Trichinella spiralis-Secreted Products Promote Collagen Capsule Formation through TGF-ß1/Smad3 Pathway.

Trichinella spiralis (T. spiralis) muscle larvae colonize in the host's skeletal muscle cells, which are surrounded by collagen capsules. The mechanism underlying muscle stage larva-induced collagen capsule formation remains unknown. To clarify the mechanism, a T. spiralis muscular-infected mouse model was established by a single lateral tail vein injection with 20,000 T. spiralis newborn larvae (NBL). The infected mice were treated with or without SB525334 (TGF-ß1 receptor type I inhibitor). Diaphragms were obtained post-infection, and the expression levels of the TGF-ß1/Smad3 pathway-related genes and collagen genes (type IV and VI) were observed during the process of collagen capsule formation. The changes in myoblasts under stimulation of the excretory-secretory (ES) products of NBL with or without SB525334 were further investigated. Results showed that the expression levels of type IV collagen gene, type VI collagen gene, Tgfb1, and Smad3 were significantly increased in infected mice muscle cells. The expression levels of all the above genes were enhanced by the products of NBL in myoblast cells. These changes were reversed by co-treatment with SB525334 in vivo and in vitro. In conclusion, the TGF-ß1/Smad3 pathway can be activated by T. spiralis infection in muscle cells. The activated TGF-ß1/Smad3 pathway can stimulate the secretion of collagens by myocytes and plays a promoting role in the process of collagen capsule formation. The research has the limitation that the protein identification of the products of NBL has yet to be performed. Therefore, the specific components in the T. spiralis ES products that induce collagen synthesis should be further investigated.

Protein deficiency during Trichinella spiralis infection impairs lung immunity against newborn larvae.

The goal of this study was to analyse the effects of a protein-deficient (PD) diet on antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro against newborn larvae (NBL) of Trichinella spiralis in the lungs of infected rats. Two groups of weaning Wistar rats received a PD diet (6.5% casein) and other two received a control diet (C, 20% casein). After ten days, one group of each diet was infected (PDI and CI ) with muscle larvae. Lung tissue extracts (LTE) and lung cell suspension (LCS) were obtained. PDI had lower titres of anti-NBL antibodies in LTE than CI . In ADCC assays using control cells, NBL mortality percentage was lower with LTE from PDI than LTE from CI (P < .01). In assays using control cytotoxic sera, ADCC was exerted by LCS from CI at all days post-infection (p.i.), but only by LCS from 13 days p.i. from PDI . ADCC assays combining LTE and LCS from the same group showed a lower response for PDI than for CI (P < .0001). LCS from PDI contained lower numbers of neutrophils, eosinophils and FcεRI+ cells than CI . PD may diminish ADCC activity against T spiralis NBL in lungs through alterations in specific antibodies and effector cells.

Larvas recién nacidas de Trichinella spiralis: cinética de agregación eritrocitaria / Newborn larvae of Trichinella spiralis: erythrocyte aggregation kinetics

El objetivo de este trabajo fue estudiar la cinética de agregación eritrocitaria producida por dos concentraciones de larvas recién nacidas (LRN) de Trichinella spiralis. Se trabajó con 5 suspensiones eritrocitarias incubadas con 500 y 1.000±100 LRN/mL durante 120 minutos, con tomas de muestra al tiempo inicial y cada 15 minutos. Los respectivos controles se incubaron de la misma manera con solución salina. Se aplicaron el método de titulación por Polibrene calculando el CexpST y la técnica de análisis de la variancia con las comparaciones múltiples según Tukey. Los resultados mostraron para ambas concentraciones de LRN, que el coeficiente promedio disminuyó con el aumento del tiempo de incubación. En el tratamiento con 1.000 LRN/mL, el coeficiente promedio no presentó diferencias significativas a tiempo 0 y 15 minutos, ni entre 60 y 75 minutos, mientras que con 500 LRN/mL no hubo diferencias entre los tiempos 0, 15 y 30 minutos. Todas las restantes diferencias fueron significativas para ambas concentraciones larvales. El valor promedio de CexpST no difirió significativamente entre los dos tratamientos a tiempo 0 y 15 minutos, pero a todos los otros tiempos fue menor a mayor concentración de larvas. La experiencia realizada indicaría que in vivo, la cantidad de LRN y el tiempo que permanecen en circulación determinan el grado de desializacion eritrocitaria, y por lo tanto el riesgo de activación T y de alteraciones hemorreológicas en el hospedador.

