144 research outputs found
Development and Validation of a Copro-Enzyme–Linked Immunosorbent Assay Sandwich for Detection of Echinococcus granulosus–Soluble Membrane Antigens in Dogs
No full textCystic echinococcosis (CE) is a parasitic zoonosis caused by the larval stage of the tapeworm Echinococcus
granulosus. Detection of the adult stage in the canine definitive host is essential for estimating infection rates,
surveillance and monitoring of CE control programs. This study sought to develop and validate a coproantigen sandwich
enzyme–linked immunosorbent assay (copro-ELISA), based on antibodies against E. granulosus–soluble membrane
antigens (EGMA), that is capable of distinguishing infected and noninfected dogs. Anti-E. granulosus polyclonal immunoglobulin
G antibodies were obtained from rabbit antiserum against EGMA. Optimization of the test was performed with
51 positive and 56 negative stool samples of canine echinococcosis. Specificity, sensitivity, cross-reactivity, intra- and
inter-assay precision, and over time detection were evaluated. According to the receiver operating characteristic analysis,
the diagnostic sensitivity and specificity were 96.1% (CI: 85.9–99.6) and 98.2% (CI: 89.5–100), respectively. Negative and
positive predictive values were 96.5% (CI: 91.7–100) and98%(CI: 94.1–100), respectively.Nocross-reactivity with Taenia
hydatigena, Dipylidium caninum, or Toxocara canis was observed. Intra- and inter-assay repeatability showed values of
less than15%of the variation coefficient. The over time detection was from 20 to 27 days postinfection with E. granulosus.
The copro-ELISA based on EGMA detection offers a simplified in-house development of diagnostic testing. This assay
showed high specificity and sensitivity and had no cross-reactivity with other parasites. Further studies and development
of this test in a kit format may be useful for the detection of active infection in dogs living in CE endemic regions.This article is published as Jara, Luis M., Magaly Rodriguez, Faride Altamirano, Antonio Herrera, Manuela Verastegui, Luis G. Gímenez-Lirola, Robert H. Gilman, and Cesar M. Gavidia. "Development and validation of a copro-enzyme–linked immunosorbent assay sandwich for detection of Echinococcus granulosus–soluble membrane antigens in dogs." The American Journal of Tropical Medicine and Hygiene 100, no. 2 (2019): 330. DOI: 10.4269%2Fajtmh.18-0645. Copyright 2018 The American Society of Tropical Medicine and Hygiene. Posted with permission
Neurocysticercal antigens stimulate chemokine secretion from human monocytes via an NF-kappaB-dependent pathway.
No full textNeurocysticercosis, infection with larval Taenia solium, is a common, serious neuroparasitic infection. Larval degeneration results in inflammatory cell influx and granuloma formation which leads to clinical symptomatology. The role of chemokines in such cell influx is unknown. We demonstrate that monocyte stimulation by T. solium larval antigen (TsAg) results in a differential profile of CXCL8/IL-8 (146.5+/-8.5ng/ml after 24h), CCL2/MCP-1 (267+/-4 ng/ml after 48 h) and CCL3/MIP-1alpha (1.72+/-0.43 ng/ml after 8 h) secretion. There was coordinate mRNA accumulation reaching maximum at 1h for CCL3 and 2 h for CXCL8 and CCL2. TsAg induced maximal nuclear binding of p65, p50 and c-rel subunits of the transcriptional regulator NF-kappaB by 2 h. IkappaBalpha but not IkappaBbeta was degraded within 10 min before resynthesis by 2 h. Pre-treatment with the broad-spectrum NF-kappaB inhibitor pyrrolidine dithiocarbamate caused complete abrogation of TsAg-induced CCL2 secretion (p=0.005) and 91% reduction of CXCL8 secretion (p=0.0003). TsAg was unable to induce CXCL8 promoter activity in Toll-like receptor (TLR)-2 or TLR-4/MD-2 transfected HeLa cells in the absence of lectins or other adaptor molecules. In summary, our data demonstrate that TsAg induces chemokine secretion via specific pathways dependent on NF-kappaB but not TLR-4/TLR-2, and indicate a potential mechanism whereby larval degeneration results in brain inflammation
Determinación de la expresión de citoquinas pro y anti-inflamatorias mediante RT-qPCR, en cultivos primarios astrocíticos y mixtos (neuronas/glias) incubados con antígenos e/s y totales del estadio de cisticerco de Taenia solium
