Research Grants

PSC Partners Annual Research Grant Awards

Every year, on the recommendation of the Scientific/Medical Advisory Committee and the Board of Directors, PSC Partners Seeking a Cure chooses the most promising research projects to support. In 2009, PSC Partners Seeking a Cure awarded its first set of research grants, selected on the basis that they supported research addressing an important and novel, basic or clinical question related to PSC. For PSC Partners, this was an important milestone, an important step towards taking us closer to new treatments and an eventual cure. Since the inception of our grants program, we have awarded eight major grants in 2009, five in 2010, three in 2011, three in 2012, seven in 2013, seven in 2014, six in 2015, six in 2016, seven in 2017, nine in 2018, and four in 2019 for a total of $3,434,666 allocated to promising PSC research projects.

Grant awards are made on an annual basis. The deadline for submission of 2021 grant proposals was January 31, 2021. Click here for the research proposal application materials.

We thank all of our donors throughout the years who have supported our mission of raising funds to research causes, treatments and cures for primary sclerosing cholangitis. Your contributions have made these awards possible!

All research proposals are reviewed annually by the PSC Partners’ Scientific/Medical Advisory (SMAC) Committee.

Click here for more information about the grant application.

Yearly Awards

Every year, on the recommendation of the Scientific/Medical Advisory Committee (SMAC), the Board of Directors of PSC Partners Seeking a Cure chooses the most promising research projects to support.

Awarded in 2020

• Defining the Interactome of Bile Duct Inflammation in PSC, Espen Melum, MD, PhD: Norwegian Research Center, Oslo, Norway
• The Beneficial Effects of Endothelial Progenitor Cells in Primary Sclerosing Cholangitis, Fanyin Meng, PhD: Indiana Center for Liver Research, Indianapolis, IN, USA
• Integrative Analysis of Intraductal Papillary Neoplasia of the Bile Duct in Patients with Primary Sclerosing Cholangitis, Stephanie Roessler, PhD &  Benjamin Goeppert, MD: Institute of Pathology, University of Heidelberg, Germany
• Micro-RNA Based Therapy for Primary Sclerosing Cholangitis, Pasquale Piccolo, PhD: Telethon Institute of Genetics and Medicine, Pozzuoli, Italy
• The Role of Activating Transcription Factor 4 in Primary Sclerosing Cholangitis, Xiaoying Liu, PhD: Northwestern University Feinberg School of Medicine, Chicago, IL, USA
• Treating PSC with Idebenone, Joy Xiaosong Jiang: UC Davis, California, USA
• Human Disease Model of PSC for Discovery of Effective PSC Therapy, Yoon-Young Jang, MD, PhD: Johns Hopkins University School of Medicine, Baltimore, MD, USA

Awarded in 2019

Metabolic-based prediction of prognosis in patients with primary sclerosing cholangitis and early diagnosis of cholangiocarcinoma: new non-invasive strategy

Jesus M. Banales, Ph.D., Head, Liver Diseases Group, Biodonostia Health Research Institute, San Sebastian, Spain

Amount Awarded: $60,000 over two years

Primary sclerosing cholangitis (PSC) is a chronic cholestatic biliary disease of unknown etiology, which increases the risk of developing cholangiocarcinoma (CCA) by approximately 15%. The late-diagnosis, aggressiveness, and lack of efficient therapies markedly contribute to the high mortality of this cancer. Liver transplantation remains the only potential curative option for patients with primary sclerosing cholangitis (PSC), but approximately 25% of these patients develop disease recurrence. Currently, there are no available accurate non-invasive biomarkers for PSC and CCA. In this project, we will analyze the serum metabolomic profile of patients with PSC and investigate new biomarkers to predict prognosis (risk of CCA development and disease recurrence after liver transplantation) and to earlier diagnose CCA. This is warranted to monitor disease progression and to guide therapeutic strategies.

To do this, we will study 5 distinct groups of patients:

Isolated PSC (no CCA development overtime)PSC patients before CCA development (CCA development in the follow up)PSC patients with concomitant CCA (PSC-CCA)PSC patients who underwent liver transplantation (LTX) without disease recurrencePSC patients who underwent liver transplantation with disease recurrence

This International Collaborative Study involves reference centers from the International Primary Sclerosing Cholangitis Study Group (IPSCSG, www.ipscsg.org) and TheEuropean Network for the Study of Cholangiocarcinoma (ENSCCA,www.enscca.org). These organizations represent 2 pivotal collaborative networks that are actively working for the greater good of PSC patients. In this study, more than 150 serum samples will be used.  The hope is that these samples will lead to the discovery of new diagnostic and prognostic biomarkers for PSC and CCA, and that these biomarkers can be translated into clinical practice in the near future.

Single-cell RNA sequencing of lymphocyte subsets and cholangiocytes in non-endstage PSC patients

Prof. Dr. C.Y. Ponsioen, Department of Gastroenterology & Hepatology, Amsterdam University Medical Centers, Academic Medical Center

Amount Awarded: $60,000 over two years

The cause of primary sclerosing cholangitis is unknown. This is due to several factors: 1. the rarity of the disease and 2. the fact that the diseased tissue is hidden deep in the body. Notwithstanding, in order to advance our knowledge why there is an immune mediated process going on at the interface between the lining of the bile duct and attracted white blood cells it is imperative that we study those cells directly in their microenvironment.

The newest technology to study in detail what is happening in these cells is by determining what the cells are producing. In other words: which parts of their DNA are being active in encoding new proteins. This technique is called RNA sequencing, and the science emerging from this type of analyses is called transcriptomics. The latest development in this technology is single-cell RNA sequencing. With this technique very low amounts of tissue can be dissected in their respective cell types and the gene expression that is present in the cells at the time they are caught determined. This is a very powerful technology to characterize cell types and determine how they are functioning and interacting with each other.

However, to collect bile duct cells and inflammatory cells from the larger bile ducts in early stage PSC in order to find out what went wrong that initiated a chronic inflammation is not easy. Normally tissue for research is taken from livers at the time they are explanted during a liver transplantation. These are usually end-stage disease livers with a lot of inflammation and cirrhosis, that likely will not reveal what went wrong some 20 years ago. The only way to capture inflamed bile duct tissue of early stage PSC patients is when a patient needs to undergo an ERCP. This may be for increased complaints and liver tests, or sometimes for a suspicion of malignancy. In this situation tiny biopsies with a biliary biopsy forceps or even smaller biopsies with a very fine biopsy forceps that can be advanced through a cholangioscopy system to offer the possibility to sample bile duct tissue. A cholangisocopy is called the last frontier of endoscopy. The newest systems consist of a 3.5 mm catheter with a fiberoptic channel and a <2 mm working channel. With this system the bile ducts can be intubated and relevant tissue biopsied.

At the Department of Gastroenterology & Hepatology of the Amsterdam University Medical Centers, Amsterdam, The Netherlands, a study is performed investigating the usefulness of this cholangioscopy system together with taking very small biopsies for the differentiation between benign and malignant narrowings of the bile duct in PSC. This offers the possibility of collecting relevant material for the cutting-edge technique of single-cell RNA sequencing, which is performed at the Department of Gastroenterology & Hepatology and the Department of Genetics of the University Medical Center Groningen, University of Groningen.

Genome Wide Association Study (GWAS) in Biliary Tract Cancer (BTC)

Lewis R. Roberts, MB ChB, PhD, Professor of Medicine and Consultant, Director, Hepatobiliary Neoplasia Clinic, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester MN

Amount Awarded: $60,000 over two years

Biliary tract cancers (BTCs), which include cholangiocarcinoma (CCA) and gallbladder cancer (GBC), are highly lethal cancers often diagnosed at an advanced stage with a poor prognosis. Researchers have determined that individuals with primary sclerosing cholangitis (PSC) are at a much greater risk of developing BTC than individuals without PSC, but it is not known why some people with PSC develop biliary tract cancer while many do not.  It has been proposed that those PSC patients who do in fact develop CCA are genetically predisposed. Through a Genome Wide Association Study (GWAS), we intend to verify the genetic differences that make some people more susceptible to developing CCA. We hypothesize that unique genetic factors can predict CCA risk in non-PSC cases, whereas other markers may predict CCA development in patients with PSC, and clinical outcomes in CCA. We expect that our findings will open new avenues of research into the molecular pathways involved in both non-PSC and PSC-related CCA. Furthermore, results from such a study will allow researchers to develop better strategies for prevention, screening, diagnosis and treatment of these fatal diseases. The funding provided by PSC Partners will be used specifically to dedicate personnel time to enhancing our current sample accrual, specifically enriching our validation cohort with a larger number of CCA-PSC cases.

Pathways of Injury and Repair of the Extrahepatic Biliary Tree in PSC

Orith Waisbourd-Zinman, MD, Schneider Children’s Medical Center of Israel, Felsenstein Research Medical Center, Tel-Aviv University, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel

Amount Awarded: $60,000 over two years

PSC remains a poorly understood bile duct disease without an effective therapy. There is limited data in the literature directed towards the damage of the extra hepatic biliary tree in PSC. In this project, our goal is to determine the mechanisms of extrahepatic bile duct injury. We propose to investigate the role of glutathione (GSH) a compound in the cell that has an antioxidant effect and is influenced by oxidative stress, in the mechanism of bile duct injury and repair. We are using both mouse models of PSC, as well as human bile ducts from patients undergoing liver transplantation whose bile ducts are being removed as part of the surgery. We have preliminary data suggesting that decrease in GSH can cause cholangiocyte damage, while an increase in GSH can improve and potentially reverse duct damage. We will also study the molecular pathway of injury downstream to GSH. Understanding the full pathway will enable various treatment approaches for repair, or prevention of progression.

Awarded in 2018

PSC Partners Seeking a Cure is incredibly excited to announce that it will award eight grants in 2018, and PSC Partners Seeking a Cure Canada will award one grant, for a total of 9 new research projects! For the first time in 2018, PSC Partners Seeking a Cure, along with our Canadian affiliate, are delighted to award two Young Investigator Awards to promising researchers who are at the early stages of their research career and are interested in clinical, translational or basic research on Primary Sclerosing Cholangitis. Please keep reading for a list of the titles, lead investigators, and lay summaries of all projects that will receive funding this year.

TREATMENT OF PRIMARY SCLEROSING CHOLANGITIS USING EXTRACELLULAR VESICLES

Gianfranco Alpini, PhD,Professor of Medicine, VA Senior Research Scientist, Hickam Endowed Chair, Director, Indiana Center for Liver Research, Richard L. Roudebush VA Medical Center and Indiana University, Gastroenterology, Medicine

Amount Awarded:  $60,000 over two years.

Primary sclerosing cholangitis (PSC) is characterized by expanded bile ducts and liver fibrosis.

Extracellular vesicles are membrane-surrounded particles secreted from cells. These vesicles play an important role in liver diseases because they are transferred from donor cells and regulate cell events in recipient cells. These vesicles carry cargo mediators, such as proteins and RNAs, and they can be used to control cell conditions. Our preliminary study has demonstrated that liver conditions in PSC could be improved or managed by these vesicles.

In this proposed study, we hypothesize that extracellular vesicles can be used as a novel therapeutic tool to regulate PSC by delivering cargo mediators. This study will establish novel methodology for the management of PSC using extracellular vesicles. Treatments using these vesicles will be less invasive with no or limited side effects. The technique developed in this study could be used for treatments of other disorders.