The aim of this work was to study the kinetics of erythrocyte aggregation produced by two concentrations of Trichinella spiralis newborn larvae (NL). Work was performed with 5 erythrocyte suspensions incubated with 500 and 1000 ± 100 NL/mL for 120 minutes, taking samples at the initial time and every 15 minutes. The respective controls were incubated in the same way with saline solution. The Polybrene titration method calculating the CexpST and the variance analysis technique with the multiple comparisons according to Tukey were applied. The results showed that the average coefficient decreased with the rise in incubation time for both NL concentrations. The average coefficient did not present significant differences between the initial time and 15 minutes, nor between 60 and 75 minutes in the treatment with 1000 NL/mL, while there were no differences between 0,15 and 30 minutes in the treatment with 500 NL/mL. All other differences were significant for both larval concentrations. The average value of CexpST did not differ significantly between the two time treatments at zero time and 15 minutes, but at all other times it was less at a higher concentration of larvae. The experience carried out would indicate that in vivo, the amount of NL and the time that they remain in circulation determines the degree of erythrocyte desialylation, and therefore, the risk of T activation and hemorrheological alterations in the host.

O objetivo desse trabalho foi estudar a cinética de agregação eritrocitária produzida por duas concentrações de larvas recém nascidas (LRN) de Trichinella spiralis. O trabalho foi feito com 5 suspensões eritrocitárias incubadas com 500 e 1.000 ± 100 LRN/mL por 120 minutos, colhendo amostras no tempo inicial e a cada 15 minutos. Os respectivos controles foram incubados da mesma forma com solução salina. Foi aplicado o método de titulação por Polibreno e se calculou CexpST. e a técnica de análise da variância com as comparações múltiplas de acordo com Tukey. Os resultados mostraram, para ambas as concentrações de LRN, que o o coeficiente médio diminuiu com o aumento do tempo de incubação. No tratamento com 1.000 LRN/mL o coeficiente médio não mostrou diferenças significativas no tempo 0 e 15 minutos ou entre 60 e 75 minutos, ao passo que não houve diferenças com 500 LRN/mL entre tempos 0, 15 e 30 minutos. Todas as restantes diferenças foram significativas para ambas as concentrações de larvas. O valor médio de CexpST não diferiu significativamente entre os dois tratamentos no tempo de 0 e 15 minutos, mas em todos os outros tempos foi menor em maior concentração de larvas. A experiência realizada indicaria que in vivo a quantidade de LRN e o tempo que permanecem em circulação determina o grau de dessialização dos eritrócitos e, portanto, o risco de ativação T e de alterações hemorreológicas no hospedeiro.

Desialización eritrocitaria por larvas recién nacidas de Trichinella spiralis incubadas in vitro / Erythrocyte desialylation by Trichinella spiralis newborn larvae incubated in vitro / Dessialização eritrocitária por larvas recém-nascidas da Trichinella spiralis incubadas in vitro

Trichinella spiralis es la especie que causa la mayoría de los casos de infección humana en todo el mundo. Se comunicó que el contacto de los eritrocitos con concentrados de larvas recién nacidas (LRN) no viables provoca la disminución de ácido siálico globular. El objetivo del trabajo fue estudiar la desialización eritrocitaria producida por LRN mantenidas en cultivo. Se realizaron 2 experiencias en las que se incubaron 80 larvas con 100 μL de eritrocitos en 1 mL de medio RPMI suplementado durante 1, 2, 3, 4 y 24 horas a 37 ºC. Se aplicó el Método de Titulación de la Agregación por Polibrene y se calculó Título, Score Total y CexpCASP en los eritrocitos control e incubados con LRN. Los resultados mostraron que en la primera y segunda hora la captación de ácido siálico fue moderada. A las 3 horas el título disminuyó significativamente en relación al del control y el CexpCASP (0,14±0,014) indicó la pérdida casi total de ácido siálico globular. Ambos valores se mantuvieron a las 4 y 24 horas. Al comparar con estudios similares realizados con larvas infectantes, se sugiere que, in vivo, las LRN captarían más rápidamente el ácido siálico que las larvas musculares.