No full textLa neurocisticercosis (NCC) es una infección parasitaria del sistema nervioso central (SNC) causada por la forma larval de Taenia solium. Esta enfermedad provoca una respuesta inflamatoria que se intensifica cuando el parásito muere, y se asocia con la epilepsia de los pacientes. El presente estudio tuvo como objetivo evaluar la expresión de citoquinas pro y antiinflamatorias en cultivos primarios de astrocitos y mixtos (neuronas/glías) de cerebro de rata expuestos a antígenos de cisticerco de T. solium. Se usaron cultivos primarios de astrocitos y mixtos (neuronas/glías) postnatales de ratas de 3 días, incubados durante 24 horas con antígenos Excretor/Secretor (E/S) y Totales. La expresión de citoquinas (IL-10, IL-1β, TGFβ-1) se evaluó mediante RT-qPCR, para lo cual se extrajo el RNAm de las células incubadas. Los resultados mostraron un incremento significativo en la expresión de IL-10 e IL-1β en ambos tipos de cultivo primario. Sin embargo, se observó una disminución en la expresión de la citoquina TGFβ-1 en cultivo primario mixto evaluando la misma condición. Por otro lado, el antígeno Total indujo una mayor expresión de la citoquina IL-1β en cultivo primario astrocítico. En conclusión, los resultados sugieren que los antígenos E/S del cisticerco podrían asociarse con una tendencia hacia la respuesta inmunoreguladora mixta de ambas citoquinas (IL-10 e IL-1β), diferente al antígeno Total que muestra un perfil proinflamatorio. Se requieren más evaluaciones para confirmar esta observación. Estos hallazgos sientan una base para futuras investigaciones acerca del papel de las citoquinas en la inmunoregulación asociada a la NCC en un modelo murino.Neurocysticercosis (NCC) is a parasitic infection of the central nervous system (CNS) caused by the larval stage of Taenia solium. This disease triggers an inflammatory response that intensifies upon parasite death and is associated with epilepsy in affected patients. This study aimed to evaluate the expression of pro- and anti-inflammatory cytokines in primary cultures of rat astrocytes and mixed neuronal/glial cells exposed to T. solium cysticercus antigens.Primary cultures of astrocytes and mixed neuronal/glial cells were obtained from three-day-old neonatal rats and incubated for 24 hours with Excretory/Secretory (E/S) and Total cysticercus antigens. Cytokine expression (IL-10, IL-1β, TGFβ-1) was assessed by RT-qPCR after extracting mRNA from the treated cells.The results showed a significant increase in IL-10 and IL-1β expression in both types of primary cultures. However, there was a decrease in TGFβ-1 expression observed in the mixed primary culture under the same condition. In addition, the Total antigen induced higher IL-1β expression in the primary astrocyte culture.In conclusion, these results suggest that the E/S cysticercus antigens may be associated with a tendency toward a mixed immunoregulatory response involving both IL-10 and IL-1β, in contrast to the Total antigen, which appears to promote a more pro-inflammatory profile. Further studies are needed to confirm this observation. These findings provide a foundation for future research into the role of cytokines in the immunoregulation associated with NCC in a murine model
Evaluación en la expresión de biomarcadores predictivos asociados a la vía de TGF-β para cardiomiopatía chagásica crónica en Cavia porcellus
No full textLa enfermedad de Chagas(EC) es causada por Trypanosoma cruzi y presenta la fase aguda que puede avanzar hacia fase indeterminada y crónica. Si el individuo infectado no recibe tratamiento durante las dos primeras fases, entre el 20% a 40% desarrollarán la fase crónica. El tratamiento es eficiente solo para la fase aguda. Específicamente al desarrollar cardiomiopatía chagásica crónica(CCC), causante de la mayor morbilidad en la EC, no existe droga que revierta el daño causado por el parásito a nivel del miocardio. Estudios recientes han asociado la vía de señales inducida por TGF-β en la progresión de la EC. Sin embargo, aún no se conoce la variación en la expresión de biomarcadores proteicos involucrados a lo largo de esta vía que estén asociados con la progresión hacia CCC y sirvan como predictores de esta. Para llenar este vacío, se utilizarán los biomarcadores