Protective efficacy of Toxoplasma gondii infected cells-derived exosomes against chronic murine toxoplasmosis.

Exosomes were isolated from T. gondii infected human hepatoblastoma cells using the exosome isolation kit and characterized by electron microscopy and Western blotting. Exosomes adsorbed to alum adjuvant were evaluated as a potential immunizing agent against murine chronic toxoplasmosis compared to excretory secretory antigens (ESA)-alum. Mice were immunized at days 1, 15 and 29. The levels of IgG, IFN-γ, IL-4 and IL-10, CD4+ and CD8+ T cells were determined using sandwich enzyme-linked immunosorbent assay (sandwich ELISA) at days 14, 28 and 56 of the experiment. Then mice were infected orally with 10 cysts of T. gondii. The protective efficacy of the antigens were evaluated by counting the brain cysts and measuring the aforementioned humoral and cellular parameters 60 days post infection. The results showed that alum increased the protective efficacy of the exosomes. Immunization with exosome-alum induced both humoral and mixed Th1/Th2 cellular immune responses. Exosome-alum gave higher levels of the humoral and cellular parameters, compared to ESA-alum. After challenge infection, exosome-alum significantly reduced the brain cyst burden by 75 % while ESA-alum gave 42 % reduction and evoked higher humoral and cellular immune responses. Therefore, the possibility of using T. gondii infected cells-derived exosome-alum as a vaccine is a new perspective in toxoplasmosis.

Emerging roles for extracellular vesicles in Schistosoma infection.

The co-evolution of Schistosoma and its host necessitates the use of extracellular vesicles (EVs) generated by different lifecycle stages to manipulate the host immune system to achieve a delicate balance between the survival of the parasite and the limited pathology of the host. EVs are phospholipid bilayer membrane-enclosed vesicles capable of transferring a complex mixture of proteins, lipids, and genetic materials to the host. They are nano-scale-sized vesicles involved in cellular communication. In this review, the author summarized the proteins involved in the biogenesis of schistosome-derived EVs and their cargo load. miRNAs are one cargo molecule that can underpin EVs functions and significantly affect parasite/host interactions and immune modulation. They skew macrophage polarization towards the M1 phenotype and downregulate Th2 immunity. Schistosoma can evade the host immune system's harmful effects by utilizing this strategy. In order to compromise the protective effect of Th2, EVs upregulate T regulatory cells and activate eosinophils, which contribute to granuloma formation. Schistosomal EVs also affect fibrosis by acting on non-immune cells such as hepatic stellate cells. These vesicles drew attention to translational applications in diagnosis, immunotherapy, and potential vaccines. A deep understanding of the interaction of schistosome-derived EVs with host cells will significantly increase our knowledge about the dynamics between the host and the worm that may aid in controlling this debilitating disease.

Praziquantel-encapsulated niosomes against Schistosoma mansoni with reduced sensitivity to praziquantel / Niosomas encapsulados en prazicuantel contra Schistosoma mansoni con sensibilidad reducida al prazicuantel

Introduction: Praziquantel (PZQ) is the only commercially available drug for schistosomiasis. The current shortage of alternative effective drugs and the lack of successful preventive measures enhance its value. The increase in the prevalence of PZQ resistance under sustained drug pressure is, therefore, an upcoming issue. Objective: To overcome the tolerance to PZQ using nanotechnology after laboratory induction of a Schistosoma mansoni isolate with reduced sensitivity to the drug during the intramolluscan phase. Materials and methods: Shedding snails were treated with PZQ doses of 200 mg/kg twice/ week followed by an interval of one week and then repeated twice in the same manner. The success of inducing reduced sensitivity was confirmed in vitro via the reduction of cercarial response to PZQ regarding their swimming activity and death percentage at different examination times. Results: Oral treatment with a single PZQ dose of 500 mg/kg in mice infected with cercariae with reduced sensitivity to PZQ revealed a non-significant reduction (35.1%) of total worm burden compared to non-treated control mice. Orally inoculated PZQ- encapsulated niosomes against S. mansoni with reduced sensitivity to PZQ successfully regained the pathogen's sensitivity to PZQ as evidenced by measuring different parameters in comparison to the non-treated infected animals with parasites with reduced sensitivity to PZQ. The mean total worm load was 1.33 ± 0.52 with a statistically significant reduction of 94.09% and complete eradication of male worms. We obtained a remarkable increase in the percentage reduction of tissue egg counts in the liver and intestine (97.68% and 98.56%, respectively) associated with a massive increase in dead eggs and the complete absence of immature stages. Conclusion: PZQ-encapsulated niosomes restored the drug sensitivity against laboratory- induced S. mansoni adult worms with reduced sensitivity to PZQ.