2019 Update:

Primary sclerosing cholangitis (PSC) is characterized by expanded bile ducts and liver fibrosis.  Extracellular vesicles are membrane-surrounded particles secreted from cells. Accumulating evidence suggests that these vesicles play an important role in liver diseases because they are transferred from donor cells and regulate cell events in recipient cells. In the previous study, we have demonstrated that bile duct epithelial cells (i.e., cholangiocytes) communicate each other via extracellular vesicles at disease conditions. These vesicles carry cargo mediators, such as proteins and RNAs, and they can be transferred into other cholangiocytes to regulate gene expression and cell events. Our preliminary data also suggest that extracellular vesicles isolated from modified cholangiocytes improve liver conditions in a mouse model of PSC.  We isolated extracellular vesicles from cholangiocytes and injected into mice of PSC model. We found that injected vesicles were taken up by cholangiocytes and other liver cells, and these vesicles regulate activation and fibrogenesis of cholangiocytes showing improved liver conditions of PSC mouse. Current results suggest that those isolated vesicles have therapeutic effects against PSC. We will continue our study to develop novel therapies for PSC using extracellular vesicles.

Background: Primary sclerosing cholangitis (PSC) is characterized by robust cholangiocyte proliferation and liver fibrosis. Previous studies have demonstrated that the Sct/SR axis is an important signaling pathway for cholangiocyte proliferation and functions. Knockout of SR attenuates PSC-induced bile duct hyperplasia as well as liver fibrosis in Mdr2-/- mice, which are a mouse model of PSC. These results indicate that the Sct/SR axis can be a therapeutic target of treatments of PSC. Extracellular vesicles (EVs) are membrane-bound vesicles secreted from various types of cells. EVs play a key role in liver pathophysiology because EVs regulate cell events and functions by delivering mediators carried inside as cargo including microRNAs (miRNAs). In our previous study, we have demonstrated that EVs secreted from cholangiocytes with bacterial infection drive inflammatory responses in other cholangiocytes at normal conditions. In addition, EVs isolated from cholangiocytes lacking Sct or SR did not induce inflammatory responses in other cholangiocytes. These results raised a hypothesis that EVs secreted from cholangiocytes lacking the Sct/SR axis may have therapeutic effects on other cholangiocytes at disease conditions. Our preliminary data have demonstrated that injection of those EVs decreases bile duct hyperplasia and liver fibrosis in Mdr2-/- mice, showing promising therapeutic effects against PSC.

AN INVESTIGATION OF BILIARY-IMMUNE INTERACTIONS AND CANDIDATE DRUG-SCREENING USING NOVEL BILE-DERIVED ORGANOID TECHNOLOGY

David N. Assis, MD, Assistant Professor of Medicine, Section of Digestive Diseases, Yale University School of Medicine

Amount Awarded:  $60,000 over two years.

Primary Sclerosing Cholangitis (PSC) is a rare disease with numerous challenges. It is hard to study PSC due to a lack of adequate human samples for research, especially since bile duct cells do not grow well once removed from the body. An additional challenge is to understand the immunologic imbalance in this autoimmune disease and to generate effective treatments to target the immune system. Our project will use organoids, which are collections of stem cells grown in the lab that resemble bile duct cells, to study PSC and its immune basis. We will develop organoids from human bile, which is a new method that allows us to easily take a small amount of bile when a patient undergoes an ERCP procedure as part of their clinical care. We will grow the organoids from patients with PSC and see how they interact with white cells (T-lymphocytes) obtained from the blood of patients with PSC seen at our clinics. We will also perform screening of potential drugs to treat PSC by exposing organoids to these compounds and observing if they can reduce inflammation. Lastly, we will relate individual organoid findings with the clinical characteristics of the patient who donated the bile. Through this project we hope to better understand, in a personalized way, the nature of the immune imbalance in PSC and search for effective drugs using organoids grown from bile.

2019 Update:

In the first year of this project we have made significant progress on developing bile-derived organoids (BDOs) as a new approach to study PSC. These BDOs are derived from bile collected from PSC patients at the time of ERCP. They can be stored and later grown and tested over time. BDOs react to stimulation with inflammatory molecules that are important in PSC, such as IL-17, and secrete inflammatory molecules such as CCL20 that can recruit immune cells such as T-cells. Our results over the past year show that BDOs from PSC patients can directly interact with and activate T-cells and that T-cells. T-cells, in turn, can make BDOs significantly more inflamed by increasing their secretion of inflammatory marker CCL20 and by increasing their expression of CCL20 and IL-8 genes. In the next year we will move forward with these findings to test candidate inhibitor drugs for their ability to prevent BDOs and T-cells from pathologically interacting with each other. Through this project we will develop BDOs as a novel method to study PSC, and as a vehicle to test new treatments on BDOs from individual patients in a personalized medicine approach.

PSCATOR – ATORVASTATIN FOR THE TREATMENT OF PRIMARY SCLEROSING CHOLANGITIS (PSC) – A RANDOMIZED CONTROLLED STUDY

Annika Bergquist, MD, Karolinska University Hospital, Division of Hepatology, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden

Amount Awarded:  $60,000 over two years.

Primary sclerosing cholangitis (PSC) is a chronic progressive liver disease with a high risk of death, need of liver transplantation (LTx) or development of cholangiocarcinoma (CCA) within 15-20 years after diagnosis. There is no medical treatment that can halt PSC progression. Treatment with statins has shown to be beneficial in other chronic liver diseases than PSC. Therefore, we tested the hypothesis that statins improve prognosis in PSC. In a population-based Swedish register study, including nearly 3000 PSC patients, we found that the risk for death or LTx in statin-users with PSC, was markedly reduced, hazard ratio, HR 0.50 (95%CI 0.28-0.66). Since there is no pharmacological treatment available in PSC, a study with statins seems highly motivated. We want to perform a long-term randomized double-blind controlled trial of atorvastatin versus placebo in patients with PSC. The primary outcome measures are CCA, LTx, variceal bleeding or death. To reach adequate power 700 patients will be included and the study period is 5 years. We currently have an ongoing, observational study in PSC that evaluate surveillance strategy with imaging in PSC. Through this observational study performed by the network for liver studies in Sweden (SweHep), we have an infrastructure for PSC studies already in place.

2019 Update:

We tested the hypothesis that statins improve prognosis in PSC. In a population-based Swedish register study, including nearly 3000 PSC patients, we found that the risk for death or LTx in statin-users with PSC, was markedly reduced, hazard ratio, HR 0.50 (95%CI 0.28-0.66). Since there is no pharmacological treatment available in PSC, a study with statins seems highly motivated.

We plan a long-term randomized double-blind controlled trial of simvastatin versus placebo in patients with PSC. The primary outcome measures are CCA, LTx, variceal bleeding or death. To reach adequate power 700 patients will be included and the study period is 5 years. We have made preparations for this study during the last year. We have received ethical approval and in preparation. We have a fruitful dialogue with the Swedish patient’s organization (PSCSverige) and they have helped us to make the written information of this study as good as possible.

Due to extensive costs for production of active drug and placebo we have decided to change our initially planned use of statin from atorvastatin to simvastatin and the study name has changed accordingly to PiSCATIN. An application to the Swedish Medical Agency is pending and we have contracted a company for the drug/placebo production. The first patient will be included in May 2020.

NOVEL USE OF MICRORNAs TO ATTENUATE PSC INDUCED LIVER FIBROSIS

Michael Choi, MD, Assistant Professor of Medicine, Harvard Medical School, Affiliate Faculty Member, Harvard Stem Cell Institute, Boston, MA

Amount Awarded:  $60,000 over two years.

Primary sclerosing cholangitis (PSC) is an insidious liver disease that can lead to cirrhosis and death through progressive scarring of the liver. Hepatic stellate cells (HSCs) play a central role in driving this scar formation once they become activated, but our research has discovered a way to force activated HSCs back toward the quiescent state using a special type of genes called microRNA. When we used specific microRNAs to revert activated HSCs back toward quiescence in mice afflicted with liver scarring, their liver inflammation and scarring improved. The proposed research expands on our previous work by attempting to use these microRNAs and their associated target gene to treat mouse models of PSC that lead to liver scarring. This technology is applicable to human disease and has the potential to become novel therapies since HSCs also drive progressive liver scarring in human PSC.

2019 Update:

Our initial experimental finding identified two microRNAs that can revert activated hepatic stellate cells (HSCs) back toward quiescence. Since HSCs play a central role in promoting liver fibrosis once they become activated, forcing their reversion back toward quiescence may be a way to derive novel therapies for progressively fibrotic liver disease such as primary sclerosing cholangitis (PSC). Our new data further demonstrate the function of these microRNAs in generating quiescent-like HSCs. More exciting result came from successfully treating an animal model of PSC using these quiescent-like HSCs. When mice challenged with biliary obstruction that simulate PSC received these quiescent-like HSCs, the liver injury and scarring caused by biliary obstruction were attenuated. Furthermore, the gene targeted by a candidate microRNA displayed an appropriate function of inducing HSC activation. This result is in line with our hypothesis that our candidate microRNAs achieve their function by repressing target genes that promote HSC activation, hence, preventing activation. We plan to further define the function and treatment potential of our candidate microRNAs and one of its gene target.

CHARACTERIZATION OF CLINICAL AND IMMUNOLOGIC PHENOTYPES OF PATIENTS WITH RECURRENT PSC AFTER LIVER TRANSPLANTATION: AN INTERNATIONAL NETWORK STUDY    

Aliya Gulamhusein, MD, MPH, FRCPC, Assistant Professor, University of Toronto, Clinical Investigator, Toronto General Hospital, Toronto Centre for Liver Disease, Toronto, Ontario, Canada

Amount Awarded:  $80,000 over two years.

Primary sclerosing cholangitis, PSC, is a rare, chronic, and progressive cholestatic liver disease that can be complicated by biliary infection, hepatobiliary malignancy, and end stage liver disease. There is no known effective therapy and while the natural history of PSC is variable, in general, patients tend towards progressive liver disease and transplantation. Recurrent PSC (rPSC) is a distressing complication that occurs in some PSC patients post liver transplantation and is associated with a four-fold increased risk of graft failure or death.

This research is directed at developing an improved understanding of clinical and immunopathogenic pathways underpinning recurrent PSC. We aim to comprehensively characterize (phenotype) a prospective cohort of patients with PSC post liver transplant and use newly emergent mass cytometry technology to understand clinical, serologic, microbiological, and immunologic changes associated with development of rPSC. In addition, we will establish an international network among liver transplant centers worldwide to characterize clinical risk factors associated with rPSC.

Results of this research will improve understanding of disease pathogenesis, and may also illuminate “immune signatures” associated with outcomes and thus has potential to identify targets for therapy. This disease is devastating to patients and their families, is costly to the health care system, and is a critical unmet need. Identifying clear risk factors for rPSC requires a systematic and comprehensive phenotypic evaluation of patients to identify those in greatest need of targeted early intervention.