asociados a la vía TGF-β: LOXL2, CTGF, Angll, y P38MAPK. La sobreexpresión de estos biomarcadores amplifica la activación de los fibroblastos cardiacos del miocardio en respuesta a la infección, siendo cruciales para el desarrollo de CCC. Nos preguntamos cómo varían los niveles de abundancia relativa de estos biomarcadores a lo largo de la EC. Hipotetizamos que el cambio en la expresión desde la fase aguda hasta el desarrollo de CCC de por lo menos una de las moléculas, permitirá usarla como potencial biomarcador predictor específico para CCC. Para ello, se usará a Cavia porcellus, modelo animal que desarrolla la EC de manera similar al humano, haciendo posible analizar los biomarcadores a nivel de tejido cardiaco y suero a diferentes tiempos post infección. Esto se hará con la finalidad de que en el futuro se puedan usar como biomarcadores predictores de riesgo de progresión a CCC en humanos.Chagas disease (CD) is caused by Trypanosoma cruzi and has an acute phase that can progress to the indeterminate and chronic phases. If the infected individual is not treated during the first two phases, 20% to 40% will develop the chronic phase. Treatment is effective only for the acute phase. Specifically, when developing chronic chagasic cardiomyopathy (CCC), which causes the highest morbidity in CD, there is no drug that reverses the damage caused by the parasite at the myocardial level. Recent studies have linked the TGF-β induced signalling pathway to the progression of CD. However, the variation in the expression of protein biomarkers involved along this pathway associated with progression to CCC and serve as predictors of CCC is not yet known. To fill this gap, the TGF-β pathway-associated biomarkers LOXL2, CTGF, Angll, and P38MAPK will be used. Overexpression of these biomarkers amplifies the activation of cardiac myocardial fibroblasts in response to infection and is crucial for the development of CCC. We asked how the relative abundance levels of these biomarkers vary across CD. We hypothesise that the change in expression from the acute phase to the development of CCC of at least one of the molecules will allow it to be used as a potential specific biomarker predictor for CCC. For this purpose, Cavia porcellus, an animal model that develops CD in a similar way to humans, will be used, making it possible to analyse biomarkers at the level of cardiac tissue and serum at different post-infection times. This will be done with the aim that in the future they can be used as biomarkers for predicting the risk of progression to CCC in humans
Evaluación de daño en el transporte axonal en un modelo animal de neurocisticercosis
No full textLa neurocisticercosis es una enfermedad parasitaria del SNC que causa epilepsia adquirida y es generada por la forma larvaria del cestodo Taenia solium. Es de curso crónico y en pacientes sintomáticos se caracteriza por convulsiones y epilepsia. Estudios en ratas de laboratorio con NCC, reportan daño axonal caracterizado por el acúmulo de proteínas (neurofilamento, APP) en forma de hinchazones axonales, que podría estar relacionado con deterioro en el transporte axonal. Objetivo: Evaluar el patrón de inmunorreactividad de las proteínas motoras kinesina y dineína como marcadores de daño en el transporte axonal en tejido cerebral de ratas Holtzman con NCC tratadas y no tratadas con antiparasitarios; y su asociación con el daño axonal. Metodología: Se utilizó tejido cerebral de ratas con NCC tratadas y no tratadas con antiparasitarios y ratas sanas sacrificadas a diferentes tiempos postratamiento (48 horas, 5 días, 2, 8 y 12 meses). Las muestras se procesaron por inmunohistoquímica, para evaluar el patrón de inmunorreactividad de las proteínas. Resultados: En ratas con NCC el patrón de inmunorreactividad de kinesina y dineína se caracterizó por la acumulación patológica de estas proteínas en forma de esferoides y varicosidades axonales en el tejido alrededor del cisticerco con ausencia de esta alteración en ratas sanas (valor p<0.05). Al comparar los valores de los grupos de ratas con NCC tratadas y no tratadas, no se encontraron diferencias estadísticas, aunque en el grupo no tratado hubo tendencia a incrementarse en el tiempo. Las hinchazones axonales prevalecieron en materia gris. Se