murine-precision-cut-liver-slices-as-an-ex-vivo-model-of-liver-biology

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Queen's University Belfast

Acetylcholinesterase (AChE) is an important metabolic enzyme of schistosomes present in the musculature and on the surface of the blood stage where it has been implicated in the modulation of glucose scavenging from mammalian host blood. As both a target for the antischistosomal drug metrifonate and as a potential vaccine candidate, AChE has been characterised in the schistosome species Schistosoma mansoni, S. haematobium and S. bovis, but not in S. japonicum. Recently, using a schistosome protein microarray, a predicted S. japonicum acetylcholinesterase precursor was significantly targeted by protective IgG1 immune responses in S. haematobium-exposed individuals that had acquired drug-induced resistance to schistosomiasis after praziquantel treatment.

Functional characterisation of Schistosoma japonicum acetylcholinesterase.

Cystic fibrosis liver disease (CFLD) in children causes progressive fibrosis leading to biliary cirrhosis, however its cause(s) and early pathogenesis are unclear. We hypothesised that a bile acid-induced Ductular Reaction (DR) drives fibrogenesis. We evaluated the DR by cytokeratin-7 immunohistochemistry in liver biopsies, staged for fibrosis, from 60 children with CFLD and demonstrated that the DR was significantly correlated with hepatic fibrosis stage and biliary taurocholate levels. To examine the mechanisms involved in DR induction liver progenitor cells (LPCs) were treated with taurocholate and key events in DR evolution were assessed: LPC proliferation, LPC biliary differentiation and hepatic stellate cell (HSC) chemotaxis. Taurocholate induced a time-dependent increase in LPC proliferation and expression of genes associated with cholangiocyte differentiation (cytokeratin-19, connexin-43, integrin-β4 and γ-glutamyltranspeptidase [GGT]), while the hepatocyte specification marker HNF4α was suppressed. Functional cholangiocyte differentiation was demonstrated via increased acetylated α-tubulin and SOX9 proteins, the number of primary cilia(+) LPCs and increased active GGT enzyme secretion. Taurocholate induced LPCs to release MCP-1, MIP1α and RANTES into conditioned medium causing HSC chemotaxis, which was inhibited by anti-MIP1α. Immunofluorescence confirmed chemokine expression localized to CK7(+) DR and LPCs in CFLD liver biopsies. This study suggests that taurocholate is involved in initiating functional LPC biliary differentiation and the development of the DR, with subsequent induction of chemokines that drive HSC recruitment in CFLD.

Taurocholate induces biliary differentiation of liver progenitor cells causing hepatic stellate cell chemotaxis in the Ductular Reaction: Role in pediatric cystic fibrosis liver disease.

Schistosomiasis japonica remains a major public health and socioeconomic concern in Southeast Asia. Sensitive and accurate diagnostics can play a pivotal role in achieving disease elimination goals.

Droplet Digital PCR Diagnosis of Human Schistosomiasis: Parasite Cell-Free DNA Detection in Diverse Clinical Samples.

Hepatic fibrosis is a common pathology in various liver diseases. Hepatic stellate cells (HSCs) are the main cell type responsible for collagen deposition and fibrosis formation in the liver. Schistosomiasis is characterised by granulomatous fibrosis around parasite eggs trapped within the liver and other host tissues. This response is facilitated by the recruitment of immune cells and the activation of HSCs. The interactions between HSCs and schistosome eggs are complex and diverse, and a better understanding of these interactions could lead to improved resolution of fibrotic liver disease, including that associated with schistosomiasis. Here, we discuss recent advances in HSC biology and the role of HSCs in hepatic schistosomiasis.

Schistosome-Induced Fibrotic Disease: The Role of Hepatic Stellate Cells.

Cercarial elastase (CE) secreted from cercariae is evinced to play a pivotal role in initial skin penetration of mammalian host. SjCE-2b, a Schistosoma japonicum CE orthologous to SmCE-2b in S. mansoni, was previously found present in cercarial stage to aid skin invasion, but its enzyme activity has not been validated due to the insolubility and altered conformation when expressed recombinantly in bacteria as inclusion bodies. We report here for the first time a bioactive and soluble recombinant SjCE-2b recovered successfully from inclusion bodies by refolding approaches, enabling our biochemical and immunological investigation of this enzyme. Using a "two-step-denaturing and refolding" method, we recovered an 83% yield with 90% purity of refolded protein. Proteolytic activity of rSjCE-2b was demonstrated and characterized by enzymatic assay, showing a K of 0.116 mM and a specific activity of 1900 nmol p-nitroaniline/min/mg protein. A significant immunoprotective response was evidenced in mice immunized with refolded rSjCE-2b. The result of immunoprotection test is at apparent variance with previously reported findings using S. mansoni CE preparation, which was poorly immunogenic in immunized animals. This work extends the knowledge of schistosome cercarial protease, and presents a bioactive form of S. japonicum recombinant CE with high yield and good quality. This will allow further biochemical and biological investigations to explore schistosome CE activity and better understand the molecular mechanisms associated with cercarial skin invasion of the mammalian host.

