Trichinella spiralis y Trichinella patagoniensis: estudio de la carga globular aplicando el método de Azul Alcian / Trichinella spiralis and Trichinella patagoniensis: study of globular charge applying Alcian Blue method / Trichinella spiralis e Trichinella patagoniensis: estudo da carga globular pelo método de Azul de Alcian

Resumen Los residuos de ácido siálico de las glicoproteínas de superficie son los principales responsables de la carga negativa eritrocitaria. El objetivo de este trabajo fue estudiar alteraciones de carga globular producidas por Trichinella spiralis y Trichinella patagoniensis. Se trabajó con concentrados de larvas musculares de ambas especies y con eritrocitos frescos. Se incubó el sedimento globular con igual volumen de concentrado larval (1 y 2 horas). Los controles fueron incubados con solución salina. Se aplicó el método de Azul Alcian y se determinó el coeficiente experimental de carga aniónica eritrocitaria (CexpCAE). Los resultados mostraron que la carga disminuyó con el aumento del tiempo de tratamiento para ambas especies. Los valores de CexpCAE de las suspensiones incubadas con T. spiralis fueron menores que con T. patagoniensis, indicando que T. spiralis produjo mayor disminución de carga que T. patagoniensis. Se concluye que la desialización producida por ambas especies no es la misma, lo que sugiere que la relación hospedador-parásito que se establecería in vivo sería distinta.

Abstract The sialic acid residues of the surface glycoproteins are mainly responsible for the erythrocyte negative charge. The objective of this work was to study alterations of globular charge produced by Trichinella spiralis and Trichinella patagoniensis. Work was carried out on muscle larvae concentrates of both species and fresh erythrocytes. The treatment was performed by incubating the globular pellet with equal volume of larval concentrate (1 and 2 hours). Controls were incubated with saline solution. The Alcian Blue method was applied and the experimental coefficient of erythrocyte anion charge (expCEAC) was determined. The results showed that the globular charge decreased with increasing treatment time for both species. The expCEAC values of the suspensions incubated with T. spiralis were lower than with T. patagoniensis, indicating that T. spiralis produced a greater decrease in charge than T. patagoniensis. It is concluded that the desialization produced by both species is not the same, suggesting that the host-parasite relationship that would be established in vivo would be different.

Resumo Os resíduos de ácido siálico das glicoproteínas de superfície são os principais responsáveis pela carga negativa dos eritrócitos. O objetivo desse trabalho foi estudar as alterações da carga globular produzidas por Trichinella spiralis e Trichinella patagoniensis. Trabalhamos com concentrados de larvas musculares de ambas as espécies e com eritrócitos frescos. O tratamento foi realizado incubando o sedimento globular com igual volume de concentrado larval (1 e 2 horas), Os controles foram incubados com solução salina. Foi aplicado o método de Azul de Alcian e se determinou o coeficiente experimental de carga aniônica de eritrócitos (CexpCAE). Os resultados mostraram que a carga diminuiu com o aumento do tempo de tratamento para ambas as espécies. Os valores de CexpCAE das suspensões incubadas com T. spiralis foram menores que com T. patagoniensis, indicando que T. spiralis produziu uma diminuição maior na carga que T. patagoniensis. Conclui-se que a dessalinização produzida por ambas as espécies não é a mesma, sugerindo que a relação hospedeiro parasita que seria estabelecida in vivo é diferente.

Analysis of Sulfated Glycosaminoglycans in ECM Scaffolds for Tissue Engineering Applications: Modified Alcian Blue Method Development and Validation.

Accurate determination of the amount of glycosaminoglycans (GAGs) in a complex mixture of extracellular matrix (ECM) is important for tissue morphogenesis and homeostasis. The aim of the present study was to investigate an accurate, simple and sensitive alcian blue (AB) method for quantifying heparin in biological samples. A method for analyzing heparin was developed and parameters such as volume, precipitation time, solvent component, and solubility time were evaluated. The AB dye and heparin samples were allowed to react at 4 ℃ for 24 h. The heparin-AB complex was dissolved in 25 N NaOH and 2-Aminoethanol (1:24 v/v). The optical density of the solution was analyzed by UV-Vis spectrometry at 620 nm. The modified AB method was validated in accordance with U.S. Food and Drug Administration guidelines. The limit of detection was found to be 2.95 µg/mL. Intraday and interday precision ranged between 2.14-4.83% and 3.16-7.02% (n = 9), respectively. Overall recovery for three concentration levels varied between 97 ± 3.5%, confirming good accuracy. In addition, this study has discovered the interdisciplinary nature of protein detection using the AB method. The basis for this investigation was that the fibrous protein inhibits heparin-AB complex whereas globular protein does not. Further, we measured the content of sulfated GAGs (sGAGs; expressed as heparin equivalent) in the ECM of decellularized porcine liver. In conclusion, the AB method may be used for the quantitative analysis of heparin in ECM scaffolds for tissue engineering applications.