encontró correlación positiva entre el patrón de inmunorreactividad de las proteínas motoras y daño axonal (neurofilamento). Conclusiones: En ratas con NCC existe acumulación patológica las proteínas kinesina y dineína en forma de hinchazones axonales en el tejido que rodea al cisticerco indicando presencia de daño en el transporte axonal y se asociado con daño axonal.Neurocysticercosis is a parasitic disease of the CNS that causes acquired epilepsy and is caused by the larval form of the cestode Taenia solium. It has a chronic course and symptomatic patients are characterized by seizures and epilepsy. Studies in laboratory rats with NCC report axonal damage characterized by the accumulation of proteins (neurofilament, APP) in the form of axonal swellings, which could be related to impaired axonal transport. Objective: To evaluate the immunoreactivity pattern of the motor proteins kinesin and dynein as markers of damage to axonal transport in brain tissue of Holtzman rats with NCC treated and untreated with antiparasitics; and its association with axonal damage. Methodology: Brain tissue from NCC rats treated and untreated with antiparasitics and healthy rats sacrificed at different post-treatment times (48 hours, 5 days, 2, 8 and 12 months) was used. The samples were processed by immunohistochemistry, to evaluate the immunoreactivity pattern of the proteins. Results: In rats with NCC, the immunoreactivity pattern of kinesin and dynein was characterized by the pathological accumulation of these proteins in the form of spheroids and axonal varicosities in the tissue around the cysticercus, with an absence of this alteration in healthy rats (p-value <0.05). When comparing the values of the groups of rats with treated and untreated NCC, no statistical differences were found, although in the untreated group there was a tendency for them to increase over time. Axonal swellings prevailed in gray matter. A positive correlation was found between the immunoreactivity pattern of motor proteins and axonal damage (neurofilament). Conclusions: In rats with NCC there is pathological accumulation of kinesin and dynein proteins in the form of axonal swellings in the tissue surrounding thecysticercus indicating the presence of damage in axonal transport and is associated with axonal damage
Identification and culture of proliferative cells in abnormal Taenia solium larvae: Role in the development of racemose neurocysticercosis.
No full textRacemose neurocysticercosis is an aggressive disease caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord resulting in a mass effect and chronic inflammation. Although expansion is likely caused by the proliferation and growth of the parasite bladder wall, there is little direct evidence of the mechanisms that underlie these processes. Since the development and growth of cysts in related cestodes involves totipotential germinative cells, we hypothesized that the expansive growth of the racemose larvae is organized and maintained by germinative cells. Here, we identified proliferative cells expressing the serine/threonine-protein kinase plk1 by in situ hybridization. Proliferative cells were present within the bladder wall of racemose form and absent from the homologous tissue surrounding the vesicular form. Cyst proliferation in the related model species Taenia crassiceps (ORF strain) occurs normally by budding from the cyst bladder wall and proliferative cells were concentrated within the growth buds. Cells isolated from bladder wall of racemose larvae were established in primary cell culture and insulin stimulated their proliferation in a dose-dependent manner. These findings indicate that the growth of racemose larvae is likely due to abnormal cell proliferation. The different distribution of proliferative cells in the racemose larvae and their sensitivity to insulin may reflect significant changes at the cellular and molecular levels involved in their tumor-like growth. Parasite cell cultures offer a powerful tool to characterize the nature and formation of the racemose form, understand the developmental biology of T. solium, and to identify new effective drugs for treatment
Relationship between serum antibodies and Taenia solium larvae burden in pigs raised in field conditions.
