: El contenido de hemoglobina de reticulocitos (CHr), es un parámetro en la biometría hematológica automatizada que proporciona información sobre el contenido de hierro, por ello se ha utilizado como un marcador de la biodisponibilidad del hierro en la eritropoyesis, permite su detección en una etapa temprana de la anemia ferropénica y otras patologías como inflamación crónica, enfermedad renal crónica; además realizar monitoreo de terapias con eritropoyetina y hierro. Objetivo: Exponer la aplicabilidad de la CHr como un parámetro en el diagnóstico precoz de la anemia por deficiencia de hierro, así como su medición e interpretación. Materiales y métodos: Se realizó la revisión de artículos científicos en inglés y español en las bases de datos PubMed, ScienceDirect, LILACS y Medline, usando descriptores validados en Medical Subject Headings (MeSH), considerando periodo de publicabilidad del 80% inferior a 5 años. Resultados: Se describe la importancia, aplicabilidad, determinación e interpretación de este parámetro como biomarcador específico hemático temprano en el diagnóstico de deficiencia de hierro antes de presentarse cambios morfológicos eritroides. Conclusiones: La CHr es un parámetro de gran utilidad en el diagnóstico temprano de anemia ferropénica y otras patologías como deficiencia funcional de hierro, estados de inflamación crónica y enfermedad renal crónica. Abstract Introduction: The reticulocyte hemoglobin content (CHr) is a parameter in automated hematological biometrics, which can provide information on the iron content. So it has been used as a marker of the bioavailability of iron in the erythropoiesis, it allows its detection at an early stage of iron deficiency anemia and other pathologies such as chronic inflammation, chronic kidney disease; in addition to monitoring therapy with erythropoietin and iron. Objective: To expose the applicability of CHr as a parameter in the early diagnosis of iron deficiency anemia, as well as its measurement and interpretation. Materials and methods: The review of scientific articles in English and Spanish was carried out in the PubMed, ScienceDirect, LILACS and Medline databases, using descriptors validated in Medical Subject Headings (MeSH), considering the publication period of 80% less than 5 years. Results: The importance, applicability, determination and interpretation of this parameter is described as an early specific biomarker in the blood in the diagnosis of iron deficiency before presenting morphological changes occurring during terminal erythroid differentiation. Conclusions: CHr is a very useful parameter in the early diagnosis of iron deficiency anemia and other pathologies such as functional deficiency, chronic inflammation states and chronic renal disease.

The purpose of this work was to evaluate the genotoxic capacity of AgNPs Argovit™ with the micronucleus technique in mouse peripheral blood reticulocytes. Argovit™ is administered orally in therapeutic doses (indicated for dogs as treatment for distemper) for three days to an experimental group with seven mice. Another two groups with an equal number of mice were administered with injectable water and arabinose C, respectively. The specimens were measured and weighed before and after the experiment. Peripheral blood samples were also taken daily by a cut at the tip of the tail and then extended on slides for micronucleus test in reticulocytes. The smears were fixed in ethanol at 80 % and stained with acridine orange, after that, the presence of micronuclei was identified in 1000 reticulocytes counted by fluorescence microscopy. Preliminary results show significant differences in the number of micronuclei in reticulocytes between the three groups, so it’s necessary to compare this result with another type of in vitro models.