2019 Update:

The goal of this project is to understand biologic and clinical variables associated with recurrent Primary Sclerosing Cholangitis (rPSC) post liver transplantation. To understand the biological basis for rPSC, patients managed at the Toronto Centre for Liver Disease  will be recruited prior to liver transplant and biologic samples including blood and liver cells will be collected pre and post-transplant to identify changes that occur within  the same patient over time that may lead to recurrent PSC. To achieve this goal, a well-defined team of research assistants, fellows and clinicians has been recruited and a dedicated clinic managing patients in the peri-transplant (pre and after 6 months post-transplant) setting has been operationalized to facilitate recruitment of patients which is expected to be complete over the next calendar year based on clinic volumes. To understand clinical variables associated with recurrent PSC post liver transplant an international network of 26 liver transplant centers from 14 countries has been developed to collect retrospective data from more than 1500 PSC patients that have had a liver transplant since 2000. A standard data collection form has been developed for widespread use and local data collection begun. A dedicated working group has been developed that will meet bi-annually at The Liver Meeting and the International Liver Congress.  Ultimately, this will be the largest retrospective dataset on clinical variables associated with recurrent PSC post transplantation that has ever been developed.

VITAMIN D, NUCLEAR RECEPTOR-INDEPENDENT ACTIONS IN PRIMARY SCLEROSING CHOLANGITIS: POTENTIAL ROLE OF PROTEIN DISULFIDE ISOMERASE FAMILY A MEMBER 3 (PDIA3) AS A THERAPEUTIC TARGET

Chantal Housset, MD, PhD, Sorbonne Université & INSERM, Saint-Antoine Research Center, Paris, France.

Amount Awarded:  60,000 over two years.

Primary sclerosing cholangitis (PSC) is a chronic liver disease of unknown cause and pathogenesis with an unmet medical need. Thus, the development of novel therapies is eagerly awaited. Vitamin D exerts anti-inflammatory and anti-fibrotic effects by the activation of the vitamin D nuclear receptor (VDR). To evaluate the implication of VDR in PSC, we have invalidated Vdr expression in Abcb4 knockout mice (lacking a protein named ABCB4), a widely utilized model of PSC. The lack of Vdr aggravated PSC features in Abcb4 knockout mice. Unexpectedly, the treatment with a vitamin D analog, or vitamin D supplementation in the diet alleviated PSC features in these mice suggesting the existence of a Vdr-independent mechanism activated by vitamin D. A protein called disulfide isomerase family A member 3 (PDIA3) that mediates some rapid actions of Vitamin D could orchestrate these effects. Our aim in this project is to test the potential of PDIA3 as a new therapeutic target in PSC. The role of PDIA3 will be studied in the primary targets of injury in PSC, i.e. cholangiocytes and hepatic stellate cells, of human origin or obtained from mice lacking Abcb4 or Abcb4 and Vdr. The potential beneficial effect of a PDIA3 activator on PSC features will also be tested in mice lacking Abcb4 or Abcb4 and Vdr. This project will allow us to better define Vitamin D signaling in PSC and determine the potential of PDIA3 as a therapeutic target in this disease.

2019 Update:

Primary sclerosing cholangitis (PSC) is a chronic liver disease of unknown cause and pathogenesis with an unmet medical need. Thus, the development of novel therapies is eagerly awaited. Vitamin D exerts anti-inflammatory and anti-fibrotic effects by the activation of the vitamin D nuclear receptor (VDR). To evaluate the implication of VDR in PSC, we have invalidated Vdr expression in Abcb4 knockout mice (lacking a protein named ABCB4), a widely utilized model of PSC. Lack of Vdr aggravated PSC features in Abcb4 knockout mice. Unexpectedly, treatment with calcipotriol, a vitamin D analog, or vitamin D supplementation in the diet alleviated PSC features in these mice suggesting the existence of a Vdr-independent mechanism activated by vitamin D. A protein called disulfide isomerase family A member 3 (PDIA3) that mediates some Vitamin D rapid actions could orchestrate these effects. The studies conducted during this year demonstrate that in human cholangiocytes inflammatory response induced by TNFa is reduced by calcipotriol. The protective effect of calcipotriol is maintained in VDR-silenced cells, but is not in cells additionally invalidated PDIA3, indicating that part of calcipotriol anti-inflammatory effects are mediated by this receptor. These results suggest that modulation of PDIA3 signaling may present a potential interest in the treatment of PSC.

DISSECTING THE ROLE OF T REGULATORY CELL POPULATIONS IN PRIMARY SCLEROSING CHOLANGITIS (PSC) WITH A MOUSE PSC MODEL 

Wei-Yu Lu, Birmingham Fellow, Centre for Liver Research, the University of Birmingham, Birmingham, UK.

Amount Awarded:  $80,000 over two years.

Primary Sclerosing Cholangitis (PSC) is a disease affecting the bile ducts, causing obstructive flow of bile into the intestines. PSC is characterized by increased inflammation, scarring and reduced bile duct regeneration. The pathogenesis of PSC is poorly understood but has been suggested to be immune mediated. T regulatory cells (Treg), a cell type specialized in controlling immune response play an important role in the pathogenesis of autoimmune liver disease such as PSC. However, there are multiple Treg populations and it is difficult to differentiate them due to their similarities. With the support of the PSC Partners Seeking a Cure, we plan to use a ‘’traffic light’’ system with transgenic mice to investigate the dynamics and functions of different Treg populations during PSC. This will enable us to mark different Treg populations in order to understand the role of Treg in PSC disease progression and identify targets for potential treatments. We hope this study in mice will complement what is currently lacking in human PSC research.

2019 Update:

We have successfully generated a new model to monitor different population of T regulatory cells. This model will be the flagship model for this study to investigate the fate of T regulatory cells during PSC disease progression. Furthermore, we have identified bile ducts communicate with immune cells through the expression of Ox40L, a protein which helps to activate immune cells during injury. In our preliminary finding, the lack of this mode of communication between bile ducts and immune cells slows down disease progression. The identification of this link of communication reveals potential target for intervention with targeting antibodies.

UPREGULATION OF BILIARY EPITHELIA INNATE REPONSES IN PSC

Mario Strazzabosco, MD, PhD, Department of Internal Medicine, Section of Digestive Diseases, Yale University, New Haven, CT, USA

Amount Awarded:  $30,000 for one year.

The pathogenesis of PSC is still unknown. The association of PSC with inflammatory bowel disease (IBD) and the enhanced response of the biliary epithelium to gut-derived endotoxins suggest that aberrant innate immune pathways in PSC cholangiocytes play a primary role in the progression of the disease. The lack of a satisfactory animal or cellular model orthologous to human PSC is one of the major gaps in the PSC research field and so far a limitation for the pipeline development of new therapies. Our lab has been focusing on the generation of patient-derived biliary cell models to model in vitro PSC disease. With the previous support of PSC Partners, we have generated a platform that combines induced pluripotent stem cell (iPSC) derived cholangiocytes, liver organoids and primary cholangiocytes obtained from liver tissue of PSC patients. In this project we will perform a transcriptomic analysis of the different models to optimize the experimental platform models and discover changes that are common among the different cell models. Furthermore, we will investigate the role played by activation of aberrant innate immune pathways in the different models of PSC cholangiocytes. Our study will generate important clues to understand the pathogenesis of PSC and specifically will provide a cell-based platform for drug development and testing.

2019 Update:

The pathogenesis of PSC is still unknown. The association of PSC with inflammatory bowel disease (IBD) and the enhanced response of the biliary epithelium to gut-derived endotoxins suggest that aberrant innate immune pathways in PSC cholangiocytes play a primary role in the progression of the disease. The lack of a satisfactory animal or cellular model representative human PSC is one of the major gaps in the PSC research field and so far a limitation for the pipeline development of new therapies. Our lab has been focusing on the generation of patient-derived biliary cell models to model in vitro PSC disease and other cholangiopathies. With the support of PSC Partners, we have generated a platform that combines induced pluripotent stem cell (iPSC) derived cholangiocytes, liver organoids and primary cholangiocytes obtained from liver tissue of PSC patients. We have investigated the role played by activation of aberrant innate immune pathways in the different models of cholangiocytes and shown that human PSC cholangiocytes express higher levels of inflammatory cytokines involved in the recruitment of immune cells and in the cross-talk with mesenchymal cell. This aberrant response can be further enhanced by exposure to specific bacterial products (i.e flagellin).  In addition, we have devised new systems for the 3D culture in-vitro of human biliary organoids and their co-culture with mesenchymal and immune cells. Our study, albeit conducted in a very limited number of cases, has generated an important experimental approach and methodology to better study the function of biliary cells in primary sclerosing cholangitis and facilitate drug development.

ESTABLISHING PSC ENDPOINTS FOR CLINICAL TRIALS 

Veronica Miller, UC Berkeley, School of Public Health, Berkeley, CA, USAThe Forum For Collaborative Research

Amount Awarded:  $60,000 over two years.

The Forum for Collaborative Research convenes the PSC Forum to address hurdles in drug development with the goal of advancing medical treatment for PSC.  One key complication for PSC clinical trials is determining study endpoints, which are predictive of health outcomes and can be measured over a reasonable period of time.  To meet this challenge, the PSC Forum will convene a working group to research PSC endpoints, such as fluctuations in alkaline phosphatase (ALP), and provide recommendations which can be used to advance the field.

2019 Update:TBA

Awarded in 2017

PSC Partners Seeking a Cure awarded six grants in 2017, and PSC Partners Seeking a Cure Canada awarded one grant, for a total of 7 new research projects.

A Multicenter Collaboration to Study PSC in Children

Mark Deneau, MD, MS, Assistant Professor of Pediatrics, Division of Pediatric Gastroenterology, Hepatology and Nutrition, University of Utah, Primary Children’s Hospital, Salt Lake City, UT

Amount Awarded: $60,000 over two years.

Researching PSC in children is a challenge. The disease affects enough children that it is a regular problem encountered in liver and inflammatory bowel disease clinics, but is rare enough that no single hospital or physician can gather enough patients to become experts in managing the disease. We all see 5 or 6 patients with PSC, but we need to see hundreds to become true experts, something no single center can do. We must work together to learn how PSC behaves in children, and how to treat it.

2018 Update:

Our team of pediatric centers has expanded, allowing us to get more data and build a better study of IBD and PSC, and of liver transplant in PSC.  We are now collaborating with John’s Hopkins, UCSD, Lurie Children’s, Indiana University, Seattle Children’s, USC, totaling 44 centers. To date, our secure online data repository is up and running and several hundred patient charts have been reviewed and entered.  We are on our way to accomplishing our research goals in the coming year!

2019 Update:

Funding from PSC Partners Seeking A Cure was crucial to the Pediatric PSC Consortium to enroll more patients and centers. We ultimately added 500 patients to our research database, and a wealth of data that will be used to find out more about PSC in children for years to come. For now, we used the data to explore inflammatory bowel disease in PSC, and liver transplant in PSC.

One unexplored area of primary sclerosing cholangitis in children is what link exists between the severity of colonic inflammation in inflammatory bowel disease, and the liver inflammation in PSC, when the two diseases occur together. One potential drug therapy for PSC and IBD is vedolizumab, an infusion that prevents the immune system from migrating into the colon and liver. We evaluated use of vedolizumab in an international group of children and unfortunately found that it does not appear to alter the course of liver inflammation. Similar to adult data on this topic, vedolizumab does not impart a specific benefit on PSC in children with PSC and IBD. The drug has a role in IBD and is effective for colonic inflammation in some patients, but should not be chosen as a first biologic simply because of a perceived possible benefit in the liver. We are now working on further characterizing the inflammation of PSC and IBD to identify which IBD patients will progress to bad liver outcomes, the best anti-inflammatory drugs of IBD in PSC-IBD, and which biochemical markers track the disease progression in this important group of patients.