No full textGAVIDIA, César M., VERASTEGUI, Manuela R., GARCIA, Hector H. [et al.]. Relationship between serum antibodies and Taenia solium larvae burden in pigs raised in field conditions. PLOS Neglected Tropical Diseases [en línea]. 2013, vol. 7, no. 5, p. 1-8. ISSN 1935-2727.Background: Serological tests have been used for the diagnosis of Taenia solium infection in pigs. However, those serological results do not necessarily correlate with the actual infection burden after performing pig necropsy. This study aimed to evaluate the Electro Immuno Transfer Blot (EITB) seropositivity with infection burden in naturally infected pigs.
Methodology/Principal Findings: In an endemic area of Peru, 476 pigs were sampled. Seroprevalence was 60.5±4.5% with a statistically higher proportion of positive older pigs (>8 months) than young pigs. The logistic model showed that pigs >8 month of age were 2.5 times more likely to be EITB-positive than ≤8 months. A subset of 84 seropositive pigs were necropsied, with 45.2% (38/84) positive to 1–2 bands, 46.4% (39/84) to 3 bands, and 8.3% (7/84) to 4+ bands. 41 out of 84 positive pigs were negative to necropsy (48.8%) and 43 (51%) had one or more cysts (positive predictive value). Older pigs showed more moderate and heavy infection burdens compared to younger pigs. In general, regardless of the age of the pig, the probability of having more cysts (parasite burden) increases proportionally with the number of EITB bands.
Conclusions/Significance: The probability of being necropsy-positive increased with the number of bands, and age. Therefore, the EITB is a measure of exposure rather than a test to determine the real prevalence of cysticercosis infection
Utility of PCR to detect Campylobacter jejuni directly from stool samples
No full textPara facilitar la identificación rápida y especifica de Campylobacter jejuni se desarrolló un Test de Reacción en Cadena de la Polimerasa (PCR) a partir de muestras de heces de niños, con problemas gastrointestinales, menores de 24 meses de edad. Los cebadores (primers) elegidos, CAMPYJ y CAMPYJ2, para la realización del PCR se diseñaron sobre la base de una secuencia específica del gen MapA, este gen codifica a la proteína A (24KD), que es parte estructural de la membrana celular, presente en todas las cepas de Campylobacter jejuni. Para la extracción de ADN directamente de heces se evaluaron 04 protocolos diferentes, mostrando el Método de Franket y col. un ADN genómico más puro y conservado. Para la estandarización del PCR se utilizaron cepas de Campylobacter jejuni, cuyo límite de detección con el par de cebadores fue 15 bacterias, equivalente a 100 fentogramos de ADN de cepa pura de C. jejuni y de 1.5 x10 bacterias, equivalente a 1picogramo de ADN en muestra de heces. Para determinarla sensibilidad y especificidad del PCR se utilizaron 33 muestras de heces positivas y 56 muestras de heces negativas a Campylobacter jejuni; asimismo el PCR no evidenció reacción cruzada con otros enteropatógenos. Determinándose una sensibilidad y especificidad de 88% y 84% respectivamente; resultados que demuestran que el PCR es una buena alternativa de diagnóstico para la rápida detección de Campylobacter jejuni directamente de muestras de heces.To facilitate the rapid and specific identification of Campylobacter jejuni, a Polymerase Chain Reaction (PCR) test was developed from stool samples of children with gastrointestinal problems under 24 months of age. The primers chosen, CAMPYJ and CAMPYJ2, for PCR were designed on the basis of a specific sequence of the MapA gene, this gene encodes protein A (24KD), which is a structural part of the cell membrane, present in all Campylobacter jejuni strains. For DNA extraction directly from feces, 04 different protocols were evaluated, with the Franket et al. method showing a purer and more conserved genomic DNA. For PCR standardization, Campylobacter jejuni strains were used, whose limit of detection with the primer pair was 15 bacteria, equivalent to 100 phentograms of DNA of pure strain of C. jejuni and 1.5 x10 bacteria, equivalent to 1 picogram of DNA in stool sample. To determine the sensitivity and specificity of the PCR, 33 stool samples positive and 56 stool samples negative for Campylobacter jejuni were used; likewise, the PCR did not show cross-reactivity with other enteropathogens. A sensitivity and specificity of 88% and 84%, respectively, were determined; results that demonstrate that PCR is a good diagnostic alternative for the rapid detection of Campylobacter jejuni directly from stool samples