En medicina actualmente las radiaciones ionizantes son utilizadas como una herramienta para el diagnóstico precoz y tratamientos oportunos de varias patologías, sin embargo, este tipo de energía también afecta al personal que la utilizan y al que se encuentra alrededor produciendo alteraciones del ADN de las células de la médula ósea y traduciéndose en patologías hematológicas leves hasta alteraciones graves como es el cáncer. Los resultados se muestran mediante medidas de tendencia central y dispersión; el comportamiento de los datos resultó normal en la mayoría de las variables según la prueba de normalidad Kolmogorov Smirnov (p > 0.05) por lo que se emplearon pruebas paramétricas; la prueba de comparación de medias para dos grupos T-Student y el coeficiente de correlación r de Pearson; las decisiones fueron tomadas con una significancia del 5% (p < 0.05). Se determinó que los valores hematológicos y de reticulocitos se encuentran dentro de parámetros normales para todo el personal en estudio. Se obtuvo valores elevados de eritrocitos, hemoglobina, hematocrito y plaquetas en los hombres en relación con las mujeres y una disminución de las plaquetas mientras la edad del trabajador aumenta, siendo posiblemente variaciones fisiológicas, sin embargo, nos deja la puerta abierta para futuras investigaciones. Magíster en Salud Ocupacional y Seguridad en el Trabajo

The RBSA protein is encoded by a gene described in Plasmodium species having tropism for reticulocytes. Since this protein is antigenic in natural infections and can bind to target cells, it has been proposed as a potential candidate for an anti-Plasmodium vivax vaccine. However, genetic diversity (a challenge which must be overcome for ensuring fully effective vaccine design) has not been described at this locus. Likewise, the minimum regions mediating specific parasite-host interaction have not been determined. This is why the rbsa gene's evolutionary history is being here described, as well as the P. vivax rbsa (pvrbsa) genetic diversity and the specific regions mediating parasite adhesion to reticulocytes. Unlike what has previously been reported, rbsa was also present in several parasite species belonging to the monkey-malaria clade; paralogs were also found in Plasmodium parasites invading reticulocytes. The pvrbsa locus had less diversity than other merozoite surface proteins where natural selection and recombination were the main evolutionary forces involved in causing the observed polymorphism. The N-terminal end (PvRBSA-A) was conserved and under functional constraint; consequently, it was expressed as recombinant protein for binding assays. This protein fragment bound to reticulocytes whilst the C-terminus, included in recombinant PvRBSA-B (which was not under functional constraint), did not. Interestingly, two PvRBSA-A-derived peptides were able to inhibit protein binding to reticulocytes. Specific conserved and functionally important peptides within PvRBSA-A could thus be considered when designing a fully-effective vaccine against P. vivax. © 2018 Camargo-Ayala, Garzón-Ospina, Moreno-Pérez, Ricaurte-Contreras, Noya and Patarroyo.

Understanding the life cycle of Plasmodium vivax is fundamental for developing strategies aimed at controlling and eliminating this parasitic species. Although advances in omic sciences and high-throughput techniques in recent years have enabled the identification and characterization of proteins which might be participating in P. vivax invasion of target cells, exclusive parasite tropism for invading reticulocytes has become the main obstacle in maintaining a continuous culture for this species. Such advance that would help in defining each parasite protein's function in the complex process of P. vivax invasion, in addition to evaluating new therapeutic agents, is still a dream. Advances related to maintenance, culture medium supplements and the use of different sources of reticulocytes and parasites (strains and isolates) have been made regarding the development of an in vitro culture for P. vivax; however, only some cultures having few replication cycles have been obtained to date, meaning that this parasite's maintenance goes beyond the technical components involved. Although it is still not yet clear which molecular mechanisms P. vivax prefers for invading young CD71+ reticulocytes [early maturation stages (I-II-III)], changes related to membrane proteins remodelling of such cells could form part of the explanation. The most relevant aspects regarding P. vivax in vitro culture and host cell characteristics have been analysed in this review to explain possible reasons why the species' continuous in vitro culture is so difficult to standardize. Some alternatives for P. vivax in vitro culture have also been described. © 2018 The Author(s).

