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Alvaro Martínez-moreno - One of the best experts on this subject based on the ideXlab platform.
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Characterization of dendritic cells and follicular dendritic cells in the Hepatic Lymph Nodes and liver of sheep experimentally infected with Fasciola Hepatica
Veterinary Research, 2020Co-Authors: María Teresa Ruiz-campillo, Alvaro Martínez-moreno, R Zafra, L. Buffoni, F.j. Martínez-moreno, Verónica Molina-hernández, María José Bautista, Isabel L. Pacheco, Jose PerezAbstract:AbstractFasciola Hepatica has been shown to have a high capacity for immunomodulation of the host response, making the development of protective vaccines extremely difficult. One of these immunomodulation mechanisms is the impairment of dendritic cells (DC) maturation and, therefore, suppression of antigenic presentation. The aim of this study was to evaluate the pathological changes as well as the characterization of two antigen presenting cells, DC (CD1b, CD83 and MHC-II positive) and follicular dendritic cells (FDC) (CNA.42, S100 and CD83 positive) by immunohistochemistry in the Hepatic Lymph Nodes (HLN) and livers of sheep during the early stages of infection with F. Hepatica [9 and 18 days post-infection (dpi)], compared with an uninfected group (UC) as a control. The results revealed a marked hyperplasia of HLN germinal centres at 9 and, in particular, 18 dpi, with respect to the UC group, with coincidental increased expression of CNA.42 in FDC of Lymphoid follicles and CD1b in the DC of paracortical areas at 18 dpi. However, the expression of MHC-II and CD83 decreased at 9 and, particularly, at 18 dpi in HLN compared with that in the UC group. Since both markers are related to active presentation of antigens by DC and FDC, the results of the present study suggest that, despite the marked hyperplasia of HLN and increase in DC and FDC numbers during early stages of infection, the DC and FDC antigenic presentation capacity, as suggested by the expression of the markers MHC-II and CD83, is suppressed by the parasite. This suppression was not observed in the liver, probably because of the low number of DC. This is the first study of the immunophenotype of DCs and FDC in sheep infected with F. Hepatica.
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Th1/Th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola Hepatica.
Veterinary parasitology, 2017Co-Authors: I. L. Pacheco, Alvaro Martínez-moreno, R Zafra, N. Morales-prieto, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Distribution of Foxp3+ T cells in the liver and Hepatic Lymph Nodes of goats and sheep experimentally infected with Fasciola Hepatica.
Veterinary parasitology, 2016Co-Authors: A. Escamilla, Jose Perez, R Zafra, I. L. Pacheco, L. Buffoni, T.n. Mcneilly, F.j. Martínez-moreno, Verónica Molina-hernández, Alvaro Martínez-morenoAbstract:Abstract Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola Hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. Hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and Hepatic Lymph Nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n = 6) and three groups of sheep (n = 6) were used in this study. Goats in groups 1–2 and sheep in groups 4–5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin–biotin–complex method was used for the immunohistochemical study. At nine dpi in acute Hepatic lesions, the number of both Foxp3+ and CD3+ T Lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15–19 wpi), the number of Foxp3+ and CD3+ T Lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of Hepatic Lymph Nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and Hepatic Lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic Hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of Hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.
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Evaluation of Hepatic damage and local immune response in goats immunized with native glutathione S-transferase of Fasciola Hepatica.