Evaluación de la asociación entre la densidad de oligodendrocitos y el daño axonal en diferentes tiempos post-infección en un modelo de ratas con Neurocisticercosis
No full textLa neurocisticercosis (NCC) es una infección producida por el cisticerco de Taenia solium cuando se posiciona en el sistema nervioso central (SNC), causando epilepsia tardía en regiones endémicas como Latinoamérica, Asia y África. En algunas enfermedades neurodegenerativas como la esclerosis múltiple, hay una disminución en la población de oligodendrocitos (OLs) en el SNC, los cuales son células encargadas de producir mielina para el recubrimiento axonal permitiendo que las señales sean saltatorias. La disminución de OLs, puede producir daño axonal, no obstante, esto se desconoce en Neurocisticercosis. El objetivo del estudio fue evaluar la asociación entre la densidad de oligodendrocitos y el daño axonal en el modelo de ratas con neurocisticercosis en diferentes tiempos post infección. Para ello, se utilizó tejidos de cerebros de ratas Holtzman que fueron infectados por la vía intracraneal con oncosferas activadas de T. solium y sacrificadas en 5 meses post-infección (mpi) (un mes, un mes y medio, tres meses, seis meses y doce meses). La cuantificación de OLs e hinchazones axonales se realizó mediante inmunohistoquímica usando los biomarcadores olig2 y NFP. Además, se determinó el porcentaje de OLs en apoptosis mediante inmunofluorescencia indirecta doble usando los biomarcadores Olig2 y caspasa-3.La población de OLs en ratas con neurocisticercosis disminuyó significativamente a partir del 6.0 mpi en materia blanca, específicamente en cuerpo calloso. Además, se observó una asociación entre la densidad de OLs y la formación de hinchazones axonales a partir de los 3.0 mpi. Por otro lado, se observó un pequeño porcentaje de OLs que entran en apoptosis en ratas infectadas, sugiriendo que la disminución de la densidad de OLs podría deberse a otro tipo de muerte celular. Estos hallazgos proporcionan una base para futuras investigaciones sobre la patología en neurocisticercosis.Neurocysticercosis is a parasitic infection caused by the larval stage of Taenia solium when it is positioned in the central nervous system (CNS). This disease is the main cause of epilepsy in endemic regions such as Latin America, Asia and Africa. In some neurodegenerative diseases such as multiple sclerosis, there is a decrease in the population of oligodendrocytes (OLs) in the CNS, which are cells responsible for producing myelin to coat axons allowing signals to jump. Decrease in Ols may result in axonal damage can occur; however, this is unknown in neurocysticercosis. Therefore, the aim of the present study was to evaluate the association between oligodendrocyte density and axonal damage in brain tissues from rats with Neurocysticercosis sacrificed at different post-infection times. For this purpose, 10-14 day old Holtzman rats were infected intracranially with activated T. solium oncospheres and then sacrificed at 5 post-infection times (one month, one and a half months, three months, six months and twelve months). Identification of the number of oligodendrocytes and axonal swellings was performed by immunohistochemistry. In addition, to determine the proportion of OLs in apoptosis, double indirect immunofluorescence was performed. The population of OLs showed a statistically significant decrease at sixth month post-infection (mpi) in white matter, specifically in corpus callosum. No significant differences were shown in gray matter. In addition, an indirect proportional association between OLs and axonal swelling (axonal damage) was observed from 6 mpi onwards. On the other hand, there is a small proportion of OLs that enter apoptosis in the presence of the parasite. These findings provide a basis for future research on pathology in neurocysticercosis
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