Background Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury. Aims Determine if Hedgehog-pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved. Methods Immunohistochemistry was used to characterize the cells that generate and respond to Hedgehog ligands in 28 liver biopsies from patients with different grades of schistosomiasis fibrosis staged by ultrasound. Cultured macrophages (RAW264.7 and primary rat Kupffer cells) and primary rat liver sinusoidal endothelial cells (LSEC) were treated with schistosome egg antigen (SEA) and evaluated by qRT-PCR. Inhibition of the Hedgehog-pathway was used to investigate its role in alternative activation of macrophages (M2) and vascular tube formation. Results Patients with schistosomiasis expressed more ligands (Shh and Ihh) and target genes (Patched and Gli2) than healthy individuals. Activated LSEC and myofibroblasts were Hedgehog-responsive (Gli2(+)) and accumulated in parallel with fibrosis stage (p<0.05). Double IHC for Ihh/CD68 showed that Ihh(+) cells were macrophages. In vitro studies demonstrated that SEA stimulated macrophages to express Ihh and Shh mRNA (p<0.05). Conditioned media from such macrophages induced luciferase production by Shh-LightII cells (p<0.001) and Hedgehog inhibitors blocked this effect (p<0.001). SEA-treated macrophages also up-regulated their own expression of M2 markers, and Hh-pathway inhibitors abrogated this response (p<0.01). Inhibition of the Hedgehog pathway in LSEC blocked SEA-induced migration and tube formation. Conclusion SEA stimulates liver macrophages to produce Hh-ligands, which promote alternative activation of macrophages, fibrogenesis, and vascular remodeling in schistosomiasis.

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Introduction Schistosomiasis is a major cause of portal hypertension worldwide. It associates with portal fibrosis that develops during chronic infection. The mechanisms by which the pathogen evokes these host responses remain unclear. We evaluated the hypothesis that schistosome eggs release factors that directly stimulate liver cells to produce Osteopontin (OPN), a profibrogenic protein that stimulates hepatic stellate cells to become myofibroblasts. We also investigated the utility of OPN as a biomarker of fibrosis and/or severity of portal hypertension. Methods Cultured cholangiocytes, kupffer cells and hepatic stellate cells were treated with soluble egg antigen (SEA); OPN production was quantified by qRTPCR and ELISA; cell proliferation assessed by BrdU. Mice were infected with S. mansoni for 6 or 16 weeks to cause early or advanced fibrosis. Liver OPN was evaluated by qRTPCR and immunohistochemistry, and correlated with liver fibrosis and serum OPN. Livers from patients with schistosomiasis mansoni (early fibrosis n=15; advanced fibrosis n= 72) or healthy adults (n=22) were immunostained for OPN and fibrosis markers. Results were correlated with plasma OPN levels and splenic vein pressures. Results SEA induced cholangiocyte proliferation and OPN secretion (p<0.001 vs. controls). Cholangiocytes were OPN (+) in schistosoma-infected mice and humans. Liver and serum OPN levels correlated with fibrosis stage (mice: r=0.861; human r=0.672, p=0.0001) and myofibroblast accumulation (mice: r=0.800; human: r=0.761, p=0.0001). Numbers of OPN (+) bile ductules strongly correlated with splenic vein pressure (r=0.778; p=0.001). Conclusions S. mansoni egg antigens stimulate cholangiocyte proliferation and OPN secretion. OPN levels in liver and blood correlate with fibrosis stage and portal hypertension severity.

Few publications have compared ultrasound (US) to histology in diagnosing schistosomiasis-induced liver fibrosis (LF); none has used magnetic resonance (MR). The aim of this study was to evaluate schistosomal LF using these three methods. Fourteen patients with hepatosplenic schistosomiasis admitted to hospital for surgical treatment of variceal bleeding were investigated. They were submitted to upper digestive endoscopy, US, MR and wedge liver biopsy. The World Health Organization protocol for US in schistosomiasis was used. Hepatic fibrosis was classified as absent, slight, moderate or intense. Histology and MR confirmed Symmers' fibrosis in all cases. US failed to detect it in one patient. Moderate agreement was found comparing US to MR; poor agreement was found when US or MR were compared to histology. Re-classifying LF as only slight or intense created moderate agreement between imaging techniques and histology. Histomorphometry did not separate slight from intense LF. Two patients with advanced hepatosplenic schistosomiasis presented slight LF. Our data suggest that the presence of the characteristic periportal fibrosis, diagnosed by US, MR or histology, associated with a sign of portal hypertension, defines the severity of the disease. We conclude that imaging techniques are reliable to define the presence of LF but fail in grading its intensity

This report describes the first time that patients from an outbreak of acute schistosomiasis have been compared to controls. Five subjects (10%) had severe manifestations of schistosomiasis. Diagnosis of the disease and its severity was delayed because physicians did not consider that an epidemic of schistosomiasis might emerge in a nonendemic area.