Background: Reticulocytes are immature red blood cells containing RNA remnants. Their population kinetics has been documented under various in vivo and in vitro conditions, including after storage of red blood cells in blood banks. The purpose of this study was to describe the influence of blood bank storage on the kinetics of reticulocyte disappearance by in vitro culturing. Method: Samples of reticulocyte-enriched fractions (Percoll density-gradient) were obtained over different storage times from six red blood cell units stored in additive solution-1 (AS-1). Reticulocyte fractions were then cultured in enriched media at 37 °C and analyzed by flow cytometry with thiazole orange taking into account hemolysis. Results: Density-gradient enriched reticulocyte fractions were obtain from standard red blood cell units with

La asimilacion deficiente de alimentos por el organismo conduce a un estado patologico con distintos grados y manifestaciones clinicas, conocido como desnutricion. En los ultimos anos ha surgido un nuevo ensayo in vivo de deteccion de mutacion somatica basado en el gen glucosilfosfatidilinositol de clase A ( Pig-a ). Este gen es necesario para la sintesis de glicosilfosfatidilinositol (GPI) que une proteinas especificas a la superficie de la celula. Cuando ocurre una mutacion en Pig-a , no se puede sintetizar el GPI, ya que, las proteinas no se encuentran en la membrana celular, dando origen al fenotipo GPI negativo, el cual es de facil deteccion por medio del analisis con citometria de flujo. En este estudio se utilizo el ensayo Pig-a para examinar el dano genico en ratas desnutridas expuestas al mutageno N-etil-N-nitrosourea (ENU). Se efectuo el marcaje diferencial de reticulocitos (RET) y eritrocitos (E) de sangre periferica de rata de la cepa Wistar. Se tomo como indicador de dano al material genetico de las celulas, el incremento en las frecuencias de mutantes (FM) Pig-a, que fueron analizadas en un 1 X 10 6 celulas, durante 8 semanas. Los resultados muestran que la desnutricion grave por si misma tiene un efecto danino sobre la integridad del material genetico. La frecuencia de mutantes se incremento con base en la dosis de ENU y el tiempo de exposicion en los grupos con desnutricion (DN).

Background: The Plasmodium vivax Duffy binding protein (PvDBP) has been the most studied ligand binding human reticulocytes to date. This molecule has a cysteine-rich domain in region II (RII) which has been used as control for evaluating the target cell binding activity of several parasite molecules. However, obtaining rPvDBP-RII in a soluble form using the Escherichia coli expression system usually requires laborious and time-consuming steps for recovering the molecule's structure and function, considering it is extracted from inclusion bodies. The present study describes an easy and fast method for expressing and obtaining several PvDBP fragments which should prove ideal for use in protein-cell interaction assays. Results: Two PvDBP encoding regions (rii and riii/v) were cloned in pEXP5-CT vector and expressed in E. coli and extracted from the soluble fraction (rPvDBP-RIIS and rPvDBP-RIII/VS) using a simple freezing/thawing protocol. After the purification, dichroism analysis enabled verifying high rPvDBP-RIIS and rPvDBP-RIII/VS secondary structure α-helix content, which was lowered when molecules were extracted from inclusion bodies (rPvDBP-RIIIB and rPvDBP-RIII/VIB) using a denaturing step. Interestingly, rPvDBP-RIIS, but not rPvDBP-RIIIB, bound to human reticulocytes, while rPvDBP-RIII/VS and rPvDBP-RIII/VIB bound to such cells in a similar way to negative control (cells incubated without recombinant proteins). Conclusions: This research has shown for the first time how rPvDBP-RII can be expressed and obtained in soluble form using the E. coli system and avoiding the denaturation and refolding steps commonly used. The results highlight the usefulness of the rPvDBP-RIII/VS fragment as a non-binding control for protein-cell target interaction assays. The soluble extraction protocol described is a good alternative to obtain fully functional P. vivax proteins in a fast and easy way, which will surely prove useful to laboratories working in studying this parasite's biology. © 2018 The Author(s).