We also investigated recurrence of PSC after liver transplantation. There are no large studies of children who undergo liver transplant for PSC. We wanted to define how often PSC comes back in the new liver, and what if anything can be done to prevent it. Multiple possible risk factors around the time of liver transplant surgery were evaluated regarding the choice of surgical technique to connect the liver to the bowel, choice of organ donor, and initial choice of immune suppressing drugs after transplant. None were associated with a higher or lower risk of PSC affecting a patient’s transplanted liver. While this is bad news in that we did not find a specific way to prevent recurrent PSC, it is good news in that choice of liver transplant surgery technique and organ donor for a PSC patient can be the same as for any other patient. We did find that patients who get recurrent PSC in a liver transplant seem to be different from PSC patients who never get recurrence. They have a more aggressive immune system, they are diagnosed with PSC earlier in life, they progress from diagnosis to transplant more quickly, and they have more episodes of transplant rejection. With further research this may lead to identification of patients at high risk of recurrence of PSC, who can then be watched more closely.

Identification of Concrete Gene Targets for Therapeutic Intervention by Characterizing Expressional Changes in Primary Sclerosing Cholangitis and Ulcerative Colitis

David Ellinghaus, PhD, Institute of Clinical Molecular Biology (IKMB), Kiel University, Germany

Amount Awarded: $60,000 over two years.

Genome-wide association studies (GWAS) have identified 23 genome-wide significant risk loci for primary sclerosing cholangitis (PSC); however, the PSC-associated genetic variants explain only 5.5-7.3% of variance in PSC heritability. Systematic transcriptome-wide sequencing studies are needed in addition to genetic studies to identify genes and pathways whose gene expression levels are associated with PSC risk. The key goal of this project is to decipher the mechanisms of action of disease-predisposing GWAS loci of the gut-liver axis to identify concrete gene targets for future experimental studies, for example for therapeutic reprogramming of gene expression. To this end we will complement our RNA-Sequencing data for ulcerative colitis (UC) patients and healthy individuals by total RNA-Sequencing of whole-blood samples from PSC patients. Significant gut-liver axis expression-disease associations will then be determined through cross-disease expression analyses. We aim to understand the molecular master switches which lead to rewiring of gene regulatory networks in PSC and UC.

2018 Update:

Genome-wide association studies (GWAS) have identified 23 genome-wide significant risk loci for primary sclerosing cholangitis (PSC), however, the PSC-associated genetic variants explain only 5.5-7.3% of variance in PSC heritability. We aim to decipher the mechanisms of action of disease-predisposing loci of the gut-liver axis to identify concrete gene targets for therapeutic intervention. For this purpose, we have generated full transcriptomes from RNA sequencing (RNA-seq) experiments of whole-blood samples of 225 PSC patients for combination with GWAS SNP array data available from international disease consortia.

With second year’s funding, we aim to complete our RNA-seq experiments data by RNA sequencing of whole-blood samples of additional 225 PSC patients, thus validating our initial findings from first year’s RNA-seq experiments and increasing the power for combined statistical analysis with transcriptome data of whole-blood samples from 500 ulcerative colitis (UC) patients. The overall scientific goal is the construction of a cross-disease PSC-UC interaction map by derivation of a common regulatory core and prediction of disease gene signatures by utilizing RNA-seq data sets from PSC and UC patients.

2019 Update:

With second year’s funding, we completed our RNA-seq experiments data by RNA-sequencing of whole-blood samples of additional 225 PSC patients, thus validating our initial findings from first year’s RNA-seq experiments and increasing the power for combined statistical analysis with transcriptome data of whole-blood samples from 500 ulcerative colitis (UC) patients. In our combined analyses utilizing RNA-seq data sets and performing transcriptome-wide association studies (TWAS) by means of in silico gene expression imputation analysis we revealed 295 significant genes for UC and 165 significant genes for PSC before cross-tissue conditional analysis. Cross-tissue conditional analysis followed by data driven mathematical-model-based network analyses will deliver a PSC-UC interaction map of genetic, expression and regulatory changes associated with PSC and UC.

The Role of Hepatic XBP1 in Cholestatic Liver Injury

Richard M. Green, MD,Professor of Medicine, Division of Gastroenterology and Hepatology, Northwestern University Feinberg School of Medicine, Chicago, IL

Amount Awarded: $60,000 over two years.

Primary Sclerosing Cholangitis (PSC) is a disease of the bile ducts that can progress to fibrosis (scarring), cirrhosis, end-stage liver disease and liver cancer. Although the initial damage in PSC typically occurs in the large bile ducts, disease progression is characterized by injury to the hepatocytes (primary liver cells) and other cells in the liver. PSC is a form of cholestatic liver disease. Cholestasis is a term used to describe the impairment of bile secretion, and can damage the liver due to bile acid toxicity. Bile acids are made by the liver and secreted into bile, but they can injure the liver when secretion is impaired and bile acid levels increase. A protein termed X-box binding protein-1 (XBP1) is a protective liver protein that may be impaired in many liver diseases. However, the role of XBP1 in PSC and other cholestatic liver diseases is essentially unexplored. We have developed mice that lack XBP1 in hepatocytes and we will utilize these mice to determine the effect of XBP1 on cholestatic liver injury. Mice will undergo a bile duct ligation (obstruction of the bile duct), which is a widely utilized model of cholestatic diseases such as Primary Sclerosing Cholangitis. We will determine whether diminished XBP1 in liver cells causes the rapid development of liver injury and cirrhosis. In addition, the MDR2-knockout mouse (mice lacking a protein termed MDR2) is a frequently utilized murine model of PSC. We will develop MDR2-knockout mice with diminished XBP1 in the liver. These mice will be studied to determine if they are more susceptible to develop progressive liver disease, liver malignancies and bile duct cancers (cholangiocarcinoma). By determining the role of hepatic XBP1 in Primary Sclerosing Cholangitis, we can develop novel pharmacologic and other therapies targeting XBP1 for the treatment of patients with PSC.

2018 Update:

Primary Sclerosing Cholangitis (PSC) is a disease of the bile ducts that can progress to fibrosis (scarring), cirrhosis, end-stage liver disease and liver cancer. Although the initial damage in PSC typically occurs in the large bile ducts, the disease may progress due to injury throughout the liver. PSC is a form of cholestatic liver disease, a term used to describe liver diseases that have impaired bile secretion from the liver into the intestine. When this occurs, bile acids (naturally occurring substances produced by the liver) can markedly increase and cause progressive liver disease.  A protein termed X-box binding protein-1 (XBP1) is a protective liver protein that may be impaired in many liver diseases. However, the role of XBP1 in PSC and other cholestatic liver diseases is unknown. We have developed mice that lack XBP1 in the liver and have utilize these mice to determine the effect of XBP1 on cholestatic liver injury. Using several models of PSC and bile duct obstruction, we have and will continue to determine whether diminished XBP1 in liver cells increases the susceptibility to develop progressive liver disease. We are also in the process of developing new models of cholestasis.  Over the past year, we have published several of these findings and are currently performing human studies to better define the importance of XBP1 in PSC and other liver diseases. These studies may allow us to develop novel pharmacologic therapies targeting XBP1 for the treatment of patients with PSC.

2019 Update:

Patients with Primary Sclerosing Cholangitis can develop bile duct narrowing called strictures, which can result in reduced bile flow from the liver to the intestine. In PSC patients, the impaired ability to secrete the bile can cause progressive liver disease. The strictures can also cause bile duct destruction by increasing liver inflammation and a form of liver injury termed ER stress. XBP1 is a protein in the liver that is important in reducing this ER stress, and therefore is important in helping the liver to heal. Using mice with strictures in their bile ducts and mice with a liver disease similar to PSC, we have determined that XBP1 is important in protecting the liver. We have investigated the mechanisms of how the liver turns-on and turns-off XBP1 in the liver in the PSC-like conditions. In addition, we have determined how the inability to turn on XBP1 can cause liver scarring that can progress to cirrhosis. We have also studied a mouse called the MDR2-knockout mouse that develops a PSC-like liver disease. When we remove XBP1 from the livers of these mice, their development of PSC is altered. We are now investigating XBP1 in the bile ducts of PSC patients, and are investigating whether the changes of XBP1 and other inflammatory genes may be causing disease progression in PSC. With this enhanced understanding of the role of XBP1 in Primary Sclerosing Cholangitis, we believe that we can better develop XBP1-related drugs and other therapies to treat patients with PSC.

Apical and Basolateral Exosome Signaling in Normal and PSC Cholangiocytes

David J. Katzmann, PhD, Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN

Amount Awarded: $60,000 over two years.

Changes in cholangiocyte cell-to-cell communication likely contribute to development of PSC. Exosomes, a type of extracellular vesicle, represent a newly appreciated mode of intracellular communication. Preliminary studies indicate that: i) PSC cell lines and senescent cholangiocyte models of PSC secrete more exosomes than normal cholangioctes, and ii) exosomes from PSC cell lines and senescent cholangiocyte models of PSC activate cholangiocytes and macrophages more potently than exosomes from normal cholangiocytes. As polarized epithelia, cholangiocytes would be expected to release distinct exosomes from their apical and basolateral domains to mediate communication to targets via the bile duct (apical targets: cholagiocytes or intestinal epithelial cells) or targets within the peribiliary milieu (basolateral targets: macrophages, hepatic stellate cells). Preliminary studies supports this model as: i) normal polarized cholangiocytes release exosomes both apically and basolaterally, ii) apical and basolateral exosomes differ in the number, protein contents, and RNA contents, and iii) apical and basolateral exosomes exhibit distinct signaling activities. Our initial observations have led to the hypothesis that cholangiocytes release domain-specific (i.e., apical versus basolateral) subpopulations of exosomes containing cargo that differentially influence the phenotype of target cells in normal and diseased states, thereby contributing to liver homeostasis or development of PSC. This hypothesis will be tested by two aims: 1) define the protein contents of apical and basolateral exosomes released from normal and PSC cholangiocyte models and address the contributions of these protein constituents to exosome-induced signaling in apical and basolateral target cells; 2) define the RNA contents of normal and PSC cholangiocyte apical and basolateral exosomes and investigate the contributions of miRNA in polarized exosome signaling in normal and diseased states.

2018 Update:

Primary sclerosing cholangitis (PSC) involves perturbed intercellular communication between cholangiocytes and other cells within the liver leading to chronic inflammation and fibrosis. However, the specific signals emanating from cholangiocytes leading to disease are unknown.  We are interested in the role that exosomes, a type of extracellular vesicle, play in cholangiocyte biology during PSC as well as during normal liver function.  Cholangiocytes secrete factors from their apical surface into the bile duct or from their basolateral surface into the peribiliary environment.  Exosomes secreted into the bile duct or into the peribiliary environment would mediate communication with distinct target cells and thus, would be predicted to contain different contents to confer unique signals.  Our characterization of exosomes from polarized normal human cholangiocytes supports this model. We have proposed and continue to undertake studies to address the role of exosome communication in normal cholangiocyte function and how this process changes in PSC.  Through these studies, we will define normal and diseased exosome signaling by cholangiocytes.  Insights gleaned from these studies have the potential to identify therapeutic rationales for disrupting PSC-associated exosome signaling.