Introducción. El prazicuantel es el único fármaco disponible comercialmente para la esquistosomiasis. La escasez actual de medicamentos alternativos y la falta de medidas preventivas eficaces aumentan su valor. La creciente prevalencia de la resistencia al prazicuantel bajo una presión prolongada del fármaco es, por tanto, un tema emergente. Objetivos. Superar la tolerancia al prazicuantel mediante nanotecnología después de la inducción en laboratorio de un aislamiento de Schistosoma mansoni con sensibilidad reducida al fármaco durante la fase intramolusco. Materiales y métodos. Los caracoles que liberaban cercarias se trataron con prazicuantel en dosis de 200 mg/kg dos veces por semana, seguidas de un intervalo de una semana, y luego se repitieron dos veces de la misma manera. La inducción exitosa de la sensibilidad reducida se confirmó in vitro mediante la reducción de la reacción de las cercarias al prazicuantel con respecto a su actividad de natación y el porcentaje de muerte en diferentes momentos de examen. El éxito en inducir una menor sensibilidad se confirmó in vitro mediante la reducción de la reacción de las cercarias al prazicuantel. Resultados. El tratamiento oral con una dosis única de prazicuantel de 500 mg/kg en ratones infectados con cercarias con sensibilidad reducida al prazicuantel, reveló una reducción no significativa (35,1 %) de la carga total de gusanos en comparación con los ratones de control no tratados. Los niosomas encapsulados en prazicuantel inoculados por vía oral contra S. mansoni con sensibilidad reducida al prazicuantel, permitieron reestablecer con éxito la sensibilidad del patógeno al medicamento, como lo demostró la medición de diferentes parámetros en comparación con los animales infectados no tratados con parásitos con sensibilidad reducida a prazicuantel. La carga media total de gusanos fue de 1,33 ± 0,52, con una reducción estadísticamente significativa del 94,09 %, y la erradicación completa de los gusanos machos adultos. Se obtuvo un aumento notable en el porcentaje de reducción del recuento de huevos en el tejido del hígado y el intestino (97,68 % y 98,56 %, respectivamente), asociado con un aumento masivo de huevos muertos y ausencia total de estadios inmaduros. Conclusión. Los niosomas encapsulados en prazicuantel restauraron la sensibilidad al fármaco contra gusanos adultos de S. mansoni con sensibilidad reducida al prazicuantel inducida en el laboratorio.

Schistosoma mansoni egg-derived extracellular vesicles: A promising vaccine candidate against murine schistosomiasis.

Extracellular vesicles (EVs) are protein-loaded nano-scaled particles that are extracellularly released by eukaryotes and prokaryotes. Parasite's EVs manipulate the immune system, making them probable next-generation vaccines. Schistosomal EVs carry different proteins of promising immunizing potentials. For evaluating the immune-protective role of Schistosoma mansoni (S. mansoni) egg-derived EVs against murine schistosomiasis, EVs were isolated from cultured S. mansoni eggs by progressive sequential cooling ultra-centrifugation technique. Isolated EVs were structurally identified using transmission electron microscope and their protein was quantified by Lowry's technique. Experimental mice were subcutaneously immunized with three doses of 20 µg EVs (with or without alum adjuvant); every two weeks, then were challenged with S. mansoni cercariae two weeks after the last immunizing dose. Six weeks post infection, mice were sacrificed for vaccine candidate assessment. EVs protective efficacy was evaluated through parasitological, histopathological, and immunological parameters. Results showed significant reduction of tegumentally deranged adult worms, hepatic and intestinal egg counts reduction by 46.58%, 93.14% and 93.17% respectively, accompanied by remarkable amelioration of sizes, numbers and histopathology of hepatic granulomata mediated by high interferon gamma (IFN γ) and antibody level. Using sera from vaccinated mice, the molecular weight of EVs' protein components targeted by the antibody produced was recognized by western immunoblot. Results revealed two bands of ~ 14 KDa and ~ 21 KDa, proving that EVs are able to stimulate specific antibodies response. In conclusion, the present study highlighted the role of S. mansoni-egg derived EVs as a potential vaccine candidate against murine schistosomiasis mansoni.

Review: Schistosoma mansoni phosphatidylinositol 3 kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway.