Resumen Introducción. La cuantificación de la inestabilidad cromosómica es un parámetro importante para evaluar la genotoxicidad y la radiosensibilidad. Las técnicas convencionales requieren cultivos celulares o laboriosos análisis microscópicos de cromosomas o núcleos. La citometría de flujo en reticulocitos ha surgido como una alternativa para los estudios in vivo, ya que reduce los tiempos de análisis e incrementa hasta en 20 veces el número de células analizables. Objetivos. Estandarizar los parámetros de citometría de flujo requeridos para seleccionar y cuantificar reticulocitos micronucleados (RET-MN) a partir de muestras de sangre periférica, y cuantificar la frecuencia de esta subpoblación anormal como medida de inestabilidad citogenética en sendas poblaciones de voluntarios sanos (n=25) y pacientes (n=25) recién diagnosticados con gliomas de alto grado antes de iniciar el tratamiento. Materiales y métodos. Las células sanguíneas se marcaron con anti-CD71-PE para reticulocitos, anti- CD61-FITC para la exclusión de plaquetas y yoduro de propidio para detectar el ADN en reticulocitos. La fracción celular MN-RETCD71+ se seleccionó y se cuantificó con un citómetro de flujo automático. Resultados. Se describió detalladamente la estandarización de los parámetros citométricos, con énfasis en la selección y la cuantificación de la subpoblación celular MN-RETCD71+. Se establecieron los niveles basales de MN-RETCD71+ en la población de control y en los pacientes se encontró un incremento de 5,2 veces antes de iniciar el tratamiento (p

O lúpus eritematoso sistêmico (LES) é uma doença autoimune e inflamatória caracterizada pela produção de diversos autoanticorpos e depósitos teciduais de complexos antígeno-anticorpo circulantes. A descoberta e validação de métodos para determinação de produtos de ativação do complemento ligados a células do sangue (CBCAPs= Cell-bound complement activation products), tais como C4d ligados a reticulócitos (R-C4d) e a plaquetas (P-C4d), como potenciais biomarcadores do estado de atividade e do potencial trombótico do LES, respectivamente, podem ser importantes ferramentas para o melhor entendimento da fisiopatologia e do manejo clínico dessa doença. Outro fator importante na patogênese do LES, são os eventos consequentes a anormalidades do sistema hemostático que necessitam ser melhor entendidos. Dessa forma, o objetivo principal desse estudo foi avaliar se, no LES, os níveis de CB-CAPs podem ser preditores de ativação da doença (R-C4d) e de predisposição trombótica (PC4d). Além dos níveis de CB-CAPs, investigou-se o potencial trombótico por meio da determinação dos níveis plasmáticos de importantes componentes do sistema hemostático, tais como dímero D (DDi), trombomodulina (TM), proteína S (PS) e proteína ligadora de C4b (C4BP), buscando-se uma possível associação entre estes parâmetros e os outros investigados. Para alcançar esse objetivo, foram incluídas um total de 60 pacientes com LES, sob tratamento, selecionadas no Serviço de Reumatologia da Santa Casa de Belo Horizonte, sendo 30 pacientes classificadas com doença em baixa atividade (SLEDAI-2K 4) e 30 com a doença em moderada/alta atividade (SLEDAI-2K > 4), enquanto simultaneamente foram selecionadas 30 mulheres sem LES (controles), pareadas por idade e do mesmo nível sócio econômico. As amostras de sangue coletadas das participantes do estudo foram utilizadas para a determinação dos níveis de CB-CAPs por meio da citometria de fluxo e para a obtenção de plasma para a realização dos ensaios previstos neste estudo. Foi observado aumento dos níveis de R-C4d, P-C4d, TM e DDi em pacientes com LES com moderada/alta atividade, cujos níveis correlacionaram com o aumento do índice SLEDAI-2K. Os níveis de PS e C4BP foram semelhantes entre os pacientes com LES moderada/alta atividade, baixa atividade e controles. A análise conjunta dos dados permitiu concluir que níveis elevados de R-C4d e P-C4d correlacionaram com o índice SLEDAI-2K e, portanto, com piora clínica em pacientes com LES. Além do mais, a combinação de RC4d e P-C4d permitiu o estabelecimento de uma proposta de cutoff como indicativo de doença em atividade. Finalmente, foi possível ainda confirmar a relação entre doença em atividade e estado de hipercoagulabilidade, provavelmente associado à dano endotelial e inflamação. Systemic lupus erythematosus (SLE) is