2019 Update:

Primary sclerosing cholangitis (PSC) involves perturbed intercellular communication between cholangiocytes and other cells within the liver leading to chronic inflammation and fibrosis. However, the specific signals emanating from cholangiocytes leading to disease are unknown.  We are interested in the role that exosomes, a type of extracellular vesicle, play in cholangiocyte biology during PSC as well as during normal liver function.  Cholangiocytes secrete factors from their apical surface into the bile duct or from their basolateral surface into the peribiliary environment.  Exosomes secreted into the bile duct or into the peribiliary environment would mediate communication with distinct target cells and thus, would be predicted to contain different contents to confer unique signals.  Our characterization of exosomes from polarized normal human cholangiocytes supports this model. We have proposed and continue to undertake studies to address the role of exosome communication in normal cholangiocyte function and how this process changes in PSC.  Through these studies, we will define normal and diseased exosome signaling by cholangiocytes.  Insights gleaned from these studies have the potential to identify therapeutic rationales for disrupting PSC-associated exosome signaling.

Dissecting the Genomic Variants of PSC Outcomes

Konstantinos Lazaridis, MD, Consultant Gastoenterology and Hepatology, Professor of Medicine, Mayo Clinic College of Medicine, Rochester, MNProfessor Tom Hemming Karlsen, MD, Department of Transplantation Medicine, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

Amount Awarded: $60,000 over two years.

Primary sclerosing cholangitis (PSC) is a heterogeneous disease with variable outcomes. At the present time, we are unable to distinguish which patients will rapidly progress to a poor outcome (i.e., need for liver transplant, development of cholangiocarcinoma, etc.) from those likely to experience a relatively benign disease course. Our research into PSC over the last 5 years has significantly advanced the field and has begun to characterize the genetic predisposing elements underlying the disease development. However, we lack studies about the importance of genetic factors in predicting long-term clinical outcomes of PSC such as disease progression and its complications. We hypothesize that genetic variation plays a key role in the clinical expression, and outcomes of PSC. The objective of this application is to discover the genomic deterninants of PSC outcomes (i.e., orthotopic liver transplantation, cholangiocarcinoma, and colon cancer development). Successful outcome of this study will lead to better prediction of PSC progression and introduction of individualized approaches for risk stratification of disease outcomes and ultimately benefit the patients that suffer from PSC.

2018 Update:

Primary Sclerosing Cholangitis (PSC) is a slowly progressive liver disease with variable outcomes. Over time, some PSC patients will develop hepatic decompensation leading to liver transplantation, colon cancer or cholangiocarcinoma. During the past decade, genome wide association studies (GWAS) have been performed on patients with PSC to identify those genetic variants associated with disease pathogenesis. However, no study has been carried out to date in the US to discover the genetic variants associated with PSC progression or development of cancer. To this end, we have gathered available genotyping and clinical follow-up information from a total of 1,179 US PSC patients to address these important questions on PSC outcome. Our preliminary work identified four genetic variants suggestive of association, one in liver transplantation analysis and three in the liver-decompensation analysis. Following the ongoing update of our clinical database and genomic imputation we plan to perform a more comprehensive GWAS for development of the above end-points and also plan to perform analyses looking at development of colorectal carcinoma and cholangiocarcinoma followed by a replication analysis of our findings. This novel study is an important step forward in identifying genetic variants contributing to PSC outcomes, thus, bringing individualized medicine approaches to PSC.

2019 Update: 

The study assessed genomics and tissue characteristics of an unprecedented panel of 186 tumor samples from cholangiocarcnoma and gallbladder cancer from patients with primary sclerosing cholangitis (PSC). Irrespective of the anatomical localization, cholangiocarcinoma in PSC showed uniform characteristics similar to extrahepatic cholangiocarcinoma, even for the tumors that were located intrahepatically. Furthermore, a total of 116 (62.4%) tumors had mutations within one or more potentially “druggable” genes. These findings advance our understanding of the development of PSC-associated cholangiocarcinoma and holds promise for future therapeutic opportunities of cholangiocarcnoma and gallbladder cancer in PSC.

Exploring the PSC Matrisome: Discovery of Serum Biomarkers for the Stratification and Classification of Patients with Primary Sclerosing Cholangitis

Massimo Pinzani,MD, PhD, FRCP, Professor of Medicine and Director of the University College London (UCL), Institute of Liver and Digestive Health (ILDH), Sheila Sherlock Chair of Hepatology, London, UK

Amount Awarded: $60,000 over two years.

Primary sclerosing cholangitis (PSC) is a disease affecting biliary ducts through which the bile flows from the liver into the intestine to ensure key digestive tasks. PSC is characterized by progressive scarring around the bile duct leading to obstruction and accumulation of bile in the liver. With time, scarring and fibrosis involve more and more portions of the liver causing cirrhosis and liver function insufficiency. Importantly, PSC is a silent disease and it is often discovered when the disease is already advanced. Also, when the disease is diagnosed in an early phase it is extremely difficult to establish the stage of disease evolution without performing a liver biopsy. This causes many difficulties in the management of patients with PSC and greatly hampers the possibility of efficient clinical trials with drugs specifically designed for this liver disease. Our laboratory has proposed a new technology based on the analysis of the scarring tissue obtained from the liver of patients with PSC. We have discovered that some proteins are specific from the scarring tissue of PSC and have an extremely good correlation with the disease stage. The presence and the amount of these proteins could be tested with a simple blood test and therefore become a diagnostic biomarker. The funding provided by PSC Partners will be used to support our efforts to further characterize these biomarkers and perform a clinical validation in our cohort of patients with PSC.

2018 Update:

The main aim of this funded research project is to identify proteins detectable in the blood of patients with PSC and reflecting the chronic process of liver scarring typical of this disease. This approach will be able to allow a more precise diagnosis of PSC together with the possibility of predicting the stage of evolution with a simple blood test. In the first portion of the planned work, we used samples of normal human liver (from healthy donors but not suitable for transplantation) and samples from PSC cirrhotic liver removed from patients at the time of liver transplantation. Both types of samples were cut is small cubic fragments and subjected to a process called decellularization consisting in the removal of all the liver cells. The remaining material termed “the scaffold” consists of the natural protein constituting the three-dimensional structure of the normal and of the PSC diseased liver. Material extracted from these scaffolds was subjected to a powerful type of analysis called “proteomics” that is able to identify with high accuracy the protein composition. This analysis revealed that the PSC scaffold is enriched with protein typical of liver scarring in general but also with proteins that appears unique to PSC fibrosis (i.e. never found in other chronic diseases of the liver). Therefore, these results confirm that liver scarring in PSC is characterized by unique proteins and this represents an important basis for the identification of PSC-specific blood markers.

In agreement with the study design, we then proceeded to simulate what happens in a scarred PSC liver during a process called “remodeling” in which scar tissue is continuously attacked by enzymes (called metalloproteinase) aimed at degrading and eliminating the scar, a process that is however ineffective due to the persistence of the chronic insult to the liver. Regardless, the process of remodeling is associated with the release of small fragments of scar that could represent suitable blood markers. The analysis of these fragments revealed that a fragment called C3M was originated from the PSC scar at significantly higher levels when compared with the scar tissue obtained from patients with other forms of chronic liver disease (i.e. alcoholic cirrhosis). Interestingly, increased blood levels of C3M were also found in the blood of a large group of patients with PSC in an independent study that we recently performed in collaboration with our international PSC clinical collaboration network (results published in 2018, reference 1).

In the next group of experiments the simulation mentioned before was performed by allowing the cells responsible for the progressive scarring occurring in PSC (called hepatic stellate cells or HSC) to grow in the scaffold obtained from normal and PSC cirrhotic liver. These cells grow and invade the scar tissue by promoting more scar deposition and, at the same time, by inducing scar remodeling. As a consequence, the proteins measured reflect both the progression of the scar formation and its remodeling. Hepatic stellate cells were prompted to more produce scar proteins (like pro-collagen type I) when growing in the PSC cirrhotic scaffolds (i.e. scar calls for more scar). Several scar fragments indicating active scar deposition were detected when considering both the normal scaffold and the PSC scaffold. Importantly, we observed a significant increase of C3M only in PSC scaffolds further confirming the absolute relevance of this biomarker.

References

Nielsen MJ, Thorburn D, Leeming DJ, Hov JR, Nygård S, Moum B, Saffioti F, Gilja OH, Boberg KM, Mazza G, Røsjø H, Pinzani M, Karlsen TH, Karsdal MA, Vesterhus M. Serological markers of extracellular matrix remodeling predict transplant-free survival in primary sclerosing cholangitis. Aliment Pharmacol Ther 2018; 48:179-189.

2019 Update:

Primary sclerosing cholangitis (PSC) is a chronic liver disease affecting bile ducts and is characterized by progressive scarring leading to obstruction and accumulation of bile in the liver. Importantly, PSC is a silent disease and it is often discovered when the disease is already advanced. Also, when the disease is diagnosed in an early phase it is extremely difficult to establish the stage of disease evolution without performing a liver biopsy. This greatly hampers the possibility of efficient clinical trials with drugs specifically designed for this liver disease.

In our laboratory we have developed new technology based on the analysis of the scarring tissue obtained from the liver of patients with PSC. We have discovered that 14 proteins are specific from the scarring tissue of PSC when compared to the scarring tissue obtained by other chronic liver diseases. To establish whether these proteins are effectively released from the scar tissue and become a specific biomarker for PSC we have performed experiments that simulate the scar remodeling occurring during the progression of the disease.

The results of this study funded by PSC Partners represent the first ever characterisation of the composition and also the function of the scar typical of PSC. The finding detailed in the final report constitute a solid and innovative basis for the additional work to be performed in large group of patients with PSC to confirm that these new markers can substitute liver biopsy to understand the clinical stage and favour the experimentation of new medicines to cure this disease.

Integrin AVB6 as a Therapeutic Target for Primary Sclerosing Cholangitis – Associated Cholangiocarcinoma

Yury V. Popov, MD, PhD, Director, Liver Fibrosis Research, Assistant Professor of Medicine, Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Amount Awarded: $60,000 over two years.

Cholangiocarcinoma (CCA) is a liver and bile duct cancer of unknown cause and rising incidence worldwide. CCA is a devastating complication of PSC, difficult to diagnose and associated with high mortality. Treatment options are extremely limited as PSC-associated CCA patients often do not qualify for liver transplantation. In the absence of drug options in the treatment of PSC-associated CCA, poor prognosis and high mortality, new therapeutic approaches are urgently needed.

Integrin αvβ6 is an epithelial tissue cell surface receptor which is virtually absent from normal livers. It has been shown to be markedly upregulated on activated bile duct epithelial cells and liver cells with features of ductular transformation, correlating with fibrosis stage, both in rodent models of fibrosis and in patients with chronic liver disease. Integrin αvβ6 binds and activates transforming growth factor beta and is expressed on liver progenitor cells that are linked to both biliary fibrosis and the formation of cancer.

This study proposes to develop a model of CCA in a mouse model of PSC (mdr2-/- ) and to assess the impact of integrin αvβ6 on the development of CCA and possible therapeutic potential of drugs targeting this pathway. Since several αvβ6-specific inhibitors are currently at various stages of drug development for other indications, rapid translation of findings into clinical practice is feasible.