Trichinella spiralis is the species that causes most human cases of infection in the world. It was reported that contact of erythrocytes with concentrates of non-viable newborn larvae (NL) causes the decrease in erythrocyte sialic acid. The objective was to study erythrocyte desialylation produced by NL maintained in culture. Two experiments were conducted, in which 80 larvae were incubated with 100 μL of erythrocytes in 1 mL of supplemented RPMI medium for 1, 2, 3, 4 and 24 hours at 37 ºC. Titration of Aggregation by Polybrene Method was used and Title, Total Score and CexpCASP were calculated in Control erythrocytes and erythrocytes incubated with NL. The results showed that the sialic acid capture was moderated in the first and second hour. At three hours of incubation, the Title decreased significantly in relation to Control and CexpCASP (0.14±0.014) indicated almost total loss of erythrocyte sialic acid. Both values were maintained at 4 and 24 hours. When compared to similar studies conducted with infective larvae, it is suggested that, in vivo, NL would capture sialic acid faster than muscle larvae.

Trichinella spiralis é a espécie que causa a maioria dos casos de infecção humana em todo o mundo. Comunicou-se que o contato dos eritrócitos com concentrados de larvas recém-nascidas (LRN), não viáveis, provoca a diminuição de ácido siálico globular. O objetivo do trabalho foi estudar a dessialização eritrocitária produzida por LRN mantidas em cultivo. Foram realizadas duas experiências nas quais se incubaram 80 larvas com 100 μL de eritrócitos em 1 mL de meio RPMI suplementado durante 1, 2, 3, 4 e 24 horas a 37 °C. Foi aplicado o Método de Titulação da Agregação por Polibreno e se calculou Título, Pontuação Total (ST) e CexpCASP nos eritrócitos. Os resultados mostraram que na primeira e segunda hora a captação de ácido siálico foi moderada. Às 3 horas o título diminuiu significativamente em relação ao do controle e o CexpCASP (0,14±0,014) indicou a perda quase total de ácido siálico globular. Ambos os valores se mantiveram às 4 e 24 horas. Ao comparar com estudos similares realizados com larvas infetantes, sugere-se que, in vivo, as LRN captariam mais rapidamente o ácido siálico que as larvas musculares.

Desialización eritrocitaria con y sin exposición del antígeno T producida por Trichinella spiralis / Erythrocyte desialylation with and without T antigen exposure produced by Trichinella spiralis / Desialylation eritrocitaria com e sem exposição antigénio T produzida por Trichinella spiralis

El objetivo del trabajo fue estudiar la desialización de eritrocitos que desenmascaran el antígeno T y de los glóbulos que no lo exponen, por contacto con larvas recién nacidas (LRN) de T. spiralis. Se trabajó con 15 suspensiones eritrocitarias en medio enzimático, incubadas con LRN. Los GR control se incubaron con solución salina. Para analizar el efecto del parásito sobre la exposición del antígeno T, se aplicó el Método de Aglutinación anti-T con antígeno T y se estudió la agregación por los métodos de Polibrene y de Análisis Digital de Imágenes, determinando CexpST, CCA y Distribución de los agregados eritrocitarios (DAE). La media de los coeficientes en los eritrocitos que expusieron el antígeno T fueron CexpST=0,22±0,179 y coeficiente de células aisladas (CCA)=0,73±0,108 y en los que no 0,86±0,125 y 0,205±0,163 respectivamente. La DAE mostró que los GR que lo expusieron, presentaron marcada disminución de células aisladas y pronunciado aumento de grandes agregados en relación a los controles, mientras que los GR en los que no hubo exposición, la disminución de células aisladas con respecto a los controles fue menor y el aumento de agregados estuvo homogéneamente distribuido entre todas las categorías. La experiencia realizada concluye que los valores obtenidos de los coeficientes difieren significativamente en los GR que exponen el antígeno T y en los que no, por lo que estas técnicas podrían ser predictivas para detectar desenmascaramiento T.