Schistosoma mansoni worms are under a milieu of external and internal signaling pathways. The life-cycle stages are exposed to enormous stimuli within the mammalian and the snail hosts and as free-living stages in the fresh water. Furthermore, there is a unique interplay between the male and the female worms involving many stimuli from the male essential for full development of the female. PI3K/Akt/mTOR is an evolutionarily divergent signal transduction pathway universal to nearly every multicellular organism. This work reviews the Schistosoma mansoni PI3K/Akt/mTOR signal pathways and the involvement of the signal in the worms' physiology concerning the uptake of glucose, reproduction and survival. The inhibitors of the signal pathway used against Schistosoma mansoni were summarized. Given the importance of the PI3K/Akt/mTOR signal pathway, its inhibition could be a promising control strategy against schistosomiasis.

Schistosoma mansoni sarco/endoplasmic reticulum Ca2+ ATPases (SERCA): role in reduced sensitivity to praziquantel.

Praziquantel leads to increase Ca2+ influx and disrupts Ca2+ homeostasis in adult Schistosoma. However, calcium influx is only one component in a series of molecular events leading to the drug effect and some downstream constituents of the cascade that is initiated by this interaction differ between worms with different degrees of susceptibility to praziquantel. Extensive use of the drug raises the concern regarding the selection of drug resistant parasites. SERCA participates in maintenance of Ca2+ homeostasis. Up-regulation of SERCA has been found in Schistosoma mansoni worms with reduced sensitivity to praziquantel. This could be due to increase cytosolic Ca2+, activation of calmodulin kinase II or may be due to SR/ER stress generated from oxidative stress that leads to impaired protein degradation. The significance of SERCA up-regulation is related to counter action of the drug effect by increasing the worm capacity to restore Ca2+ homeostasis, reducing cytosolic Ca2+ followed by lowering mitochondria Ca2+ and consequently inhibition of apoptosis beside its relation to P-glycoprotein. In schistosomes with reduced sensitivity to praziquantel, the agitations produced by Ca2+ influx and the downstream component of the cascade that is initiated by this interaction may be opposed by up-regulation of SERCA and possibly by certain elements of Ca2+ signaling which modulate the process determining cells entrance in the apoptotic state. Revealing the principal mechanisms of up-regulation of SERCA and its significance in reducing the effect of the drug could lead to possible strategies to reverse drug resistance or develop alternative therapies.

Heat shock protein 70 (Hsp70) in Schistosoma mansoni and its role in decreased adult worm sensitivity to praziquantel.

Schistosoma mansoni is the most common species causing schistosomiasis. It has a complex life cycle involving a vertebrate definitive host and a snail intermediate host of the genus Biomphalaria. Each stage encounters a plethora of environmental stresses specially heat stress. Another sort of stress arises from repeated exposure of the parasite to praziquantel (PZQ), the only drug used for treatment, which leads to the development of resistance in the fields and the labs. Heat shock protein 70 (Hsp70) is found in different developmental stages of S. mansoni. It is immunogenic and regulate cercarial invasion besides its chaperone function. In the Biomphalaria/S. mansoni interaction, epigenetic modulations of the Hsp70 gene underscore the susceptibility phenotype of the snail. Hsp70 is up-regulated in adult S. mansoni with decreased sensitivity to PZQ. This could be due to the induction of oxidative and endoplasmic reticulum stress, induction of apoptosis, exposure to the stressful drug pressure and increase influx of calcium ions. Up-regulation of Hsp70 might help the worm to survive the schistosomicidal effect of the drug mainly by dealing with misfolded proteins, inhibition of apoptosis, induction of autophagy, up-regulation of the P-glycoprotein transporter and attenuation of the signalling from G protein coupled receptors.

Biological and proteomic studies of Schistosoma mansoni with decreased sensitivity to praziquantel.

The impact of the laboratory induced Schistosoma mansoni with decreased PZQ sensitivity on the biological performance of its different developmental stages and the concomitant structural changes of adult worms' total proteins were investigated. PZQ exposed snails showed stoppage of cercarial shedding for eight weeks followed by progressive significant reduction of cercarial production along four successive weeks. In the vertebrate host, in comparison to Schistosoma mansoni susceptible isolate, inoculated cercariae with decreased PZQ sensitivity led to an evident decrease in male to female ratio associated with significant reduction in tissue egg counts and significant increase in dead egg percentage. Significant reduction in the fecundity was also determined. Interestingly, eggs from adult worms with decreased PZQ sensitivity showed two unique features as they found to be smaller and more spherical in addition to the observation of hourglass shaped miracidium in about 10% of the detected mature eggs. Proteomic analysis of adult worms with decreased sensitivity to PZQ using mass spectrometry revealed up-regulation of Ca2+ ATPase 2 and Hsp70. This study can point to the increase incidence of the neuroschistosomiasis due to the small size eggs of Schistosoma mansoni with reduced PZQ sensitivity. These worms can also impact the epidemiology in the field. The study can also provide help to elucidate underlying potential molecular mechanisms of resistance that could lead to possible strategies to reverse drug resistance.