2018 Update:

A new mouse model of cholangiocarcinoma (CCA) was established on PSC-like Mdr2-/- background. This new mouse model is termed “SB CCA.Mdr2-/-” model. Susceptibility to CCA in this system is driven by the biliary injury and fibrosis in Mdr2-/- mice, similar to the predisposition to biliary cancer in human PSC. Most of CCAs arising in SB CCA.Mdr2-/- mice express various levels of αvβ6 integrin. In the second year, we will interrogate this molecule as a potential therapeutic target in PSC-associated CCA using genetic and pharmacologic approaches in the SB CCA.Mdr2-/- model.

2019 Update:

In the first year, a new mouse model of cholangiocarcinoma (CCA) was established on PSC-like Mdr2-/- background, termed SB CCA.Mdr2-/- model. Susceptibility to CCA in this system is driven by the biliary injury and fibrosis in Mdr2-/- mice, recapitulating the predisposition to biliary cancer in human PSC. Most of CCAs arising in SB CCA.Mdr2-/- mice express various levels of αvβ6 integrin.

In the second year, we refined the model characterization by replication (and thus increasing the samples size to at least 9 mice/group), and performing long-term survival analysis and interrogated the influence of common laboratory mouse genetic backgrounds (FVB, C57Bl/6, BALB/c) on CCA susceptibility and growth. These tests confirmed 1) model reproducibility, 2) major differences in tumorigenesis observed between healthy and fibrotic hosts, and 3) established the surrogate and hard end-points for future therapeutic efficacy tests. Further, we utilized T- and B- cell deficient Mdr2-/-;Rag1-/- double-mutants to demonstrate that adaptive immunity functionally restricts CCA growth, suggesting that our model is suitable for testing novel immunotherapies.

Ongoing experiments interrogate the role of αvβ6 and related integrins as a potential therapeutic target in PSC-associated CCA using genetic and pharmacologic approaches in the SB CCA.Mdr2-/- model. We are preparing manuscript describing the model and develop several grant applications, both NIH and industry-sponsored, that directly stem from research supported by this grant.

Awarded in 2016

In 2016, PSC Partners funded six research projects and one PSC educational project. All of our lead investigators are of international renown and have proposed highly innovative studies to advance the diagnosis and therapy of primary sclerosing cholangitis. Each researcher has provided a lay summary for the PSC Partners community.

Macrophages in the Development of Sclerosing Cholangitis: Therapeutic Implications

Maria Eugenia Guicciardi, PhD, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA

Amount Awarded: $60,000 over two years.

Primary sclerosing cholangitis (PSC) is a cholestatic liver disease of unknown etiopathogenesis characterized by fibrosis around large and small bile ducts. Compelling evidence supports a major functional role for macrophages in liver fibrosis. We noted that interlobular cholangiocytes in human PSC express markedly reduced levels of cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2), two important regulators of inflammation and cell death. In a novel murine model developed in our laboratory, injection of a cIAP antagonist (named SMAC mimetic), which induces rapid degradation of cIAP-1 and cIAP-2, into the biliary tree of mice causes an acute fibrous cholangiopathy that resembles that of PSC. Following exposure to the SMAC mimetic in vitro and in vivo, cholangiocytes upregulate several proinflammatory cytokines and chemokines, including the monocyte chemoattractant MCP-1/CCL2. Consistently, periductular accumulation of macrophages is evident in the livers of SM-treated mice and PSC patients. This project aims to test the hypothesis that cholangiocyte damage in PSC is associated with decrease in cIAP-1 and -2 cellular levels and release of pro-inflammatory chemokines, with subsequent recruitment and activation of macrophages. In particular, this project will focus on the role of TNF-related apoptosis-inducing ligand (TRAIL), a cytokine expressed by several immune cells, including macrophages (especially the pro-inflammatory M1 macrophages), in the development of sclerosing cholangitis. These studies will allow us to verify whether inhibition of macrophage-derived TRAIL can effectively prevent or attenuate the PSC-like phenotype, and could identify TRAIL as a key molecule in the initiation and/or progression of PSC, potentially providing a rationale for the development of novel therapies for this disease.

2017 Update

The causes of primary sclerosing cholangitis are largely unknown. Macrophages, a type of white blood cells that rid the body of dead cells, debris and bacteria, and their precursors, monocytes, participate in the body’s immune response, and have been shown to strongly contribute to the pathogenesis of several liver diseases. The studies conducted in this first year demonstrate that monocyte-derived macrophages play an important role in the development of the fibro-inflammatory biliary injury in two different mouse models of sclerosing cholangitis and in human PSC, and suggests therapeutic strategies targeting macrophages and their recruitment to the liver may be beneficial in the treatment of human PSC.

2018 Update

The causes of primary sclerosing cholangitis are largely unknown. In a series of studies in animal models funded by PSC Partners Seeking a Cure, we examined the nature of the inflammation causing liver damage in PSC. We focused our studies on inflammatory cells, identifying a cell type termed macrophages as the mail culprit of liver damage. Our results demonstrated that blocking macrophage entry into the liver was therapeutic. These important observations were recently published on a prestigious scientific journal, and support macrophage-directed therapies for PSC.

The results of Dr. Guicciardi’s research have been published in the Journal of Hepatology. Please click to view the article and its supplement. Dr. Guicciardi’s research has also been published in the journal Cell Death and Disease.

Functional microbial biomarkers in primary sclerosing cholangitis

Johannes R. Hov, MD, PhD Norwegian PSC Research Center, Department of Transplantation Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway

Amount Awarded: $60,000 over two years

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts with no known cause. It may lead to end-stage liver disease and sometimes cancer. We have recently shown using genetic methods that PSC patients harbor a different set of microbes in the gut than healthy controls. This is an important starting point, but with the technology used so far, only a low-resolution profile of the gut microbes is available. In the present study, we want to sequence all bacterial DNA (the full “metagenome”) present in the stool of PSC patients and controls from two countries (Norway and Germany) to investigate the hypothesis that specific microbial functions are altered in the gut community. Identified altered microbial functions and subsequent altered metabolism of endogeneous or dietary substances may potentially be relevant for disease development and progression and provide new clues to disease pathogenesis and treatment. We believe that gut metagenomics is the first step in the translation of the basic knowledge of the gut microbiome to address some of the most critical clinical challenges in PSC.

2018 Update

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the bile ducts with no known cause. It may lead to end-stage liver disease and sometimes cancer. We have recently shown using genetic methods that PSC patients harbor a different set of microbes in the gut than healthy controls. Funded by a PSC Partners grant in 2016 we have now been able to sequence all bacterial DNA (the full “metagenome”) present in the stool of PSC patients and controls from two countries (Norway and Germany) to investigate the hypothesis that specific microbial functions are altered in the gut community. The data amounts are extensive (terabytes) but from the preliminary analyses we can already reproduce our previous results at higher resolution. The aim ahead is to identify altered microbial functions and subsequent altered metabolism of endogeneous or dietary substances, which may be relevant for disease development and progression and provide new clues to disease pathogenesis and treatment. We believe this is a feasible road ahead to utilize basic knowledge of the gut microbiome to address important clinical challenges in PSC. Final results are expected early 2019.

Mutational profiling for therapeutic targets in PSC – associated biliary tract cancer

Tom Hemming Karlsen, MD, PhD, Norwegian PSC Research Center, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway

Amount Awarded: $60,000 over two years

Cholangiocarcinoma is a dreaded complication in patients with primary sclerosing cholangitis (PSC). In personalized cancer medicine, screening for clinically relevant genetic tumor alterations is increasingly used to stratify patients for targeted cancer therapy. Dedicated genetic studies in PSC-associated cholangiocarcinoma are so far missing. This also means that personalized therapeutic approaches have not been possible to consider for this group of patients. The proposed project aims to bridge this knowledge gap, by investigating known cancer genes in cholangiocarcinoma tumor tissue from a large number of PSC patients. Pilot data from Oslo and Heidelberg shows promising results, and the funds from PSC Partners will be used to determine the validity of findings in many more samples from other German centers, Sweden, Finland, the Netherlands, Poland, the UK and the US.

2017 Update

This study provides the first comprehensive genomic characterization of PSC-associated biliary tract cancer (BTC) utilizing the largest BTC collective arising from a mono-etiological background described to date. By performing targeted mutation sequencing we have identified a significant number of novel somatic mutations not previously reported in PSC-associated BTC. Characterization of the mechanistic implications of detected mutations may provide basis for early phase clinical trials and personalized treatment and follow-up in this devastating cancer.

2018 Update

Bile duct cancer is a dreaded complication of primary sclerosing cholangitis (PSC). Much progress has been made in other cancer types regarding genetic characterization and molecular subtyping, both of which are potentially relevant for personalized treatment strategies. In PSC-related bile duct cancer, no comprehensive effort has so far been made. In this project, we aimed to overcome this knowledge gap, and collected tissue samples from approximately 200 bile duct cancers throughout 8 different countries. Through thorough histological, immunohistochemical and genomic characterization, we were able to define several molecular characteristics of the PSC-related biliary cancer. Some of these molecular characteristics may open for the inclusion of affected patients in clinical trials, thereby providing therapeutic avenues forward for a condition which is often hard to treat.

The Enterohepatic Metabolome in Primary Sclerosing Cholangitis

Steven P. O’Hara, PhD, Assistant Professor of Medicine and Biochemistry/Molecular Biology, Mayo Clinic, Rochester, MN, USA

Amount Awarded: $60,000 over two years

Emerging data, including work from our laboratory and clinical research group, suggest fundamental disease associated mechanisms in primary sclerosing cholangitis (PSC) that are centered on molecules derived in the gut and brought to the liver through portal circulation (gut-liver axis). The gut-liver axis has been proposed to play a significant role in the initiation, progression, and adverse clinical events associated with PSC; however, this has not been directly tested to date. Our goal is to determine whether there are differences in the metabolites (proteins, fats, and other chemicals) in the portal vein (which delivers blood from the intestines to the liver) and in bile between individuals with primary sclerosing cholangitis and those without PSC.  We anticipate that patients with PSC will have distinct alterations in the portal venous and bile metabolites compared to controls; and these alterations may be amenable to future therapies.

2017 Update

Blood from the intestine is circulated to the liver via the portal vein (portal blood); conversely, bile produced in the liver is deposited into the small intestine. This direct link between the gut and liver has been proposed to play a significant role in the initiation, progression, and adverse clinical events associated with primary sclerosing cholangitis (PSC). We proposed that PSC patients may have a distinct metabolomic profile (proteins, fats, and other chemicals) coming to the liver from portal blood and/or leaving the liver in bile. We have collected biological samples (portal blood, peripheral blood, and bile) from PSC patients, non-diseased controls (i.e., normal donor), and disease controls (i.e., patients with liver disease that is not PSC) obtained at the time of living donor liver transplant. We then utilized a method to determine the molecules (metabolites) in these samples. We have found that primary sclerosing cholangitis, disease control, and non-diseased control individuals differed in a number of metabolic readouts, including amino acid and lipid (e.g., sphingolipid) content in blood and bile. Future studies will specifically explore, in a targeted manner, amino acid and lipid metabolism to better quantitate differences in these patient populations.  Moreover, we intend to interrogate the mechanisms and consequences of altered amino acid and lipid metabolism on hepatobiliary health and disease.