The aim of this work was to study the desialylation of erythrocytes that expose the T antigen by contact with T. spiralis newborn larvae (NL) and of those that do not. Work was carried out with 15 red cell suspensions in enzymatic medium, which were incubated with NL. The respective Control RBC were incubated with saline solution. To analyze the effect of the parasite on the exposure of the T antigen, the Anti T- antigen T Agglutination Method was applied and the aggregation was studied by Polybrene and Digital Image Analysis Methods determining CexpTS, ICC and Distribution of Erythrocyte Aggregates (DEA). The mean of coefficients in the erythrocytes that exposed the T antigen were CexpTS= 0.22 ± 0.179 and ICC= 0.73 ± 0.108 and of those that did not it was 0.86±0.125 y 0.205±0,163 respectively. DEA showed that the RBCs that exposed it showed a marked decrease of isolated cells and a pronounced increase of large aggregates in relation to the Controls, while the RBCs in which there was no exposure, the decrease of isolated cells with respect to Controls was lower and the increase of aggregates was homogeneously distributed among all categories. The experience concludes that the obtained values of the coefficients differ significantly in the RBCs that expose the T antigen from those that do not, so these techniques could be predictive to detect T unmasking.

O objetivo do trabalho foi estudar a desialização de eritrócitos que desmarcaram o antígeno T e dos glóbulos que não o expõem, por contato com LRN de T. spiralis. Trabalhou-se com 15 suspensões de eritrócitos em meio enzimático, incubadas com LRN. Os GV controle foram incubados com solução salina. Para analisar o efeito do parasita sobre a exposição do antígeno T, foi aplicado o Método de Aglutinação anti T com antígeno T e se estudou a agregação pelos Métodos de Polibrene e de Análise Digital de Imagens, determinando CexpST, CCA e Distribuição dos agregados eritrocitários (DAE). A média dos coeficientes nos eritrócitos que expuseram o antígeno T foram CexpST= 0.22±0.179 e CCA= 0.73±0.108 e naqueles que não o expuseram 0.86±0.125 y 0.205±0,163 respectivamente. Foi demonstrado que os GV que o expuseram, apresentaram marcada diminuição de células isoladas e pronunciado aumento de grandes agregados em relação aos controles, enquanto que os GV nos quais não houve exposição, a diminuição de células isoladas com respeito aos controles foi menor e o aumento de agregados esteve homogeneamente distribuído entre todas as categorias. A experiência realizada conclui que os valores obtidos dos coeficientes diferem significativamente nos GV que expõem o antígeno T e nos que não, portanto estas técnicas poderiam ser preditivas para detectar desmascaramento T.

Exposición de criptoantígeno eritrocitario por larvas recién nacidas de Trichinella spiralis / Exposure of erythrocyte cryptic antigen by Trichinella spiralis newborn larvae / Exposição do cripto antígeno eritrocitário por larvas recém-nascidas de Trichinella spiralis

La desialización del eritrocito puede exponer determinantes antigénicas crípticas de la membrana, entre las cuales se encuentra el antígeno T. Se ha comunicado que las larvas recién nacidas de Trichinella spiralis (LRN), las cuales circulan por el torrente sanguíneo del hospedador, captan el ácido siálico eritrocitario. El objetivo de este trabajo fue estudiar in vitro la exposición del criptoantígeno T por la desialización producida por distintas concentraciones de LRN. Se trabajó con 30 suspensiones eritrocitarias en medio enzimático, que fueron incubadas en partes iguales con concentrados de LRN (GR Tratados) durante 2 horas a 37 ºC con agitación continua. Los respectivos GR Controles se pusieron en contacto con solución salina. El tratamiento de 10/30 suspensiones globulares se realizó con 500 LRN/mL, 10 con 300 LRN/mL y las últimas 10 con 150 LRN/mL. Se aplicó el Método de Aglutinación anti-T con antígeno T (en Placa y Tubo) enfrentando las suspensiones a suero de adulto y suero de cordón. El tratamiento de 9/10 de las suspensiones con 500 LRN/mL, el de 7/10 con 300 LRN/mL y el de 2/10 con 150 LRN/mL las expuso al criptoantígeno. Se concluye que en pacientes diabéticos, hipertensos o con otra patología que produzca disminución de ácido siálico eritrocitario y que cursen simultáneamente una infección por T. spiralis, en la etapa de circulación de larvas por el torrente sanguíneo podría producirse la activación T con la consecuente hemólisis, trombocitopenia y trombosis.

