Resveratrol against Echinococcus sp.: Discrepancies between In Vitro and In Vivo Responses

In an attempt to find new anti-echinococcal drugs, resveratrol (Rsv) effectiveness against the larval stages of Echinococcus granulosus and E. multilocularis was evaluated. The in vitro effect of Rsv on parasites was assessed via optical and electron microscopy, RT-qPCR and immunohistochemistry. In vivo efficacy was evaluated in murine models of cystic (CE) and alveolar echinococcosis (AE). The impact of infection and drug treatment on the mouse bone marrow hematopoietic stem cell (HSC) population and its differentiation into dendritic cells (BMDCs) was investigated via flow cytometry and RT-qPCR. In vitro treatment with Rsv reduced E. granulosus metacestode and protoscolex viability in a concentration-dependent manner, caused ultrastructural damage, increased autophagy gene transcription, and raised Eg-Atg8 expression while suppressing Eg-TOR. However, the intraperitoneal administration of Rsv was not only ineffective, but also promoted parasite development in mice with CE and AE. In the early infection model of AE treated with Rsv, an expansion of HSCs was observed followed by their differentiation towards BMCDs. The latter showed an anti-inflammatory phenotype and reduced LPS-stimulated activation compared to control BMDCs. We suggest that Rsv ineffectiveness could have been caused by the low intracystic concentration achieved in vivo and the drug’s hormetic effect, with opposite anti-parasitic and immunomodulatory responses in different doses.Fil: Loos, Julia Alexandra. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Franco, Micaela. Gobierno de la Provincia de Buenos Aires. Hospital Interzonal General de Agudos Dr. Oscar Alende de Mar del Plata.; ArgentinaFil: Chop, Maia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Rodríguez Rodrígues, Christian Fernando Ariel. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Cumino, Andrea Carina. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; Argentin

Hydatid fluid from Echinococcus granulosus induces phagophore and autophagosome formation in dendritic cells through an upregulation of Beclin-1

Background: The cestode Echinococcus granulosus (Eg) is the etiological agent of cystic echinococcosis. This parasite develops cysts filled with hydatid fluid (HF) in the viscera of the intermediate host. Autophagy is a cellular catabolic process that plays a key role in the presentation of endogenous and exogenous proteins, promoting the activation of T cells. The aim of this work is to analyze if HF, constituted by a wide range of parasite proteins, could trigger autophagy in dendritic cells. Methods: HF was punctured from the hydatid cysts collected of infected cattle slaughtered. Murine BMDCs were cultured in RPMI 1640 medium, supplemented with FLT3-L. First, lysosome activity was evaluated using Acridine Orange, a fluorophore that can be trapped in acidic vesicular organelles. Then, autophagy induction was evaluated by FACS, qPCR, Confocal and Transmission Electron Microscopy. Rapamycin (20 nM) and chloroquine (100μM) were used to modulate autophagic flux. LC3-attachment to the autophagic membrane, were analyzed by stained DCs with anti LC3-β antibody (clone H50). Results: HF significantly increased acridine orange cytoplasmic accumulation compared to control cells (***p <0.001) and enhanced the effect of rapamycin (****p<0.0001). The ultrastructural analysis of TEM showed that in the presence of HF, DCs stimulate the formation of phagophores, double lipid membrane autophagosome, MVBs and autolysosomes. Also, HF-stimulated BMDCs significantly enhanced the mean fluorescence intensity of LC3-positive structures in comparison with unstimulated cells (*p<0.05, **p<0.01, ***p<0.001 HF-stimulated cells vs controls). Finally, we have observed that HF induces a significant increase in the transcriptional expression of LC3 and Beclin-1 (n=3, **p <0.01 vs control) and enhances the expression induced by rapamycin. Conclusions: These results suggest that HF of Echinococcus granulosus regulates gene expression to increase autophagy- related structures in DCs.Fil: Chop, Maia. Universidad Nacional de Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente; ArgentinaFil: Nicolao, María Celeste. Universidad Nacional de Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente; ArgentinaFil: Loos, Julia Alexandra. Universidad Nacional de Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente; ArgentinaFil: Ledo, Camila. Universidad Nacional de Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente; ArgentinaFil: Cumino, Andrea Carina. Universidad Nacional de Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente; ArgentinaFil: Rodríguez Rodrígues, Christian Fernando Ariel. Universidad Nacional de Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente; ArgentinaLXVI Reunión Anual de la Sociedad Argentina de Investigación Clínica; LXIX Reunión Anual de la Sociedad Argentina de Inmunología; LIII Reunión Anual de la Asociación de Farmacología Experimental y XI Reunión Anual de la Asociación Argentina de NanomedicinasArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologíaAsociación de Farmacología ExperimentalAsociación Argentina de Nanomedicina