an autoimmune and inflammatory disease characterized by the production of various autoantibodies and tissue deposits of circulating antigen-antibody complexes. The discovery and validation of methods for the determination of cell bound complement activation products (CB-CAPs), such as C4d bound to reticulocytes (R-C4d) and to platelets (P-C4d), as potential biomarkers of the activity state and the thrombotic potential of SLE, respectively, may be important tools for a better understanding of the pathophysiology and clinical management of SLE patients. Another important factor in the pathogenesis of SLE are the events resulting from hemostatic system abnormalities that need to be better understood. Thus, the main objective of this study was to evaluate whether the levels of CB-CAPs in SLE patients can be predictors of disease activity (R-C4d) and thrombotic predisposition (P-C4d). In addition to the CB-CAPs levels, the thrombotic potential was investigated by determining the plasma levels of important components of the hemostatic system such as D-dimer (DDi), thrombomodulin (TM), protein S (PS) and C4b binding protein (C4BP), seeking a possible association between these parameters and the others also investigated. To achieve this goal, a total of 60 patients with SLE, under treatment, selected at the Hospital Santa Casa Rheumatology Service in Belo Horizonte was included, with 30 LES patients classified as having low-disease activity (SLEDAI-2K 4) and 30 patients with moderate/high activity (SLEDAI-2K> 4), while 30 women without SLE (controls), matched by age and socioeconomic status were simultaneously selected. Blood samples collected from the study participants were used to determine the levels of CB-CAPs by means of flow cytometry and to obtain plasma for carrying out the other tests included in this study. Compared to both controls and SLE patients with low activity disease, increased levels of R-C4d, P-C4d, TM and DDi were observed in patients with SLE with moderate/high activity disease, whose levels correlated with the increase in the SLEDAI-2K index. PS and C4BP levels were similar among all three groups. Data analysis showed that high levels of R-C4d and P-C4d correlated with the SLEDAI-2K index and, therefore, with clinical worsening in SLE patients. Moreover, the combination of R-C4d and P-C4d allowed the establishment of a cutoff proposal as indicative of disease activity. Finally, it was possible to confirm the relationship between disease activity and hypercoagulability, probably associated with endothelial damage and inflammation.

Las interacciones hospedero-patógeno y la formación de complejos macromoleculares durante el proceso de invasión de los parásitos Apicomplexa a sus células hospederas, representan un blanco de acción importante para el diseño de métodos de control contra estos microorganismos. En Plasmodium vivax, el conocimiento acerca de la biología y de las funciones de las protemas e interacciones que ocurren durante el proceso de invasión es escaso debido a la difícil propagación del parásito in vitro. En este esñidio se evaluó si las protemas RON-2. -4 y AMA-1 de P. vivax tienen la capacidad de interactuar con reticulocitos humanos. Así mismo, se evalúo si existen interacciones binarias entre las proteínas RON2. -4. -5 y AMA-1 que puedan asociarlas como un complejo proteico como ha sido sugerido para P. falciparitm. Para las interacciones receptor-ligando se encontró que los fragmentos PvAMA-DI-IL PvR0N2-RI. PvR0N2-RII y P\ R0N4 se unen a reticulocitos CD71+. De particular interés, se encontró que el dominio I de PvAMA-1 contiene una región de 20 residuos responsable de la interacción específica y de alta afinidad con receptores de reticulocitos C'D71+. Por otro lado, mediante la técnica ELISA, se encontraron interacciones relevantes entre el fragmento rPvAMA-DII-III con rPvRON2-RI. 1P1RON2-RH y rPvRON4, y entre la proteína rPvRON5 con los fragmentos de PvRON2. Estos resultados representan un avance en el conocimiento biológico de P. vivax y sugiere la participación de algunas regiones de estas proteínas en interacciones hospedero-patógeno y proteina-proteína que pueden ser esenciales para la búsqueda de métodos de control específicos contra esta especie parasitaria. Host-pathogen interactions and macromolecular complex formation during Apicomplexa parasite invasion