Erythrocyte desialylation can expose cryptic antigenic determinants of the membrane, among which is the T antigen. It has been reported that newborn larvae (NL), which circulate in the bloodstream of the host, capture erythrocyte sialic acid. The aim of this study was to study in vitro exposure of cryptic T antigen by the desialylation produced by different concentrations of NL. Work was carried out on 30 red cell suspensions in enzymatic medium, which were incubated in equal parts with NL concentrates (Treated RBC) for 2 hours at 37 ºC with continued agitation. The respective Control RBC was incubated with saline solution. Treatment of 10/30 globular suspensions was performed with 500 NL/mL, 10 with 300 NL/mL and the last 10 with 150 NL/mL. Anti T- antigen T Agglutination Tests (Plate and Tube) were made, facing the globular suspensions against adult and cord human sera. Treatment of 9/10 suspensions with 500 NL/mL, 7/10 with 300 NL/mL and 2/10 with 150 NL/mL exposed T antigen. It is concluded that in patients with diabetes, hypertension or other diseases with lower content of erythrocyte sialic acid and who simultaneously have a T. spiralis infection, T activation may occur in the stage of larvae circulating through the bloodstream, with consequent haemolysis, thrombocytopenia and thrombosis.

A dessialização do eritrócito pode expor determinantes antigênicos crípticos da membrana, entre os quais se encontra o antígeno T. Foi comunicado que as larvas recém-nascidas de Trichinella spiralis (LRN), as quais circulam na corrente sanguínea do hospedeiro, capturam o ácido siálico eritrocitário. O objetivo foi estudar in vitro a exposição do cripto antígeno T pela dessialização produzida por diferentes concentrações de LRN. Trabalhou-se com 30 suspensões eritrocitárias em meio enzimático, incubadas em partes iguais com concentrados de LRN (GV Tratados) durante 2 horas a 37 °C com agitação contínua. Os respectivos GV Controles entraram em contato com solução salina. Tratamento de 10/30 suspensões globulares foi realizado com 500 LRN/mL, 10 com 300 LRN/mL e as últimas 10 com 150 LRN/mL. Foi aplicado o Método de aglutinação anti T-antígeno T (em Placa e Tubo) enfrentando as suspensões a soro de adulto e soro de cordão. O tratamento de 9/10 suspensões com 500 NRL / mL, o de 7/10 com 300 LRN/mL e o de 2/10 com 150 LRN/mL expuseram o cripto antígeno. Conclui-se que em pacientes diabéticos, hipertensos ou com outra patologia que produza diminuição do ácido siálico eritrocitário, e que padeçam simultaneamente uma infecção por T. spiralis, na fase de circulação de larvas pela corrente sanguínea, poderia ocorrer a ativação T com a consequente hemólise, trombocitopenia e trombose.

In vitro alterations of erythrocyte aggregation by action of Trichinella spiralis newborn larvae.

Possible changes in the erythrocyte membrane, by in vitro interaction with newborn larvae of T. spiralis (NL), were evaluated analyzing the alterations in erythrocyte aggregation by digital image analysis and laser transmission in a new optical chip aggregometer. NL were obtained from CBi mice infected with T. spiralis. RBCs samples from healthy donors where in vitro exposed to NL (concentration (3000 ± 500) larvae/mL) to assess its effect on RBC aggregation. Individual cell Coefficient (CCA) and aggregation parameter (S) were calculated by digitally processing RBC aggregate images, indicating the amount and size of the erythrocyte aggregates present. Also, size distribution of aggregate was analyzed. Kinetic aggregation parameters (Amp750 and t1/2) were calculated with a new optical chip aggregometer. Results show significant alterations in erythrocyte aggregability due the in vitro action of T. spiralis larvae increasing incubation time. These results are possibly related to the loss of surface sialic acid as it is captured by NL. Obtained results suggest that NL could produce hemorheological alterations in the host, which could be related to thrombosis and anemia reported in some patients with trichinosis.

Proteomic analysis of differentially expressed proteins in the three developmental stages of Trichinella spiralis.