Mucins and polysaccharides from Echinococcus granulosus laminar layer induce a mild maturation phenotype in dendritic cells and promote gene expression of pro-inflammatory cytokines

Background: The laminar layer is an acellular structure rich in glycans that surrounds the metacestode, creating a mechanical and immunological protective barrier, crucial in the Echinococcus?host interface. The aim of this work is to analyze if purified laminar layer (pLL) could trigger maturation and cytokine production in dendritic cells by activating mTOR pathway, a central regulator of cell metabolism and environmental signals. Methods: BMDCs were cultured in complete RPMI supplemented with 100 ng/ml Flt3-L.The germinal layer was removed from pLL by washing with 2M NaCl. Flow cytometry: FITC or PE-conjugated mAbs directed to CD11c, CD40, CD80, CD86, MHC I and MHC II were used. Immunobloting and confocal microscopy: mouse anti-puromycin and rabbit anti-Phospho-mTOR antibodies were used. Gene expression analysis of IL-10, IL-12p35, IL-6, TNF-, IL-23p19, TGF- was performed on a Real Time PCR System Results: The pLL of Echinococcus induce a non-statistically significant, but a trend in the up-regulation of CD86 and MHC II and that expression change was diminished by the use of rapamycin (n=5). Conversely, no changes in the expression of CD40, CD80 or MHC I were registered. Further studies were done to analyze whether pLL was also able to stimulate the production of cytokines by BMDCs. When BMDCs were stimulated with pLL induce the expression of IL-6 and TNF-an=3 *p<0.05, **p<0.01), but not differences compared to control were observed in IL-12, IL-23, IL-10 and TGF-. Finally, we evaluated global protein synthesis and phosphorylation of mTOR using confocal microscopy or western blot. pLL (MFI:_979) showed an increase in translation levels compared to its counterpart without stimulation (MFI:_686, *p<0.05 n=3) and also induce an increase in mTOR phosphorylation levels compared to untreated BMCDs (n=3, p<0.01) Conclusions: These data suggest that Echinococcus pLL are recognized by DCs and induce activation of mTOR pathway favoring cell activation.Fil: Plá, Natalia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Química; ArgentinaFil: Chop, Maia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Nicolao, María Celeste. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Cumino, Andrea Carina. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Rodríguez Rodrígues, Christian Fernando Ariel. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaReunión de Sociedades de BiocienciasMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologíaSociedad Argentina de Fisiologí

A mannose-binding lectin from helianthus annuus recognizes GP120 from HIV-1 and promotes phonotype maturation in dendritic cells