Journal of Comparative Pathology, 2010Co-Authors: R Zafra, Alvaro Martínez-moreno, Ricardo E. Mendes, R.a. Perez-ecija, L. Buffoni, F.j. Martínez-moreno, M.e. Martínez Galisteo, J PerezAbstract:Summary Worm burden, Hepatic damage and local cellular and humoral immune responses were assessed in goats immunized with glutathione-S-transferase and challenged with Fasciola Hepatica. Infected but unimmunized and uninfected control groups were also studied. Hepatic damage was evaluated grossly and microscopically. Local immune response was evaluated by (1) microscopical examination of Hepatic Lymph Nodes (HLNs); (2) analysis of the distribution of CD2+, CD4+, CD8+, T-cell receptor γδ+ Lymphocytes and immunoglobulin (Ig) G+ plasma cells; and (3) investigation of the distribution of cells expressing interleukin (IL)-4 and interferon (IFN)-γ in the Hepatic inflammatory infiltrates and HLNs. Immunized animals did not have significant reduction in fluke number, but there was significant (P
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Evaluation of local immune response to Fasciola Hepatica experimental infection in the liver and Hepatic Lymph Nodes of goats immunized with Sm14 vaccine antigen
Memorias do Instituto Oswaldo Cruz, 2010Co-Authors: Ricardo E. Mendes, Alvaro Martínez-moreno, R Zafra, R.a. Perez-ecija, L. Buffoni, Miriam Tendler, J PerezAbstract:Protection against Fasciola Hepatica in goats immunized with a synthetic recombinant antigen from Schistosoma mansoni fatty acid-binding protein 14 (rSm14) was investigated by assessing worm burdens, serum levels of Hepatic enzymes, faecal egg count and Hepatic damage, which was evaluated using gross and microscopic morphometric observation. The nature of the local immune response was assessed by examining the distribution of CD2+, CD4+, CD8+ and γ´+ T Lymphocytes along with IgG+, IL-4+ and IFN-γ+ cells in the liver and Hepatic Lymph Nodes (HLN). The goats used consisted of group 1 (unimmunized and uninfected), group 2 [infected control - immunized with Quillaia A (Quil A)] and group 3 (immunized with rSm14 in Quil A and infected), each containing seven animals. Immunization with rSm14 in Quil A adjuvant induced a reduction in gross Hepatic lesions of 56.6% (p < 0.001) and reduced Hepatic and HLN infiltration of CD2+, CD4+, CD8+ and γ´+ T Lymphocytes as well as IL-4+ and IFN-γ+ cells (p < 0.05). This is the first report of caprine immunization against F. Hepatica using a complete rSm14 molecule derived from S. mansoni. Immunization reduced Hepatic damage and local inflammatory infiltration into the liver and HLN. However, considering that Quil A is not the preferential/first choice adjuvant for Sm14 immunization, further studies will be undertaken using the monophosphoryl lipid A-based family of adjuvants during clinical trials to facilitate anti-Fasciolavaccine development.
J Perez - One of the best experts on this subject based on the ideXlab platform.
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Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola Hepatica
Nature Publishing Group, 2019Co-Authors: I. L. Pacheco, R Zafra, N. Abril, N. Morales-prieto, Molina V. Hernández, M. T. Ruiz, R. Perez-caballero, A. Martínez-moreno, J PerezAbstract:Abstract The aim of this study was to validate reference genes for gene normalisation using qRT-PCR in Hepatic Lymph Nodes (HLN) and livers from sheep infected with Fasciola Hepatica during early and late stages of infection. To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, ΔCt method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn/Vn + 1) analysis revealed that PGK1, HSP90AA1 and GYPC were the most stable reference genes and suitable for qRT-PCR normalisation in both HLN and liver tissues. These three genes were validated against FoxP3, IL-10, TGF-β, TNF-α and IL-1β genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from F. Hepatica and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-α and IL-1β during chronic stages of infection with F. Hepatica and in uninfected controls. Our study is the first to evaluate and validate sheep reference genes in order to provide tools for monitoring cytokines in Fasciola Hepatica infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in F. Hepatica vaccinated and infected sheep
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th1 th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with fasciola Hepatica
Veterinary Parasitology, 2017Co-Authors: I. L. Pacheco, R Zafra, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, Noelia Moralesprieto, A Martinezmoreno, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Th1/Th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola Hepatica.
Veterinary parasitology, 2017Co-Authors: I. L. Pacheco, Alvaro Martínez-moreno, R Zafra, N. Morales-prieto, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Peripheral blood Lymphocyte subsets in Fasciola Hepatica infected and immunised goats.
Veterinary immunology and immunopathology, 2013Co-Authors: R Zafra, J Perez, L. Buffoni, F.j. Martínez-moreno, I Acosta, E Mozos, A. Martínez-morenoAbstract:The proportions of CD4(+), CD8(+) and WC1+ T Lymphocytes from peripheral blood using flow cytometry were investigated in goats infected with Fasciola Hepatica and previously immunised with recombinant Cathepsin-L1 (rCL1) and Glutathione-S-transferase sigma class (GST). The immunisation trial did not induce protective responses, and no significant differences were recorded between immunised and non-immunised groups. However, there was a significant decrease in the proportion of CD4(+) T Lymphocytes in the infected groups both at 5 weeks post-infection (wpi), coinciding with the migratory stage of the infection, and at 12 wpi in the biliary stage of the infection. The proportional decrease in this circulating population may be related to the recruitment of CD4(+) T cells in liver and Hepatic Lymph Nodes and also to the immunomodulatory effect of the parasite through the interaction of F. Hepatica excretory-secretory products (FhESP) with this cell population. To date, this is the first report about the effect of F. Hepatica infection in peripheral Lymphocyte subsets in goats.