Trichinella spiralis, an intracellular parasitic nematode, can cause severe foodborne zoonosis, trichinellosis. The life cycle of T. spiralis consists of adult (Ad), muscle larvae (ML) and newborn larvae (NBL). The protein profiles in different developmental stages of the parasite remain unknown. In the present study, proteins from lysates of Ad, ML and NBL were identified by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 4691 proteins were identified in all the developmental stages, of which 1067 proteins were differentially expressed. The number of up-regulated proteins in NBL was higher than that of the other two groups. The protein profiles from Ad, ML and NBL were compared in pairs. The identified proteins were involved in various functions of T. spiralis life cycle, including sexual maturity, metabolism, utilization of carbohydrates, lipids and nucleotides, and other crucial developmental processes that occur at distinct stages. Further investigation of the transcriptional levels of major sperm protein, serine protease, zinc finger protein, etc. from the different protein profiles using quantitative RT-PCR showed identical results to the iTRAQ analysis. The differentially expressed proteins that are involved in developmental regulation and host-parasite interactions should be further studied.

Regulatory parameters of the lung immune response during the early phase of experimental trichinellosis.

Parasitic infection caused by Trichinella spiralis provokes an early stimulation of the mucosal immune system which causes an allergic inflammatory response in the lungs. The present work was intended to characterize the kinetics of emergence of regulatory parameters in Wistar rat lungs during this early inflammatory response, between days 0 and 13p.i. The presence of regulatory cells such as regulatory T cells (Tregs) and alternatively activated macrophages (AAM) was analyzed in lung cell suspensions. Moreover, a regulatory cytokine (TGF-ß) was studied in lung tissue extracts. Considering that newborn larvae (NBL) travel along the pulmonary microvasculature, the ability of this parasite stage to modulate the activation of lung macrophages was evaluated. For this purpose, lung macrophages from non-infected or infected rats (day 6p.i.) were cultured with live or dead NBL. Arginase activity (characteristic of AAM) and nitric oxide (NO produced by iNOS, characteristic of classical activated macrophages) were measured after 48h. Our results revealed a significant increase in the percentage of Tregs on days 6 and 13p.i., arginase activity on day 13p.i. and TGF-ß levels on days 6 and 13p.i. Lung macrophages from non-infected rats cultured with live NBL showed a significant increase in arginase activity and NO levels. Live and dead NBL induced a significant increase in arginase activity in lung macrophages from infected rats. Only live NBL significantly increased NO levels in these macrophages. The present work demonstrates for the first time, the emergence of regulatory parameters in the early lung immune response during T. spiralis infection. The immumodulatory properties exerted by NBL during its passage through this organ could be the cause of such regulation. Moreover, we have shown the ability of NBL to activate macrophages from the lung parenchyma by the classical and alternative pathways.

Alteración de la agregación eritrocitaria por larvas recién nacidas de Trichinella spiralis / Alteration of the erythrocyte aggregation by newborn larvae of Trichinella spiralis / Alteração do agregação eritrocitária por larvas recém-nascidas da Trichinella spiralis

El objetivo del trabajo fue estudiar las alteraciones en la agregación eritrocitaria producidas por recién nacidas (LRN) de T. spiralis. Se usaron concentrados de larvas LRN incubados en partes iguales con glóbulos rojos (GR) Grupo O (GR Tratados) durante 2 horas, con y sin agitación controlada, tomando muestras al tiempo inicial, 60 y 120 minutos. Los Controles fueron incubados con igual volumen de solución salina. Se aplicó Análisis Digital de Imágenes para estudiar la distribución de los agregados eritrocitarios y calcular el valor de coeficiente de células aisladas (CCA) y la Técnica de Titulación de la Agregación para determinar el Título y el CexpST. Se utilizó ANOVA bifactorial para analizar el efecto de la agitación y del tiempo de incubación en los valores de CCA. Los resultados mostraron que el aumento del tiempo de tratamiento produjo la disminución de las células aisladas y los pequeños rouleaux, y el aumento de los agregados formados por 5 o más glóbulos, lo cual incrementó significativamente el valor de CCA. El análisis estadístico determinó que la agregación de los GR Tratados a los 60 minutos fue mayor que al tiempo 0, y a los 120 minutos mayor que a los otros dos tiempos. La Titulación de la agregación mostró la disminución del CexpST y del Título de los GR Tratados. Las metodologías empleadas no mostraron diferencias significativas en tratamientos con y sin agitación. Se concluye que la disminución de carga eritrocitaria producida por LRN podría provocar alteraciones hemorreológicas en el hospedador.