Background: Plant lectins have potential use in biomedicine as antimicrobial reagents due to their binding specificity to glycoconjugates. Helja is a mannose-binding lectin isolated from sunflower seeds. Half of the molecular mass of the HIV-1 envelope glycoprotein (gp120) is constituted by high-mannose and complex N-glycans. The aim of this work is to analyze if Helja could recognize gp120 and induce dendritic cell maturation. Methods: Helja was purified from Helianthus annuus seeds on a D-mannose-agarose affinity chromatography. Potential molecular interaction between Helja and recombinant gp120 (M Group, B Subtype, SinoBiological, USA) was evaluated by dot blot and ligand blot approaches using anti-Helja antibodies. Bone marrow dendritic cells (BMDCs) were cultured in complete RPMI medium supplemented with FLT3-L. Cellular activation state was evaluated in Helja-treated BMDCs by measuring membrane proteins (CD86, CD40, MHCII, CD11c, CD80, Clec9a, Ly6G, SIPRα, CD103, SiglecH) by FACS. Results: The interaction of Helja with gp120 was initially detected in dot blot assays by immunodetection of lectin bound to viral protein immobilized on nitrocellulose membranes. This interaction was confirmed in ligand blot assays by detecting a signal whose molecular weight corresponds to gp120 (95 kDa). On the other hand, the presence of Helja (10 µg/ml) in BMDC culture induced an upregulation of MHCII, MHCI, CD80, CD40 and CD86 (n=3, **p<0.01 Helja-treated BMDCs vs controls, *p<0.05 Helja+LPS-treated BMDCs vs LPS control). No changes in Clec9a, Ly6G, SIRPα, CD103 and SiglecH were detected. Conclusions: These results suggest that Helja induces dendritic cell maturation, important to promote immune activation, and recognizes specific glycosidic residues and arrangements on the HIV-1 envelope protein which could have a potential biomedical use as a phyto-neutralizing agent in viral infection.Fil: Radicioni, Melisa Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Chop, Maia. Universidad Nacional de Mar del Plata; ArgentinaFil: del Rio, Marianela Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Sabatte, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Regente, Mariana Clelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Rodriguez Rodrigues, Christian. Universidad Nacional de Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Producción, Sanidad y Ambiente; ArgentinaLXVII Reunión Anual de la Sociedad Argentina de Investigación Clínica; LXX Reunión Anual de La Sociedad Argentina de Inmunología; 3er Congreso Franco-Argentino De Inmunología y Reunión Anual 2022 de La Sociedad Argentina De FisiologíaMar del PlataArgentinaSociedad Argentina de Investigaciones ClínicasSociedad Argentina de InmunologíaSociedad Argentina de Fisiologí

Hydatid fluid from Echinococcus granulosus induce the autophagy process in dendritic cells and promote antigen presentation and T- cell proliferation

Background: Autophagy is an important process for the presentation of endogenous and exogenous proteins on MHC I and II molecules, promoting activation of CD8+ and CD4+ T cells respectively. The aim of this work is to analyze if hydatid fluid (HF) from Echinococcus, constituted by a wide range of parasite proteins could trigger autophagy improving antigen presentation and T cell proliferation. Methods: BMDCs were cultured in complete RPMI. Hydatid fluid (HF) was punctured from the hydatid cysts collected of infected cattle slaughtered. Antigen uptake was measured with (FITC-OVA) in BMDCs using a standard method. HF-stimulated BMDCs, were evaluated in autophagy induction and MHC II expression. For it, fixed cells were immunostained with LC3-clone H50 and analyzed them by immunofluorescence confocal microscopy. CFSE-stained splenocytes were co-incubated with BMDCs using a DC: splenocyte ratio of 1:4 Cellular proliferation was assayed after 4 days of culture by flow cytometry. Results: First, we evaluated if stimulation of HF during 18 h in BMDCs, induce different rates of antigen uptake. Effectively, the presence of Echinococcus antigens induces a markedly decreased OVA-uptake compared to control (**p <0.01, n=3). Next, we studied if stimulation with Eg antigens induces changes in the basal level of autophagy. HF-stimulated BMDCs significantly enhanced the mean fluorescence intensity of MHC II and LC3 and showed a trend in the increment of number and the average size of LC3-positive structures in comparison with unstimulated cells (*p<0.05, **p<0.01, ***p<0.001 HF-stimulated cells vs controls). Finally, we observed that culture splenocytes in the presence of stimulated DC induce their proliferation % CFSE+ cells CTRL:99%, HF:55% (***p<0.001, n=3). Conclusions: These data suggest that HF of Echinococcus induces an increase in autophagy processes promoting the presentation of exogenous antigens presented in MHC II molecules to improve T cell proliferation.Fil: Chop, Maia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Química; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Plá, Natalia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Departamento de Química; ArgentinaFil: Loos, Julia Alexandra. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Nicolao, María Celeste. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Cumino, Andrea Carina. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaFil: Rodríguez Rodrígues, Christian Fernando Ariel. Universidad Nacional de Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones En Produccion, Sanidad y Ambiente.; ArgentinaReunión de Sociedades de BiocienciasMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologíaSociedad Argentina de Fisiologí