Enzyme activity of Schistosoma japonicum cercarial elastase SjCE-2b ascertained by in vitro refolded recombinant protein.

Hepatic fibrosis is the central cause of chronic clinical pathology resulting from infection by the blood flukes Schistosoma japonicum or S. mansoni. Much has been elucidated regarding the molecular, cellular and immunological responses that correspond to the formation of the granulomatous response to trapped schistosome eggs. A central feature of this Th2 response is the deposition of collagen around the periphery of the granuloma. To date, traditional histology and transcriptional methods have been used to quantify the deposition of collagen and to monitor the formation of the hepatic granuloma during experimental animal models of schistosomiasis. We have investigated the dynamic nature of granuloma formation through the use of a transgenic mouse model (B6.Collagen 1(A) luciferase mice (B6.Coll 1A-luc)). With this model and whole-animal bioluminescence imaging, we followed the deposition of collagen during an active schistosome infection with Chinese and Philippines geographical strains of S. japonicum and after clearance of the adult parasites by the drug praziquantel. Individual mice were re-imaged over the time course to provide robust real-time quantitation of the development of chronic fibrotic disease. This model provides an improved method to follow the course of hepatic schistosomiasis-induced hepatic pathology and effectively supports the current dogma of the formation of hepatic fibrosis, originally elucidated from static traditional histology. This study demonstrates the first use of the B6.Coll 1A-luc mouse to monitor the dynamics of disease development and the treatment of pathogen-induced infection with the underlying pathology of fibrosis.

Live imaging of collagen deposition during experimental hepatic schistosomiasis and recovery: a view on a dynamic process.

In eukaryotes, effective calcium homeostasis is critical for many key biological processes. There is an added level of complexity in parasites, particularly multicellular helminth worms, which modulate calcium levels while inhabiting the host microenvironment. Parasites ensure efficient calcium homeostasis through gene products, such as the calmodulin-dependent kinases (CaMK), the main focus of this review. The importance of CaMK is becoming increasingly apparent from recent functional studies of helminth and protozoan parasites. Investigations on the molecular regulation of calcium and the role of CaMK are important for both supplementing current drug regimens and finding new antiparasitic compounds. Whereas calcium regulators, including CaMK, are well characterised in mammalian systems, knowledge of their functional properties in parasites is increasing but is still in its infancy.

Calcium and Ca/Calmodulin-dependent kinase II as targets for helminth parasite control.

Schistosomiasis, a neglected tropical parasitic disease caused by the trematode flatworms of the genus Schistosoma, affects approximately 207 million people worldwide. Among the five main species infecting humans, Schistosoma mansoni and S. japonicum are responsible for the majority of hepatointestinal schistosomiasis. Human settlements near fresh water sites that lack proper sanitary systems often contribute to the transmission of disease. This risk particularly impacts on travellers or immigrants who come into contact with larvae-contaminated water. This review discusses the central features of schistosomiasis; including clinical manifestations, diagnosis, treatments, and the preventive measures available for the control of this disease. The description of the Malaysian schistosome species Schistosoma malayensis and the current status of schistosomiasis in Malaysia including the compilation of cases diagnosed from 1904 to 2015 are also discussed in this paper.

Schistosomiasis in Malaysia: A review.

Foodborne infections caused by lung flukes of the genus Paragonimus are a significant and widespread public health problem in tropical areas. Approximately 50 Paragonimus species have been reported to infect animals and humans, but Paragonimus westermani is responsible for the bulk of human disease. Despite their medical and economic importance, no genome sequence for any Paragonimus species is available.

Whole-genome sequence of the oriental lung fluke Paragonimus westermani.

Co-parasitism is a frequent occurrence in impoverished communities in the tropics resulting in a considerable disease burden. While there are extensive reports of intestinal helminthiases, including schistosomiasis japonica, the occurrence and extent of diseases caused by intestinal protozoa (IP) have yet to be investigated in depth in the Philippines. We present a detailed analysis of polyparasitism in a rural community of Northern Samar, focusing on co-infections of IP with Schistosoma japonicum.

Co-parasitism of intestinal protozoa and Schistosoma japonicum in a rural community in the Philippines.

Mesenteric infection by the parasitic blood fluke Schistosoma bovis is a common veterinary problem in Africa and the Middle East and occasionally in the Mediterranean Region. The species also has the ability to form interspecific hybrids with the human parasite S. haematobium with natural hybridisation observed in West Africa, presenting possible zoonotic transmission. Additionally, this exchange of alleles between species may dramatically influence disease dynamics and parasite evolution. We have generated a 374 Mb assembly of the S. bovis genome using Illumina and PacBio-based technologies. Despite infecting different hosts and organs, the genome sequences of S. bovis and S. haematobium appeared strikingly similar with 97% sequence identity. The two species share 98% of protein-coding genes, with an average sequence identity of 97.3% at the amino acid level. Genome comparison identified large continuous parts of the genome (up to several 100 kb) showing almost 100% sequence identity between S. bovis and S. haematobium. It is unlikely that this is a result of genome conservation and provides further evidence of natural interspecific hybridization between S. bovis and S. haematobium. Our results suggest that foreign DNA obtained by interspecific hybridization was maintained in the population through multiple meiosis cycles and that hybrids were sexually reproductive, producing viable offspring. The S. bovis genome assembly forms a highly valuable resource for studying schistosome evolution and exploring genetic regions that are associated with species-specific phenotypic traits.