The aim of this paper was to study the alterations in the erythrocyte aggregation produced by newborn larvae (NL) of T. spiralis. Work was performed with NL concentrates, which were incubated with an equal volume of O Group RBC (Treated RBC) for 2 hours, with and without controlled agitation, taking samples at the initial time, at 60 and 120 minutes. RBC Controls were incubated with equal volume of saline solution. Digital Image Analysis was applied to study the distribution of erythrocyte aggregates and to calculate ICC values, and the Aggregation Titration Technique was used to determine the Title and TSexpC. Two-factor ANOVA was used to analyze the effect of agitation and incubation time on the ICC values. The results showed that at higher treatment time, there was a decrease of isolated cells and small rouleaux and an increase of the aggregates formed by 5 or more cells, with a significant increase of ICC values. Statistical analysis determined that Treated RBC aggregation at 60 minutes was higher than at initial time and that at 120 minutes it was higher than the other two times. Aggregation Titration showed a decrease in the Treated RBC Title and TSexpC. The methodologies employed showed no significant differences in treatments with and without agitation. It is concluded that the decrease in erythrocyte charge produced by NL could cause hemorrheologic alterations in the host.

O objetivo foi estudar as alterações na agregação de eritrócitos produzidas por larvas recém-nascidas (LRN) da T. spiralis. O trabalho foi feito com concentrados de LRN incubados em partes iguais com glóbulos vermelhos (GV) Grupo O (GV Tratados), durante 2 horas, com e sem agitação controlada, levando as amostras ao tempo inicial, 60 e 120 minutos. Os controles foram incubados com igual volume de solução salina. Análise Digital de Imagens foi aplicada para estudar a distribuição dos agregados de eritrócitos e calcular o valor do Quoficiente de Células Isoladas (QCI) e a Técnica de Titulação da Agregação para determinar o Título e CexpST. Para analisar o efeito da agitação e do tempo de incubação sobre os valores do QCI foi utilizada ANOVA bifatorial. Os resultados mostraram que o aumento do tempo de tratamento produziu a diminuição das células isoladas e os pequenos rouleaux, e o aumento dos agregados formados por 5 ou mais glóbulos, aumentando o valor de QCI significativamente. A análise estatística determinou que a agregação de GV Tratados aos 60 minutos foi maior que no tempo inicial e, aos 120 minutos era maior que durante os outros dois tempos. A Titulação da Agregação mostrou a diminuição do CexpST e do Título de GV Tratados. As metodologias usadas não mostraram diferenças significantes em tratamentos com e sem agitação. Conclui-se que a diminuição de carga de eritrócitos produzida por LRN poderia resultar em alterações hemorreológicas no hospedeiro.

Detection of Anti-Trichinella spiralis Antibody by Indirect ELISA Using Recombinant Protein as Antigen / 中国寄生虫学与寄生虫病杂志

Objective To study the specificity, sensitivity of diagnostic antigen for Trichinella spiralis(T. s). Methods T668 recombinant protein from highly efficient expression of newborn larvae stage-specific gene of T. s in E. coli as diagnostic antigen. By using negative and positive sera from rabbit, pig, human as the first antibody, and goat-anti-rabbit IgG, goat-anti-pig IgG, goat-anti-human IgG labeled with HRP as the secondary antibody, indirect ELISA method was established for detecting anti-T.s antibody, while excretory-secretory (ES) antigen of T.s muscle larvae was used as control. Results Sera from rabbit, pig and human were detected by T668 recombinant protein as antigen, which showed a positive rate of 100% with 0.016 ?g/well. There was no difference on the results between the T668 recombinant antigen and the ES antigen for diagnosing T. s-infection. Conclusion The T668 recombinant antigen is promising in substituting ES antigen in the detection of anti-T. s antibody.

ANALYSIS OF NEWBORN LARVA OF TRICHINELLA SPIRALIS BY IMMUNOBLOT / 中国寄生虫学与寄生虫病杂志

Newborn larva (NBL) antigens of Trichinella spiralis were analysed by Immuroblot,and were comparied with the adult and muscle larva antigens.The SDS-PAGE patterns of NBL somatic constituents consisted of about 40 polypeptide bands,which were obviously different from those of adult and muscle larva.Immunoblot analysis indicated that immunization with NBL could induce a stage specific immune response.The molecular weight of specific NBL antigens were 129,120,89,87,79,74,72,64,58,43,40,38,34,32,and 20kDa.But during the natural course of the infection,we could not detect the antibodies of anti-NBL in the host.