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Evaluation of Hepatic damage and local immune response in goats immunized with native glutathione S-transferase of Fasciola Hepatica.
Journal of Comparative Pathology, 2010Co-Authors: R Zafra, Alvaro Martínez-moreno, Ricardo E. Mendes, R.a. Perez-ecija, L. Buffoni, F.j. Martínez-moreno, M.e. Martínez Galisteo, J PerezAbstract:Summary Worm burden, Hepatic damage and local cellular and humoral immune responses were assessed in goats immunized with glutathione-S-transferase and challenged with Fasciola Hepatica. Infected but unimmunized and uninfected control groups were also studied. Hepatic damage was evaluated grossly and microscopically. Local immune response was evaluated by (1) microscopical examination of Hepatic Lymph Nodes (HLNs); (2) analysis of the distribution of CD2+, CD4+, CD8+, T-cell receptor γδ+ Lymphocytes and immunoglobulin (Ig) G+ plasma cells; and (3) investigation of the distribution of cells expressing interleukin (IL)-4 and interferon (IFN)-γ in the Hepatic inflammatory infiltrates and HLNs. Immunized animals did not have significant reduction in fluke number, but there was significant (P
R Zafra - One of the best experts on this subject based on the ideXlab platform.
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Characterization of dendritic cells and follicular dendritic cells in the Hepatic Lymph Nodes and liver of sheep experimentally infected with Fasciola Hepatica
Veterinary Research, 2020Co-Authors: María Teresa Ruiz-campillo, Alvaro Martínez-moreno, R Zafra, L. Buffoni, F.j. Martínez-moreno, Verónica Molina-hernández, María José Bautista, Isabel L. Pacheco, Jose PerezAbstract:AbstractFasciola Hepatica has been shown to have a high capacity for immunomodulation of the host response, making the development of protective vaccines extremely difficult. One of these immunomodulation mechanisms is the impairment of dendritic cells (DC) maturation and, therefore, suppression of antigenic presentation. The aim of this study was to evaluate the pathological changes as well as the characterization of two antigen presenting cells, DC (CD1b, CD83 and MHC-II positive) and follicular dendritic cells (FDC) (CNA.42, S100 and CD83 positive) by immunohistochemistry in the Hepatic Lymph Nodes (HLN) and livers of sheep during the early stages of infection with F. Hepatica [9 and 18 days post-infection (dpi)], compared with an uninfected group (UC) as a control. The results revealed a marked hyperplasia of HLN germinal centres at 9 and, in particular, 18 dpi, with respect to the UC group, with coincidental increased expression of CNA.42 in FDC of Lymphoid follicles and CD1b in the DC of paracortical areas at 18 dpi. However, the expression of MHC-II and CD83 decreased at 9 and, particularly, at 18 dpi in HLN compared with that in the UC group. Since both markers are related to active presentation of antigens by DC and FDC, the results of the present study suggest that, despite the marked hyperplasia of HLN and increase in DC and FDC numbers during early stages of infection, the DC and FDC antigenic presentation capacity, as suggested by the expression of the markers MHC-II and CD83, is suppressed by the parasite. This suppression was not observed in the liver, probably because of the low number of DC. This is the first study of the immunophenotype of DCs and FDC in sheep infected with F. Hepatica.
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Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola Hepatica
Nature Publishing Group, 2019Co-Authors: I. L. Pacheco, R Zafra, N. Abril, N. Morales-prieto, Molina V. Hernández, M. T. Ruiz, R. Perez-caballero, A. Martínez-moreno, J PerezAbstract:Abstract The aim of this study was to validate reference genes for gene normalisation using qRT-PCR in Hepatic Lymph Nodes (HLN) and livers from sheep infected with Fasciola Hepatica during early and late stages of infection. To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, ΔCt method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn/Vn + 1) analysis revealed that PGK1, HSP90AA1 and GYPC were the most stable reference genes and suitable for qRT-PCR normalisation in both HLN and liver tissues. These three genes were validated against FoxP3, IL-10, TGF-β, TNF-α and IL-1β genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from F. Hepatica and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-α and IL-1β during chronic stages of infection with F. Hepatica and in uninfected controls. Our study is the first to evaluate and validate sheep reference genes in order to provide tools for monitoring cytokines in Fasciola Hepatica infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in F. Hepatica vaccinated and infected sheep
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th1 th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with fasciola Hepatica
Veterinary Parasitology, 2017Co-Authors: I. L. Pacheco, R Zafra, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, Noelia Moralesprieto, A Martinezmoreno, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Th1/Th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola Hepatica.