Alteración del ácido siálico eritrocitario por efecto de Trichinella spiralis aplicando el método de Azul Alcian / Alteration of erythrocyte sialic acid by the effect of Trichinella spiralis applying the Alcian Blue method / Alteração do ácido siálico eritrocitário por efeito da Trichinella spiralis aplicando o método de Alcian Blue

Actualmente se reconoce que los ácidos siálicos están involucrados en múltiples funciones biológicas y que tienen un papel importante en la interacción parásito-hospedador. El objetivo del trabajo fue estudiar la alteración del ácido siálico eritrocitario por efecto de T. spiralis aplicando el método de Azul Alcian. Se trabajó con 11 concentrados de larvas musculares de T. spiralis (LM) y 9 de larvas recién nacidas de T. Spiralis (LRN) (total: 20) y con suspensiones de eritrocitos frescos. Se realizó el tratamiento incubando el sedimento globular con igual volumen de concentrado larval (37 ºC durante 1 hora). Los controles fueron incubados de la misma forma con solución salina. Se aplicó el método de Azul Alcian y se determinó CAE% en el control y en los glóbulos tratados. Se calculó CexpCAE. Los resultados mostraron que 5/9 de los concentrados de LRN y 9/11 de LM modificaron la carga globular. La media y la desviación estándar de los CexpCAE de las suspensiones en que la carga disminuyó por contacto con LRN y LM, fueron 0,614±0,1946 y 0,656±0,1865 respectivamente, mientras que en las que no se modificó resultó 0,955±0,0289 en el tratamiento con LRN y 0,93±0,0141 con LM. Se concluye que el Método de Azul Alcian es útil para estudiar las variaciones en la carga eritrocitaria por efecto de LRN y LM de T. spiralis.

It is now recognized that sialic acids are involved in multiple biological functions and that they have an important role in the parasite-host interaction. The objective of this work was to study the alteration of erythrocyte sialic acid by the effect of T. spiralis applying the Alcian Blue method. Work was carried out with 11 larval concentrates of ML and 9 of NL (total 20) and with fresh erythrocyte suspensions. The treatment was performed incubating the globular sediment with equal volume of larval concentrate (37 °C for 1 hour). The controls were incubated in the same way with saline solution. The Alcian Blue method was applied and CAE% was determined in the control and in the treated globules. CexpCAE was calculated. The results showed that 5/9 of the NL concentrates and 9/11 of ML modified the globular charge. The mean and standard deviation of the CexpCAE of the suspensions in which the charge decreased by contact with NL and ML were 0.614±0.1946 and 0.656±0.1865 respectively, whereas in those that did not change, it was 0.955±0.0289 in the NL treatment and 0.93±0.0141 in the ML. It is concluded that the Alcian Blue Method is useful to study the variations in erythrocyte charge due to NL and ML of T. spiralis.

Atualmente se reconhece que os ácidos siálicos estão envolvidos em múltiplas funções biológicas e que têm um papel importante na interação parasita-hospedeiro. O objetivo do trabalho foi estudar a alteração do ácido siálico eritrocitário por efeito de T. spiralis aplicando o método do Alcian Blue. Trabalhou-se com 11 concentrados de larvas musculares de T. spiralis (LM) e 9 de larvas recém-nascidas de T. Spiralis (LRN) (total: 20) e com suspensões de eritrócitos frescos. Realizou-se o tratamento incubando o sedimento globular com igual volume de concentrado larval (37 ºC durante 1 hora). Os controles foram incubados da mesma forma com solução salina. Aplicou-se o método de Azul Alcian e determinou-se CAE% no controle e nos glóbulos tratados. Calculou-se CexpCAE. Os resultados mostraram que 5/9 dos concentrados de LRN e 9/11 de LM modificaram o carga globular. A média e o desvio padrão dos CexpCAE das suspensões em que a carga diminuiu por contato com LRN e LM, foram 0,614±0,1946 e 0,656±0,1865 respectivamente, enquanto que naquelas onde não se modificou, resultou 0,955±0,0289 no tratamento com LRN e 0,93±0,0141 com LM. Conclui-se que o método de Alcian Blue é útil para estudar as variações na carga eritrocitária por LRN e LM de T. spiralis.