Total protein composition of young and adult Biomphalaria alexandrina snails with different compatibilities to Schistosoma mansoni infection / Composición total de proteína en caracoles Biomphalaria alexandrina juveniles y adultos con distintas compatibilidades a la infección por Schistosoma mansoni

Abstract:Schistosomiasis remains a disease of major global public health concern since it is a chronic and debilitating illness. The widely distributed Schistosoma mansoni that causes intestinal schistosomiasis represents a great threat. Its world-wide distribution is permitted by the broad geographic range of the susceptible species of its intermediate host, Biomphalaria, which serves as an obligatory host for the larval stage, at which humans get infected. The objectives were to identify the proteins responsible for the snails' compatibility outcome through differentiation between the total proteins among Biomphalaria alexandrina snails at different ages. The work was conducted on snails that differ in age and genetic backgrounds. Four subgroups (F1) from the progeny of self-reproduced susceptible and resistant snails (F0) were studied. Infection rates of these subgroups (young susceptible, adult susceptible, young resistant and adult resistant) were 90 %, 75 %, 40 % and 0 %, respectively. Using Sodium Dodecyl Sulphate Polyacrylamide Gel electrophoresis (SDS-PAGE), differences in protein expression were evaluated between adult and young snails of different subgroups. Dice similarity coefficient was calculated to determine the percentage of band sharing among the experimental subgroups. The results showed that the combination of similarities between age and compatibility status of the snails, lead to the highest similarity coefficient, followed by the combination of similarities of both genetic origin and age, even though they differ in the compatibility status. On the other hand, the differences in the genetic background, age and compatibility status, lead to the least similarity index. It was also found that the genetic background in young snails plays a major role in the determination of their compatibility, while the internal defense system has the upper hand in determining the level of adult compatibility. In conclusion, the findings of the present work highlight the great impact of the snail age in concomitance with the genetics and the internal defense in the determination of B. alexandrina/S.mansoni compatibility. Future works are recommended, as further characterization of the shared protein bands among the studied subgroups is needed to clarify their role in host-parasite relationship. Rev. Biol. Trop. 64 (4): 1747-1757. Epub 2016 December 01.

Resumen:La esquistosomiasis es una enfermedad crónica y debilitante que constituye una problemática de salud pública a nivel mundial. Debido a que Schistosoma mansoni está ampliamente distribuida y a que es el causante de la esquistosomiasis intestinal representa una gran amenaza. Biomphalaria es el hospedero intermedio y obligatorio para el estado larval, presenta una amplia distribución geográfica e infecta al ser humano. El objetivo fue identificar las proteínas responsables del efecto de compatibilidad en caracoles Biomphalaria alexandrina de distintos estadios a través de la diferenciación del total de proteínas en ellos. La investigación se llevó a cabo con caracoles de diferentes edades y antecedentes genéticos. Se estudiaron cuatro subgrupos (F1) de la progenie de caracoles susceptibles y resistentes reproducidos asexualmente (F0). Las tasas de infección de estos subgrupos (juvenil susceptible, adulto susceptible, juvenil resistente, adulto resistente) fueron 90 %, 75 %, 40 % y 0 %, respectivamente. Con la electroforesis en gel de poliacrilamida en presencia de dodecilsulfato sódico (SDS-PAGE) se evaluaron las diferencias en la expresión proteica entre los caracoles juveniles y adultos de los distintos subgrupos. Se calculó el coeficiente de similitud de Dice para determinar el porcentaje de bandas compartidas entre los subgrupos experimentales. Los resultados mostraron que la combinación de similitudes entre la edad y el estado de compatibilidad de los caracoles genera el mayor coeficiente de similitud seguido por el de la combinación de similitudes tanto de la edad como del origen genético aunque varían en el estado de compatibilidad. Por otra parte, las diferencias en los antecedentes genéticos, la edad y el estado de compatibilidad generan el índice de similitud más bajo. También se encontró que el antecedente genético en caracoles juveniles es importante en la determinación de su compatibilidad, mientras que el sistema de defensa interno es el que determina el nivel de compatibilidad en adultos. En conclusión, los resultados de este trabajo resaltan la importancia de la edad del caracol en conjunto con la genética y la defensa interna para determinar la compatibilidad de B. alexandrina/S.mansoni. Se recomienda realizar futuros trabajos así como una mayor caracterización de las bandas proteicas compartidas entre los subgrupos estudiados para esclarecer su papel en la relación hospedero-parásito.