Veterinary parasitology, 2017Co-Authors: I. L. Pacheco, Alvaro Martínez-moreno, R Zafra, N. Morales-prieto, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Distribution of Foxp3+ T cells in the liver and Hepatic Lymph Nodes of goats and sheep experimentally infected with Fasciola Hepatica.
Veterinary parasitology, 2016Co-Authors: A. Escamilla, Jose Perez, R Zafra, I. L. Pacheco, L. Buffoni, T.n. Mcneilly, F.j. Martínez-moreno, Verónica Molina-hernández, Alvaro Martínez-morenoAbstract:Abstract Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola Hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. Hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and Hepatic Lymph Nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n = 6) and three groups of sheep (n = 6) were used in this study. Goats in groups 1–2 and sheep in groups 4–5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin–biotin–complex method was used for the immunohistochemical study. At nine dpi in acute Hepatic lesions, the number of both Foxp3+ and CD3+ T Lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15–19 wpi), the number of Foxp3+ and CD3+ T Lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of Hepatic Lymph Nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and Hepatic Lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic Hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of Hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.
I. L. Pacheco - One of the best experts on this subject based on the ideXlab platform.
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Identification of reference genes for real-time PCR cytokine gene expression studies in sheep experimentally infected with Fasciola Hepatica
Nature Publishing Group, 2019Co-Authors: I. L. Pacheco, R Zafra, N. Abril, N. Morales-prieto, Molina V. Hernández, M. T. Ruiz, R. Perez-caballero, A. Martínez-moreno, J PerezAbstract:Abstract The aim of this study was to validate reference genes for gene normalisation using qRT-PCR in Hepatic Lymph Nodes (HLN) and livers from sheep infected with Fasciola Hepatica during early and late stages of infection. To this end, a comprehensive statistical approach (RefFinder) encompassing four different methods of analysis (geNorm, BestKeeper, ΔCt method and NormFinder) was used to validate ten candidate reference genes. Stability analysis of gene expression followed by pairwise variation (Vn/Vn + 1) analysis revealed that PGK1, HSP90AA1 and GYPC were the most stable reference genes and suitable for qRT-PCR normalisation in both HLN and liver tissues. These three genes were validated against FoxP3, IL-10, TGF-β, TNF-α and IL-1β genes in the HLN tissue of sheep vaccinated with Cathepsin L1 from F. Hepatica and unvaccinated infected and uninfected controls during early stages of infection. In the liver, the three reference genes were validated against TNF-α and IL-1β during chronic stages of infection with F. Hepatica and in uninfected controls. Our study is the first to evaluate and validate sheep reference genes in order to provide tools for monitoring cytokines in Fasciola Hepatica infected sheep target organs. Our results present an approach to elucidate the role of different cytokines in F. Hepatica vaccinated and infected sheep
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th1 th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with fasciola Hepatica
Veterinary Parasitology, 2017Co-Authors: I. L. Pacheco, R Zafra, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, Noelia Moralesprieto, A Martinezmoreno, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Th1/Th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola Hepatica.
Veterinary parasitology, 2017Co-Authors: I. L. Pacheco, Alvaro Martínez-moreno, R Zafra, N. Morales-prieto, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Distribution of Foxp3+ T cells in the liver and Hepatic Lymph Nodes of goats and sheep experimentally infected with Fasciola Hepatica.
Veterinary parasitology, 2016Co-Authors: A. Escamilla, Jose Perez, R Zafra, I. L. Pacheco, L. Buffoni, T.n. Mcneilly, F.j. Martínez-moreno, Verónica Molina-hernández, Alvaro Martínez-morenoAbstract:Abstract Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola Hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. Hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and Hepatic Lymph Nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n = 6) and three groups of sheep (n = 6) were used in this study. Goats in groups 1–2 and sheep in groups 4–5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin–biotin–complex method was used for the immunohistochemical study. At nine dpi in acute Hepatic lesions, the number of both Foxp3+ and CD3+ T Lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15–19 wpi), the number of Foxp3+ and CD3+ T Lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of Hepatic Lymph Nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and Hepatic Lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic Hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of Hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.
A. Escamilla - One of the best experts on this subject based on the ideXlab platform.
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th1 th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with fasciola Hepatica
Veterinary Parasitology, 2017Co-Authors: I. L. Pacheco, R Zafra, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, Noelia Moralesprieto, A Martinezmoreno, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Th1/Th2 balance in the liver and Hepatic Lymph Nodes of vaccinated and unvaccinated sheep during acute stages of infection with Fasciola Hepatica.