of their host cells represents an important action target for designing methods for controlling these microorganisms. Knowledge regarding the biology and functions of proteins and interactions occurring dining invasion is scarce regarding Plasmodium vivax due to the difficulty of propagating the parasite in vitro. This study was thus aimed at evaluating P. vivax RON-2, -4 and AMA-1 proteins, bearing in mind then capability for niteracting with human reticulocytes. An attempt was also made to ascertain whether binary interactions between R0N2. -4. -5 and AMA-1 proteins could be associated with a protein complex, as has been suggested for P. falciparum. Concerning receptor-ligand interactions, it was found that PiAMA-DI-II. PvR0N2-RI. PvR0N2-RII and P1R0N4 fragments bound to CD71+ reticulocytes. It was particularly interesting to find that Pi AMA-1 domain I contained a 20 residue-long region responsible for specific, high affinity interaction with CD71+reticulocyte receptors. ELISA revealed relevant interactions between the rPvAMA-DII-III fragment and iPi RON2-RI. rPiRON2-RII and rPrRON4 and between rPi RON5 and Pi RON 2 fragments. These results represent an advance regaiding knowledge concerning P. vivax biology and suggest the participation of some of these proteins’ regions in host-pathogen and protein-protein interactions which might be essential regarding the search for specific control methods against this parasite species. Magíster en Ciencias Biológicas Maestría

Tesis por compendio de publicaciones, [EN] Identifying and characterising proteins which use Plasmodium merozoites to invade host cells represents an important strategy for developing a method for controlling these parasites. However, basic P. vivax research has been delayed due to difficulties in propagating it in vitro as the parasite prefers to invade reticulocytes; there is a low percentage of these in adult human peripheral blood (1%-2%) and they are difficult to obtain with high purity, in a sufficient amount and totally viable. Consequently, knowledge is scarce regarding the amount of molecules being expressed by P. vivax and which of them represent good candidates for inclusion in an effective vaccine. This study has been aimed at evaluating the proteome of a primate-adapted P. vivax strain; antigenic molecules able to bind to human reticulocytes have been characterised. Analysing the P. vivax VCG-1 strain proteome led to detecting 734 proteins, some of them essential in key steps for establishing merozoite invasion of target cells. Furthermore, 811 A. nancymaae primate erythrocyte components (vital Plasmodium hosts) were identified; 51 of them were integral membrane proteins, 7 described as Plasmodium receptors. The presence, transcription, expression and antigenicity of genes encoding three P. vivax molecules (PvARP, PvRBSA and PvGAMA) were identified. Particularly interesting was the finding that a higher percentage of PvRBSA and PvGAMA bound to reticulocytes abundantly expressing the CD71 receptor (CD71hi), thereby suggesting that these molecules could be participating in P. vivax merozoite preferential selection for human reticulocytes. This the first study in Colombia which has determined the protein composition of a primate-adapted P. vivax strain as well as A. nancymaae erythrocytes. More importantly, P. vivax molecules were characterised which appear to be suitable candidates for being evaluated as components of a vaccine against malaria caused by the parasite species., [ES] La identificación y caracterización de proteínas que utilizan los merozoitos de Plasmodium para invadir a su célula hospedera, representan una estrategia importante para desarrollar un método de control contra estos parásitos. A pesar de ello, la investigación básica en P. vivax está retrasada por su difícil propagación in vitro, debido a la preferencia que tiene el parásito por invadir reticulocitos, los cuales se encuentran en escaso porcentaje en sangre periférica de humanos adultos (1-2%) y son difíciles de obtener con alta pureza, en suficiente cantidad y totalmente viables. Como consecuencia de lo anterior, el conocimiento del número de moléculas que expresa P. vivax y cuáles de ellas son candidatas para componer una vacuna, es escaso. En este estudio, se evaluó el proteoma de una cepa de P. vivax adaptada a primates y se caracterizaron