Defense response of susceptible and resistant Biomphalaria alexandrina snails against Schistosoma mansoni infection

In Egypt, Biomphalaria alexandrina is the intermediate host for Schistosoma mansoni. The fates of Schistosoma miracidia in the snails varies between different species of Biomphalaria. The internal defense system is one of the factors that influence the susceptibility pattern of the snails. The interaction between Biomphalaria snails and S. mansoni needs to be identified for each species, and even between the members of the same species with different degrees of susceptibility. In the present study, the first generation of susceptible and resistant parents of B. alexandrina was examined histologically at the 30th day post exposure. The study includes the characterization of the immune response, as expressed by tissue reactions, of susceptible and resistant B. alexandrina snails against S. mansoni. It was also designed to determine the impact of the resistance increase in parent snails, on the mechanisms of interaction of their offspring against infection. The results showed that the infection rate of the offspring from the susceptible parents was 92%. No susceptible offspring was produced from the resistant parents. When the parents were of equal number of susceptible and resistant snails, they gave an offspring with an infection rate of 20%. Susceptible snails that had susceptible parents showed a higher degree of susceptibility than those that had both susceptible and resistant parents. A common feature of the resistant snails was the absence of any viable parasites. The tissue reactions of the resistant snails having only resistant parents occurred at the site of miracidial penetration. In resistant snails for which susceptible ones were included in their parents, the reactions occurred in the deep tissues. These results characterized the immune response of B. alexandrina snails against Schistosoma infection which was found to occur by two different mechanisms. One type of defense occurs in highly resistant snails, and employs direct miracidial destruction soon after parasite penetration. The other type occurs in less resistant snails where a delayed resistance development occurs after the dissemination of the sporocysts in the snail tissues. It seems that B. alexandrina snails respond more or less similar to B. glabrata. The results also proved that the immune response of the internal defense system increased with increasing the number of the inherited resistant genes.

En Egipto, Biomphalaria alexandrina es el huésped intermediario de Schistosoma mansoni. La supervivencia de los miracidios de Schistosoma en los caracoles varía entre las especies de Biomphalaria. El sistema de defensa interno es uno de los factores que influyen en el patrón de susceptibilidad de los caracoles. La interacción entre los caracoles Biomphalaria y S. mansoni requiere ser identificada para cada especie e incluso, entre los miembros de la misma especie con diferente grado de susceptibilidad. En el presente estudio, la primera generación de padres susceptibles y resistentes de B. alejandrina fue examinada histológicamente al día 30, después de la exposición. El trabajo fue realizado tanto para caracterizar la respuesta inmune, según las reacciones de los tejidos, de los caracoles susceptibles y resistentes de B. alejandrina contra S. mansoni. También, el estudio se diseñó para determinar el impacto en el aumento de la resistencia en los caracoles padres, en los mecanismos de interacción de sus crías contra la infección. Los resultados mostraron que la tasa de infección para las crías, de padres susceptibles, fue del 92%. No se originaron crías susceptibles de los padres resistentes. Cuando los padres incluían un número igual de caracoles susceptibles y resistentes, dieron como resultado crías con una tasa de infección del 20%. Los caracoles susceptibles que tuvieron padres susceptibles mostraron un mayor grado de susceptibilidad que los que tenían tanto padres sensibles como resistentes. Una característica común de los caracoles resistentes fue la ausencia de parásitos viables. Las reacciones en los tejidos de los caracoles resistentes de sólo padres resistentes ocurrió en el sitio de penetración del miracidio. En los caracoles resistentes, para los que variedades susceptibles fueron incluídas entre sus padres, las reacciones se produjeron en tejidos profundos. Los resultados caracterizaron la respuesta inmune de los caracoles B. alexandrina contra la infección por Schistosoma, que ocurre por dos mecanismos diferentes. El primer tipo de defensa la cual se produce en los caracoles con alta resistencia, utiliza la destrucción directa del miracidio poco después de la penetración de los parásitos. El segundo tipo se produce en los caracoles menos resistentes, en el cual se después de la difusión de los esporocistos en los tejidos del caracol. Parece que los caracoles B. alexandrina responden de una manera más o menos similar a B. glabrata. Los resultados también demostraron que la respuesta inmune del sistema de defensa interna aumentó cuando en el número de genes de resistencia heredados es mayor.