Veterinary parasitology, 2017Co-Authors: I. L. Pacheco, Alvaro Martínez-moreno, R Zafra, N. Morales-prieto, M. T. Ruiz, A. Escamilla, Nieves Abril, M.j. Bautista, J PerezAbstract:The expression of IFNγ and IL4 was quantified using q-PCR in the liver and Hepatic Lymph Nodes (HLN) of sheep during early stages of infection with Fasciola Hepatica (1, 3, 9 and 18days post-infection, dpi). A group of animals (Group 1) were vaccinated with Fasciola Hepatica recombinant cathepsin L1 (FhCL1) in montanide 70 VG prior to infection, a second group (group 2) was used as infected control and a third (group 3) was used as uninfected control. To study vaccine efficacy three additional groups were sacrificed 19 weeks post-infection (group 4 immunized with CL1, group 5 with the adjuvant and group 6 was used as infected control). The vaccinated group did not show significant fluke reduction compared to the adjuvant group and infected control group. IL4 expression was observed to increase at 9 dpi and was further elevated at 18 dpi in the liver and HLN of vaccinated and infected control groups compared to the uninfected group. IFNγ expression exhibited different dynamics in the liver and HLN compared to IL4; thus, in the liver this cytokine increased at 9 dpi in the vaccinated and at 18 dpi in vaccinated and infected control groups, while in the HLN it decreased gradually and significantly from 1 dpi onwards. These results suggest that a marked Th2 polarization is present from 9 dpi in HLN and from 18 dpi in the liver. The increase of IFNγ in the liver may correspond with tissue damage response with granuloma formation. The FhCL1 vaccine did not alter the Th1/Th2 balance when compared to unvaccinated and infected sheep. The study of IFNγ and IL4 in the various tissue compartments in sheep could facilitate selection of new adjuvants inducing a strong Th1 response for a more rationale vaccine formulation.
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Distribution of Foxp3+ T cells in the liver and Hepatic Lymph Nodes of goats and sheep experimentally infected with Fasciola Hepatica.
Veterinary parasitology, 2016Co-Authors: A. Escamilla, Jose Perez, R Zafra, I. L. Pacheco, L. Buffoni, T.n. Mcneilly, F.j. Martínez-moreno, Verónica Molina-hernández, Alvaro Martínez-morenoAbstract:Abstract Foxp3 regulatory T cells (Tregs) are now considered to play a key role in modulation of immune responses during parasitic helminth infections. Immunomodulation is a key factor in Fasciola Hepatica infection; however, the distribution and role of Foxp3+ Tregs cells have not been investigated in F. Hepatica infected ruminants. The aim of this study was to evaluate the presence of Foxp3+ Tregs in the liver and Hepatic Lymph Nodes from experimentally infected sheep and goats during acute and chronic stages of infection. Three groups of goats (n = 6) and three groups of sheep (n = 6) were used in this study. Goats in groups 1–2 and sheep in groups 4–5 were orally infected with metacercarie of ovine origin. Groups 1 and 4 were killed during the acute stage of the infection, at nine days post infection (dpi); groups 2 and 5 were killed during the chronic stage, at 15 and19 weeks post infection respectively (wpi). Groups 3 (goats) and 6 (sheep) were left as uninfected controls. Fluke burdens and liver damage were assessed and the avidin–biotin–complex method was used for the immunohistochemical study. At nine dpi in acute Hepatic lesions, the number of both Foxp3+ and CD3+ T Lymphocytes increased significantly in goats and sheep. In the chronic stages of infection (15–19 wpi), the number of Foxp3+ and CD3+ T Lymphocytes were also significantly increased with respect to control livers, particularly in portal spaces with severely enlarged bile ducts (response to adult flukes) while the increase was lower in granulomas, chronic tracts and smaller portal spaces (response to tissue damage). Foxp3+ Tregs were increased in the cortex of Hepatic Lymph Nodes of sheep (chronic infection) and goats (acute and chronic infection). The estimated proportion of T cells which were Foxp3+ was significantly increased in the large bile ducts and Hepatic Lymph node cortex of chronically infected goats but not sheep. This first report of the expansion of Foxp3+ Tregs in acute and chronic Hepatic lesions in ruminants suggests that these cells may be involved in both parasite survival and modulation of Hepatic damage. Future studies should be focused on the investigation of parasite molecules and cytokines involved in this process.