Whole-genome sequence of the bovine blood fluke Schistosoma bovis supports interspecific hybridization with S. haematobium.

Ever since the first known written report of schistosomiasis in the mid-19th century, researchers have aimed to increase knowledge of the parasites, their hosts, and the mechanisms contributing to infection and disease. This knowledge generation has been paramount for the development of improved intervention strategies. Yet, despite a broad knowledge base of direct risk factors for schistosomiasis, there remains a paucity of information related to more complex, interconnected, and often hidden drivers of transmission that hamper intervention successes and sustainability. Such complex, multidirectional, non-linear, and synergistic interdependencies are best understood by looking at the integrated system as a whole. A research approach able to address this complexity and find previously neglected causal mechanisms for transmission, which include a wide variety of influencing factors, is needed. Systems epidemiology, as a holistic research approach, can integrate knowledge from classical epidemiology, with that of biology, ecology, social sciences, and other disciplines, and link this with informal, tacit knowledge from experts and affected populations. It can help to uncover wider-reaching but difficult-to-identify processes that directly or indirectly influence exposure, infection, transmission, and disease development, as well as how these interrelate and impact one another. Drawing on systems epidemiology to address persisting disease hotspots, failed intervention programmes, and systematically neglected population groups in mass drug administration programmes and research studies, can help overcome barriers in the progress towards schistosomiasis elimination. Generating a comprehensive view of the schistosomiasis system as a whole should thus be a priority research agenda towards the strategic goal of morbidity control and transmission elimination.

A Call for Systems Epidemiology to Tackle the Complexity of Schistosomiasis, Its Control, and Its Elimination.

Polyparasitism, involving soil-transmitted helminths (STH) and Schistosoma blood flukes, is common in low to middle income countries. These helminths impact on the gut environment and can cause changes to the gut microbiome composition. Here we examined the gut microbiome in individuals with polyparasitism from two human cohorts in the Philippines utilising DNA sequencing-based profiling. Multiple helminth species infections were high with 70.3% of study participants harbouring at least two parasite species, and 16% harbouring at least five species. Increased numbers of helminth co-infections, in particular with the gut-resident STH, were significantly associated with increased bacterial diversity; however no significant parasite-gut microbiome associations were evident for individuals infected only with Schistosoma japonicum. In general, a healthy gut is associated with high bacterial diversity, which in these human cohorts may be the result of helminth-mediated immune modulation, or due to changes in the gut environment caused by these parasitic helminths.

Article	Year
Functional characterisation of Schistosoma japonicum acetylcholinesterase. Parasites & vectors\| PubMed ID: 27283196	2016
Taurocholate induces biliary differentiation of liver progenitor cells causing hepatic stellate cell chemotaxis in the Ductular Reaction: Role in pediatric cystic fibrosis liver disease. The American journal of pathology\| PubMed ID: 28935574	2017
Droplet Digital PCR Diagnosis of Human Schistosomiasis: Parasite Cell-Free DNA Detection in Diverse Clinical Samples. The Journal of infectious diseases\| PubMed ID: 29029307	2017
Schistosome-Induced Fibrotic Disease: The Role of Hepatic Stellate Cells. Trends in parasitology\| PubMed ID: 29526403	2018
Enzyme activity of Schistosoma japonicum cercarial elastase SjCE-2b ascertained by in vitro refolded recombinant protein. Acta tropica\| PubMed ID: 30017495	2018
Live imaging of collagen deposition during experimental hepatic schistosomiasis and recovery: a view on a dynamic process. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 30401957	2019
Calcium and Ca/Calmodulin-dependent kinase II as targets for helminth parasite control. Biochemical Society transactions\| PubMed ID: 30420417	2018
Schistosomiasis in Malaysia: A review. Acta tropica\| PubMed ID: 30448471	2019
Whole-genome sequence of the oriental lung fluke Paragonimus westermani. GigaScience\| PubMed ID: 30520948	2019
Co-parasitism of intestinal protozoa and Schistosoma japonicum in a rural community in the Philippines. Infectious diseases of poverty\| PubMed ID: 30526666	2018
Whole-genome sequence of the bovine blood fluke Schistosoma bovis supports interspecific hybridization with S. haematobium. PLoS pathogens\| PubMed ID: 30673782	2019
A Call for Systems Epidemiology to Tackle the Complexity of Schistosomiasis, Its Control, and Its Elimination. Tropical medicine and infectious disease\| PubMed ID: 30699922	2019
Helminths, polyparasitism, and the gut microbiome in the Philippines. International journal for parasitology\| PubMed ID: 32135180	2020

Geoffrey N. Gobert

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