Upregulation of biliary epithelia innate immune responses in PSC

Mario Strazzabosco, MD, PhD, Department of Internal Medicine, Section of Digestive Diseases, Yale University, New Haven, CT, USA

Amount Awarded: $60,000 over two years

Primary Sclerosing Cholangitis (PSC) is a chronic inflammatory disease of the intrahepatic and/or extrahepatic biliary tract characterized by obliterative fibrosis and cholestasis. The etiology and pathophysiology of PSC remain still unclear and lack of a valid human experimental model has hampered this area of research. Association of PSC with Inflammatory Bowel Disease together with preliminary evidence showing increased responsiveness of PSC-derived cholangiocytes to endotoxins (i.e NF-kB activation, IL8 secretion, TH17 response) strongly suggest that PSC may be caused by aberrant innate immune mechanisms.

The hypothesis of this study is that the biliary epithelium of PSC patients responds with increased production of inflammatory mediators to a variety of factors released during biliary or hepatocellular damage and able to activate the innate immunity. To address our hypothesis and generate data relevant for the human disease we will use the latest advances in stem cell technology to establish two new in vitro human models of PSC, one derived from induced-pluripotent stem cells (iPSCs) and the second from human 3D liver organoids. We will compare the activation of innate immune pathways in these new models compared with primary PSC cholangiocytes isolated from liver explants and perform gene array analysis. We expect that disease-relevant mechanisms will be common to all the three models.

The results of our study will have a strong translational potential for the understanding of the pathogenesis of sclerosing cholangitis and will also generate a screening platform for new therapeutic approaches.

2017 Update

During the first part of the study we have made important progresses on the establishment of human cell models relevant to the disease.  In particular, we have generated a cellular model of PSC derived human iPSCs (induced pluripotent stem cells) and 3D liver organoids from PSC patients. We have shown that iPSC can be differentiated in mature and functional biliary cells and we have created a platform for patient-specific disease modeling as reported in our recent publication (Fiorotto et al., Hepatology 2017).

In the second part of this study, we plan to compare the innate immune responses to PAMPs and DAMPs in our human PSC models (primary PSC organoids, iPSCs-derived cholangiocytes and cultures of cholangiocytes immunoisolated from liver explants) as respects to their appropriate normal controls. We expect to identify differentially expressed genes that may account for increased TLR-dependent NF-kB activation and are expressed in all the three different cell models as a signature of the disease.

This study will improve the understanding of the pathogenesis of PSC and likely identify novel therapeutic targets.

2018 Update

The pathogenesis of PSC is still unknown; as a consequence, curative treatment is lacking. Association of PSC with Inflammatory Bowel Disease suggests the involvement of innate immune mechanisms that in normal conditions are activated by cells to protect from infections. Preliminary evidence indicate that biliary epithelial cells derived from PSC patients have an enhanced responsiveness to endotoxin bacterial product (i.e LPS) suggesting that a primary malfunction of biliary cells is the cause of PSC, rather than the consequence of the disease. We have described similar mechanisms in Cystic Fibrosis (CF), a known cause of Sclerosing Cholangitis, where negative control of Src/TLR4/NFkB signaling is lost and biliary cells have increased pro-inflammatory cytokine production in response to LPS (Fiorotto et al., Hepatology 2011, 2016, 2018).

Based on these premises we hypothesized that the central pathogenetic mechanism of PSC is an aberrant inflammatory reaction of the biliary epithelium to factors able to stimulate innate immunity, released during biliary or hepatocellular or intestinal damage.

To demonstrate the above hypothesis, we have planned to study the effect of the activation of TLR innate immune pathways in response to bacterial products (PAMPs) and other damage-associated molecule (DAMPs) released by liver cells. As there is no satisfactory animal or cellular model orthologous to human PSC, to produce data relevant for the human disease, during the first year of the study, we have generated novel in vitro human models of biliary cells that are: 1) induced pluripotent stem cell (iPSC)-derived cholangiocytes from blood samples; 2) 3D liver spheroids (organoids) (obtained from bile fluid, from liver explant tissue or from liver biopsy); 3) primary cholangiocytes immune-isolated from liver explant. During the first year of the project, we have demonstrated that iPSC can be differentiated in mature and functional biliary cells and we have created a platform for patient-specific disease modeling (Fiorotto et al., Hepatology 2018). (see figure 1,2,3)

In the second year of the project, we have studied different components of the innate immune pathway by assessing NF-kB activation and cytokines secretion in the above described different human PSC models. We also have accrued more cases, and we have now 6 cases from peripheral blood monoclueated cells.

In the following third year period we will characterize in deep the genetic profile of the different biliary models by transcriptomic expression analysis and use these models to perform gene expression arrays of genes relevant for TLR signaling. We expect to detect important genetic signatures among the different in vitro models to better define their potential to resemble the human biliary epithelium. In addition, we expect to identify differentially expressed genes that may account for increased TLR-dependent NF-kB activation and are in common between the three different cellular models as a signature of the disease.

This study will improve the understanding of the pathogenesis of PSC and will provide a cell-based platform for drug development and testing.

2019 Update

The pathogenesis of PSC is still unknown. The association of PSC with inflammatory bowel disease (IBD) and the enhanced response of the biliary epithelium to gut-derived endotoxins suggest that aberrant innate immune pathways in PSC cholangiocytes play a primary role in the progression of the disease. The lack of a satisfactory animal or cellular model representative human PSC is one of the major gaps in the PSC research field and so far a limitation for the pipeline development of new therapies. Our lab has been focusing on the generation of patient-derived biliary cell models to model in vitro PSC disease and other cholangiopathies. With the support of PSC Partners, we have generated a platform that combines induced pluripotent stem cell (iPSC) derived cholangiocytes, liver organoids and primary cholangiocytes obtained from liver tissue of PSC patients. We have investigated the role played by activation of aberrant innate immune pathways in the different models of cholangiocytes and shown that human PSC cholangiocytes express higher levels of inflammatory cytokines involved in the recruitment of immune cells and in the cross-talk with mesenchymal cell. This aberrant response can be further enhanced by exposure to specific bacterial products (i.e flagellin).  In addition, we have devised new systems for the 3D culture in-vitro of human biliary organoids and their co-culture with mesenchymal and immune cells. Our study, albeit conducted in a very limited number of cases, has generated an important experimental approach and methodology to better study the function of biliary cells in primary sclerosing cholangitis and facilitate drug development.

Development and validation of a patient-derived, disease specific, health-related quality of life (HRQoL) tool for PSC

Douglas Thorburn, MD, Royal Free Hospital, London, UK

Amount Awarded: $60,000 over two years

With limited treatment options for patients with PSC, there is an urgent need to develop new therapies. These are required to slow or stop the progression of PSC but also to treat the symptoms of disease and improve the associated quality of life. It is also important to ensure that any new treatments under evaluation are not associated with a worsening in quality of life of PSC patients. Currently there are no validated quality of life tools validated for evaluating the experience of having PSC.

The UK-PSC research collaboration is collaborating with experienced qualitative researchers and patient groups to develop a disease-specific quality of life measure for PSC and then proceed to validate it internationally in collaboration with leading international PSC research groups via the International PSC Study Group. This international validation is crucial given the rarity of PSC and the need to recruit patients into interventional clinical trials across continents.

Development will take place over three to five years, involving the identification of relevant issues for patients through scientific literature review and in-depth interviews with patients, refining and validation of the questionnaire in a large patient survey, and finally evaluation in PSC patients to ensure a scientifically sound, valid, reliable and precise tool. The team will work with the relevant regulatory authorities to ensure that the new tool is approved and suitable for multicenter international clinical trials.

2019 Update:

A team at University College London (UCL) have been working on the development of a new measure of quality of life specifically for people living with PSC in the United Kingdom. The study has been conducted by Elena Marcus, as part of her PhD project, and under the supervision of Prof Douglas Thorburn from the Royal Free Hospital London, and Prof Paddy Stone and Dr Bella Vivat from the Marie Curie Palliative Care Research Department at UCL.

Developing a questionnaire to measure quality of life is important because people with PSC can experience many quality of life and well-being issues, yet there is no validated way to measure these. This is mostly relevant to studies (clinical trials) which are testing whether new drug treatments might help people with PSC. With a PSC-specific measure of quality of life, we can assess whether such treatments are having a positive impact on well-being or whether they may in fact worsen it.

The project was conducted in two stages. In the first stage nearly 400 quality of life issues were identified from a PSC Support patient survey and a review of the scientific literature. The relevance and importance of these issues were explored with 28 people with PSC and 11 clinicians, and the list was then reduced to 89 issue. These quality of life issues were constructed into 83 questionnaire items for the provisional measure, the UK-PSC-QoL. In the second stage of the project 60 people with PSC completed the UK-PSC-QoL, of which 25 people also took part in interviews to explore whether there were any problems with the phrasing of items or with the measure as a whole. Based on the feedback of participants, 25 items were re-phrased to improve clarity. Future research with a larger group of people with PSC is necessary to make definitive decisions about which items to retain in the measure and which can be deleted.

Patient education in PSC; Development of a 3D-animation

Cyriel Ponsioen, MD, PhD, Academic Medical Center, Amsterdam, Netherlands

Amount Awarded:$9,960 over one year

Primary sclerosing cholangitis (PSC) is a very complex disease. For patients it can take a long time to understand what exactly is happening. Despite explanation by clinicians, patients knowledge is often lacking.

It is important for patients to understand the basic principles of PSC. By knowing these, they can participate more and better in the treatment process. Well educated patient have been shown to have better clinical outcomes.
The internet is providing a lot of medical information for patients, however the reliability of this information is doubtful. Until now there is no video or animation on the internet providing proper information about PSC.

In this project we want to develop a 3D- animation with information about PSC. It will be especially designed for patients. Pathophysiology, diagnosis and treatment will be discussed. A voice-over will give a clear explanation during the video.
A 3D-animation is ideal to educate patients. First of all, visual information is easy to understand and the three-dimensional aspect makes it even clearer. Secondary, it is very easy to spread (via the internet). Also, with a little extra effort an extra voice-over language can be added, whereby people can get information in their own language.

Input and feedback from patient representatives from PSC partners seeking a cure and patients from the AMC will be used to optimize the content of the video.

A survey will be administered to a group of patients one month prior to the release of the video to assess their knowledge. This survey will also contain questions on the perception and satisfaction of their knowledge. After watching the video the same questionnaire will be administered. A control group of patients will complete the second questionnaire without watching the video first. Subsequently, knowledge improvement and satisfaction can be measured.

2017 Update

Dr. Ponsioen’s video is now available online. To view, pleaseclick below.

Awarded in 2015

In 2015, PSC Partners funded five research studies. All of our lead investigators are of international renown and have proposed highly innovative studies to advance the diagnosis and therapy of primary sclerosing cholangitis. Each researcher has provided a lay summary for the PSC Partners community.

Fecal microbiota transplantation for the treatment of primary sclerosing cholangitis

Jessica Allegretti, MD, MPH, Brigham and Women’s Hospital, Boston, MA.

Amount Awarded: $60,000 over two years.

Primary sclerosing cholangitis (PSC) is a progressive, chronic cholestatic liver disease characterized by inflammatory and fibrotic destruction of the intrahepatic and/or extrahepatic bile ducts. PSC will progress to biliary cirrhosis, portal hypertension and liver failure. In up to 90% of patients, ulcerative colitis or Crohn’s disease will also be present. Medications used for the treatment of ulcerative colitis have not been effective in reducing inflammation or bringing about remission in PSC. Currently there is no medical therapy that has been shown to be effective in PSC and no therapy has won FDA approval for this indication.

