Project description:Fasciolosis remains an important food-borne tremaode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs as well as the molecular basis involved in acquiring protection is extremely important in designing and selecting new vaccine candidates. The present study gives a first report of microarray-based technology for describing changes in splenic gene expression profile for mice immunised with a highly effective, protection-inducing,multi-epitope, subunit-based, chemically-synthesised vaccine candidate. Female CD1 mice were immunised with synthetic peptides containing B- and T-cell epitopes as novel vaccine candidates and challenged with Fasciola hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. The Ingenuity Pathway Analysis tool was used to annotate bio-functions and constructing and visualising molecular interaction networks. Immunising mice with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes; 168 being up-regulated and 652 down-regulated. Functional study revealed changes in pathways related to nitric oxide and reactive oxygen species production, IL-12 signalling and production in macrophages and IL-8 signalling with up-regulation of S100A8, MMP9 and CXCR2 genes. The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against Fasciola hepatica in a murine model Three mice were used as healthy controls and remained untreated along the experiment. Three mice were subcutaneously immunised with our vaccine candidate on day 0, and two booster doses were applied on days 14 and 28, respectively. Two-weeks after the last immunisation, all of the mice used in the present study (including controls) were humanely euthanised by intraperitoneal injection of pentobarbital (60mg/Kg) and necropsied for spleen recovery and RNA isolation using standardised protocols. Splenic gene expression profile was compared between healthy and immunised groups.

Project description:Background: Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with Fasciola hepatica metacercariae using a microarray-based methodology. Methodology: A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21). Results: We found that Fasciola hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death. Conclusion: The present study provides us insights at the molecular level about the underlying mechanisms used by Fasciola hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis. We used three experimental groups each containing 3 mice. Group 1 remains untreated and served as control. Group 2 was infected with Fasciola hepatica metacercariae on day 0 and humanely necropsied at 7 days post-infection. Group 3 was infected with Fasciola hepatica metacercariae on day 0 and humanely necropsied at 21 days post- infection. At the time of necropsy, liver of each mice were removed and the RNA was isolated. We compared the gene expression profile in the liver of mice infected with Fasciola hepatica.

Project description:Background: Fasciola hepatica infection still remains one of the helminthic neglected tropical diseases (NTDs). It has a huge worldwide distribution, affecting mainly cattle and, sometimes, human beings. In addition to data reported about the immunological response induced by helminthic infections and that induced by Fasciola hepatica, little is known about the gene expression profile in its organ target, the liver, which is where adult worms are established and live for long periods of time, causing its characteristic pathology. In the present work, we study both the early and late gene expression profiles in the livers of mice infected with Fasciola hepatica metacercariae using a microarray-based methodology. Methodology: A total of 9 female-6-week-old BALB/c mice (Charles River Laboratories, Barcelona, Spain) weighing 20 to 35 g were used for the experiments. Two groups of BALB/c mice were orally infected with seven F. hepatica metacercariae, and the other group remained untreated and served as a control. Mice were humanely euthanized and necropsied for liver recovery, histological assessment of hepatic damage, RNA isolation, microarray design and gene expression analysis on the day of infection (t0), seven days post-infection (t7) and twenty-one days post-infection (t21). Results: We found that Fasciola hepatica infection induces the differential expression of 128 genes in the liver in the early stage of infection and 308 genes in the late stage, and most of them are up-regulated. The Ingenuity Pathway Analysis revealed significant changes in the pathways related to metabolism, biosynthesis and signaling as well as genes implicated in inducing liver-toxicity, injury and death. Conclusion: The present study provides us insights at the molecular level about the underlying mechanisms used by Fasciola hepatica, leading to liver damage and its subsequent pathophysiology. The expression pattern obtained here could also be used to explain the lack of association between infection with F. hepatica and cholangiocarcinoma. However, more studies should be performed to confirm this hypothesis.

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Splenic gene expression profile of mice immunized with an anti-Fasciola hepatica vaccine candidate: insights into the immunological mechanisms leading to protection

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