moléculas antigénicas y con capacidad de adhesión a reticulocitos humanos. En el análisis del proteoma de la cepa VCG-1 de P. vivax, se detectaron 734 proteínas, algunas esenciales en los pasos clave para establecer la invasión del merozoito a su célula diana. Además, se identificaron 811 componentes de eritrocitos (hospederos vitales de Plasmodium) del primate A. nancymaae, de los cuales 51 son proteínas integrales de membrana, 7 descritas como receptores de Plasmodium. Por otro lado, se identificó la presencia, transcripción y expresión de los genes codificantes de tres moléculas de P. vivax: PvARP, PvRBSA y PvGAMA, así como su antigenicidad. De particular interés, se encontró que PvRBSA y PvGAMA se unen en mayor proporción a reticulocitos que expresan el receptor CD71 de forma abundante (CD71hi), lo que sugiere que estas moléculas pueden estar participando en la selección preferencial que tienen los merozoitos de P. vivax por los reticulocitos humanos. Este es el primer estudio en Colombia donde se determina la composición proteica de una cepa de P. vivax adaptada a primates, así como la de eritrocitos de A. nancymaae. Como resultado más importante, se caracterizaron moléculas de P. vivax que son candidatos idóneos a ser evaluados como componentes de una vacuna contra la malaria causada por esta especie parasitaria.

Pdf La anemia por deficiencia de hierro es el trastorno hematológico más frecuente en la edad pediátrica. Representa un problema grave de salud que afecta a más de una tercera parte de la población mundial. A partir de los datos obtenidos se aplicó la fórmula para determinar ADE, con el objetivo de aportar al diagnóstico de la anemia ferropénica. Se realizó un estudio cuantitativo no experimental, retrospectivo, transversal, en el que se incluyeron 500 niños con orden para estudio de la anemia a los cuales se les tomó una muestra sanguínea venosa, luego de obtener el consentimiento informado de los padres. En la muestra de aquellos que consintieron su participación se realizó biometría hemática que se analizó mediante impedancia, y se determinó hierro sérico por colorimetría, en los equipos automatizados de hematología y bioquímica que se encuentran en las instalaciones del Laboratorio INTERLAB. El 23 % de los niños estudiados hematológicamente, presentaron algún tipo de anemia. El análisis diferencial de casos sospechosos de anemia por deficiencia de hierro arrojó similares resultados ya sea cuando se utiliza como parámetro la concentración de hierro sérico como cuando se realiza el tamizaje mediante el factor de amplitud de la distribución de tamaño eritrocitario. Dando como resultados un 5 % (con la concentración de hierro sérico) y un 4,8 % (con el factor ADE). En ambos tamizajes (hierro vs ADE) se confirmaron 10 (2 %) pacientes con anemia ferropénica. Los parámetros “Concentración de hierro sérico” y “ADE” mostraron que no existe asociación estadística entre ambos. Iron deficiency anemia is the most common hematological disorder in the pediatric age. It represents a serious health problem affecting more than a third of the world's population. From the data obtained the formula was applied to determine ADE, with the objective of helping the diagnosis of iron deficiency anemia. A non-experimental, retrospective, cross- sectional quantitative study was conducted in which 500 children with an order for the study of anemia were included, who were given a venous blood sample after obtaining the informed consent of the parents. In the sample of those who consented to their participation, blood count was determined by means of impedance and serum iron was determined by colorimetry in the automated hematology and biochemistry equipment at the INTERLAB Laboratory facilities. Twenty-three percent of the hematologically studied children had some type of anemia. The differential analysis of suspected cases of iron deficiency anemia showed similar results when serum iron concentration was used as the parameter when screening was performed using the amplitude factor of the erythrocyte size distribution. The results were 5% (with serum iron concentration) and 4.8% (with ADE factor). In both screenings (iron vs ADE) 10 (2%) patients were confirmed with iron deficiency anemia. The parameters "Concentration of serum iron" and "ADE" showed that there is no statistical association between both.