Alteración de la carga eritrocitaria utilizando el método de azul Alcian en extractos de Ascaris lumbricoides / Change of erythrocyte charge with the use of Alcian blue method in Ascaris lumbricoides extracts

Introducción: el estudio de las interacciones hospedador-parásito es un nuevo desafío para comprender aspectos del metabolismo parasitario, los mecanismos de invasión, escape inmunológico y daño. Ascaris lumbricoides puede provocar anemia y trombosis. Previamente se demostró que este parásito altera la carga superficial eritrocitaria, lo cual indica que puede captar ácido siálico del glóbulo rojo. Objetivo: estudiar el efecto producido por extractos del parásito adulto sobre la carga eritrocitaria, utilizando el método de azul Alcian y comparar su sensibilidad con el método de Polibrene. Métodos: se trabajó con 55 extractos parasitarios [EA] y con suspensiones de eritrocitos grupo O. Se realizó el tratamiento de los glóbulos incubando el sedimento con igual volumen de [EA] a 37 ºC durante 1 h. El Control (eritrocitos sin contacto con [EA]) fue incubado con tampón fosfato pH 7,4. Se aplicó el método de azul Alcian y se determinó la carga aniónica eritrocitaria porcentual (CAE por ciento) en el Control y en los glóbulos tratados. Se definió el coeficiente experimental de carga aniónica eritrocitaria (Cexp CAE), como el cociente entre CAE por ciento final e inicial. Resultados: se observó que 27 de los 55 [EA] (49,1 por ciento) modificaron la carga de los glóbulos rojos, el Cexp CAE para estos eritrocitos resultó 0,75 ± 0,1144 y para los que no mostraron variación de carga de 0,94 ± 0,04450. El análisis estadístico concluyó que los métodos de Polibrene y de azul Alcian tienen sensibilidades comparables (p> 0,20). Conclusiones: Ascaris lumbricoides es capaz de captar ácido siálico del eritrocito, lo que contribuiría a explicar la trombosis atribuida al parásito, pero también sugeriría que el nematodo lo podría utilizar en sus rutas metabólicas o en sus estrategias de evasión inmunológica.

Introduction: the study of the host-parasite interactions is a new challenge to understanding some aspects of the parasitic metabolism and the mechanisms of invasion, immunological evasion and damage. Ascaris lumbricoides may cause anemia and thrombosis. It was previously shown that Ascaris lumbricoides modified the superficial charge of erythrocytes, which means that the parasite can capture sialic acid from the red blood cell. Objective: to study the effect of adult parasite extracts on the erythrocyte charge using the Alcian Blue method and to compare its sensitivity with the Polybrene method. Methods: fifty five adult parasite extracts and Group O erythrocyte suspensions were used. The erythrocytes were treated by incubating the sediment with an equal volume of parasite extracts for one hour at 37 ºC. The control group (erythrocytes without any contact with the parasite extracts) was incubated with pH 7.4phosphate buffer solution. Alcian Blue method was applied and the percentage erythrocyte anionic charge was determined in the control group and in the treated red cells. The experimental coefficient of erythrocyte anionic charge was defined as the quotient between the initial and the final percentage erythrocyte anionic charge. Results: it was shown that 27 out of 55 parasite extracts (49.1 percent) modified the charge of the red blood cells, being their experimental coefficient of the erythrocyte anionic charge 0.75 ± 0.1144 whereas the same coefficient amounted to 0,94 ± 0.0445 for those which did not show any charge variation. The statistical analysis concluded that the Polybrene and Alcian Blue Methods had comparable sensitivities (p>0.20). Conclusions: A. lumbricoides is able to capture sialic acid from the erythrocyte, which would not only explain the thrombosis attributed to the parasite, but also suggest that the nematode could use this acid either in its metabolic routes or for its strategies of immunological evasion.