It has been postulated that bacterial components may stimulate an aberrant immune response resulting in the perpetuation of the biliary inflammation seen in PSC. Bacteria gain access to the liver and biliary tree through translocation across an abnormal and inflamed intestinal mucosa into the portal venous system. Animal models have demonstrated that an enteric dysbiosis can lead to hepatobiliary inflammation with features similar to PSC.

Preliminarily we have found that patients with PSC have an altered bile acid composition compared to non-cholestatic controls. Our central hypothesis is that for patients with PSC, fecal microbiota transplantation (FMT) will correct an altered bile salt profile resulting from a dysbiosis that has led to hepatobiliary inflammation and will therefore improve LFTs and slow progression to cirrhosis. Assessing the effects of FMT in this population will allow us to better understand the microbial contribution to the pathology of this disease.

2016 Update

Our study assessing fecal microbiota transplantation (FMT) for the treatment of PSC is well underway and still actively recruiting. To date we have enrolled 5 patients with plans to enroll 5 more patients with early stage PSC and concurrent inflammatory bowel disease. Our central hypothesis remains that for patients with PSC, FMT will correct an altered bile salt profile resulting from a dysbiosis that has led to hepatobiliary inflammation and will therefore improve LFTs and slow progression to cirrhosis. Among the 5 patients enrolled, 2 have completed the treatment and sample collection portion of the study, 2 are still undergoing sample collection and one has recently been enrolled and screened. Even with the limited amount of data we have to date, we are already seeing a positive trend regarding decreasing alkaline phosphatase levels after FMT. We are encouraged by these preliminary results and are excited to continue recruitment.

Entero-hepatic trafficking of lymphocytes in the pathogenesis of juvenile onset autoimmune sclerosing cholangitis

Rodrigo Liberal, MD, PhD, Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, London, UK.

Amount Awarded: $60,000 over two years.

Primary Sclerosing Cholangitis (PSC) is a chronic inflammatory liver disease characterized by progressive bile duct destruction.  In children and young adults, sclerosing cholangitis is often associated with florid autoimmune features, including high immunoglobuli G (IgG) levels, elevated autoantibody titres, and interface hepatitis on liver biopsy. This AIH/sclerosing cholangitis overlap syndrome, called autoimmune sclerosing cholangitis (ASC) has the same prevalence as autoimmune hepatitis in the juvenile population. Since ASC is associated with inflammatory bowel disease (IBD), lymphocytes of intestinal origin might be involved in its development, as suggested for PSC.

The link between the liver and bowel disease remains speculative. One of the most fascinating hypotheses accounting for this association is that long-lived memory cells effectors of damage, primed in the lymphoid tissue of the inflamed gut, reach and circulate through the liver initiating and perpetuating hepatic autoimmunity, which can persist even when intestinal inflammation has subsided.

Recirculation of T lympocytes from the gut to the liver is dependent on the β7 family of integrins, with the α4 subtype governing exit from the blood stream and the αE being responsible for localization within the intestinal epithelium. Some of these cells may persist as long-lived memory T cells after resolution of IBD, therefore providing an explanation for the sometime independent courses of bowel and liver disease.

This project will investigate the role of entero-hepatic lymphocyte recirculation in the pathogenesis of ASC. Specific aims are to: 1) investigate whether lymphocytes of gut origin are present in the inflammatory liver infiltrate of patients with ASC and characterize their immunological function in terms of cytokine production/secretion; 2) investigate the frequency and phenotype of peripheral blood and gut lamina propria T cells in patients with ASC; 3) examine the role of the α4β7/MadCAM-1 (ligand of α4β7) and αEβ7/E-caderhin (ligand of αEβ7) pathways in the trafficking of pathogenic lymphocytes from the gut to the liver.

Results from phase II/III studies on the use of monoclonal antibodies targeting the α4- and αEβ7 integrins in IBD are very promising. Whereas ‘standard’ biologic therapy is traded with the risk of infection and other side-effects, a Cochrane review pooling all four vedolizumab (a therapeutic anti-α4β7 monoclonal antibody) studies has shown no difference in adverse events, including infections, in comparison to placebo. The benefit observed in isolated IBD may translate to patients with liver disease, such as PSC and ASC, if the pathogenic hypothesis discussed herein is supported by our studies.

2016 Update

In children and young adults primary sclerosing cholangitis (PSC) often presents with symptoms and signs that suggest an autoimmune process, including high IgG levels, elevated autoantibody titre, and interface hepatitis on liver biopsy. This autoimmune hepatitis/sclerosing cholangitis overlap syndrome, called autoimmune sclerosing cholangitis (ASC) has the same prevalence as autoimmune hepatitis. Since ASC is associated with inflammatory bowel disease (IBD), lymphocytes of intestinal origin might be involved in its pathogenesis, as suggested for PSC.

The link between the liver and bowel disease remains speculative. One of the most fascinating etiopathogenic hypotheses accounting for these observations is that long-lived effector memory cells, primed in the lymphoid tissue of the inflamed gut, recirculate through the liver initiating and perpetuating hepatic autoimmunity, even when intestinal inflammation has subsided.

Recirculation of T lympocytes from the gut to the liver is dependent on 7 family of integrins, with the α4 subtype governing exit from the circulation and αE being responsible for localisation within the intestinal epithelium. Some of these cells may persist as long-lived memory T cells after resolution of IBD, therefore providing an explanation for the sometime independent courses of bowel and liver disease.

The aim of the project is to investigate the role of an entero-hepatic lymphocyte recirculation in the pathogenesis of AILD.

During the first year of the PSC Partners grant, we have used peripheral blood mononuclear cells (PBMCs) obtained from 8 AIH patients, one ASC-IBD and 8 healthy subjects. Preliminary analysis shows elevations in gut-derived lymphocytes (namely those expressing the intergrin 7) within the circulation of AIH and ASC patients compared to healthy controls. We are currently investigating under the microscope whether b7/CCR9 expressing T cells are located in proximity to aberrantly expressed gut adhesion molecules in the liver and gut biopsies of ASC patients. Additionally, we have collected and stored for later experiments samples from 19 patients with ASC (7 females; median age 15.8; range 7.4 – 29.8 years; 9 with concomitant ulcerative colitis and 3 with Crohn disease) and 8 patients with AIH (6 females; median age 14.6; range 10.4 – 26.8 years; 1 with concomitant ulcerative colitis), as well as 10 healthy subjects. We will continue to determine the frequency and phenotype of T cells by flow cytometry and by intracellular cytokine staining.

Intrahepatic bile duct tissue engineering utilizing liver matrix and 3D bioprinting

Ramille Shah, PhD, Department of Materials Science and Engineering, Department of Surgery – Transplant Division, Northwestern University, Chicago, IL.

Amount Awarded: $60,000 over two years.

Currently the only treatment for primary sclerosing cholangitis is liver transplantation, but there remains a critical shortage of donor organs. Liver tissue engineering aims at using biomaterials designed to provide the necessary biochemical and architectural cues to guide cells to organize and synthesize new functional tissue that may lead to a viable alternative therapy for those facing end stage liver disease. However, the majority of liver tissue engineering approaches do not involve regenerating the intrahepatic bile ducts, an essential component if the engineered tissue is to be a fully functioning transplant alternative. The goal of these studies is to engineer an organized ductular network by controlling both the biochemical and architectural environment of biliary epithelial cells (e.g. cholangiocytes). We propose to control ductule formation by using 3D bioprinting to spatially pattern cells and bioactive materials in a fashion that will lead to organized hierarchical ductal structures reminiscent of native liver tissue. Future studies will incorporate 3D patterned co-cultures of cholangiocytes, hepatocytes, and blood vessel forming cells to enhance the engineered tissue functionality, vascularization, and integration with host tissue.

2016 Update

We have previously demonstrated the ability of in vitro cultured biliary epithelial cells (cholangiocytes) to form complex branching network structures within gels composed of liver matrix. Within the past year, we compared the response of cholangiocytes within the liver matrix gels to commercially available matrix gels, such as Matrigel and purified type 1 collagen, and have validated that our liver matrix gels are the only material capable of inducing the complex in vitro branching formation. Additionally, we have customized traditional methods (electron microscopy) and are developing new methods (computational 3D image analysis) to evaluate and quantify the branching structures that form in vitro.

In order to control the formation of these structures in 3D, we have also begun exploring several distinct routes to 3D-print the liver matrix gels, each of which has unique advantages. Within the next year, we expect to demonstrate a degree of control over the directionality of cholangiocyte branching formation using our 3D-printable liver matrix. Additionally, we have begun sourcing and processing mouse livers in different states of regeneration after partial hepatectomy. We anticipate that liver matrix isolated from regenerating livers will impart a beneficial effect upon in vitro cultured cholangiocytes and their duct and branch forming response.

Confirmation of rare exonic variants involved in the development of primary sclerosing cholangitis

Rinse K. Weersma, MD, PhD, Department of Gastroenterology and Hepatology, University of Groningen and University Medical Centre Groningen, the Netherlands.

Amount Awarded: $60,000 over two years.

PSC is a severe and complex disease. Both environmental factors and genetics are involved in its development and course. Several genome-wide association studies have identified genetic variants in the DNA that are associated with susceptibility to PSC.

However, a large part of the heritability for PSC is considered to be still unexplained. Part of the “hidden” heritability is thought to reside in genetic variants that occur less frequently in the general population, and are located in the exons of genes, i.e. the parts of genes that are translated into proteins. To identify such variants involved in PSC pathogenesis, the applicants have used Illumina Exome Arrays including ≈250.000 genetic variants, to genotype a cohort of 1243 PSC patients and 10038 healthy controls.

For genetic studies targeting rare variants, it is mandatory to confirm the initial findings in independent case-control cohorts to exclude false positive findings and strengthen the proof that the identified variants are indeed associated to PSC.

In this study we aim to replicate and confirm exonic variants that have been identified through the exome array analysis, in large independent cohorts of PSC patients and population matched healthy controls.

Replication genotyping will be performed using the Agena Bioscience technology (formerly Sequenom). For replication we will use DNA of PSC cases and population matched healthy controls from the USA, UK, Sweden and Belgium.

Confirmed variants will potentially give important insights in disease pathogenesis. The variants on the exome array will have direct functional implications and will therefore enable additional functional studies and hopefully targets for novel therapeutic interventions.

2016 Update

In the past year, in close collaboration with the Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Kiel, Germany and The Wellcome Trust Sanger Institute, Hinxton, Cambridge, U.K. we have started genotyping 104 genetic variants in large independent cohorts from the USA, the UK, Belgium and Sweden. We are excited to report that this is expected to finish this month (Oct 2016).

Once we have collected the data, we will see whether we find the associations back in the independent cohorts and perform a meta-analysis to see whether these are robust genetic associations with PSC susceptibility.

Immunoglobulin glycosylation repertoire: Novel serological biomarkers of PSC

Weici Zhang, PhD, Division of Rheumatology, Allergy and Clinical Immunology, University of California School of Medicine, Davis, CA.

Amount Awarded: $60,000 over two years.

Early, accurate, specific and non-invasive biomarkers are needed for the differential diagnosis, disease monitoring, and treatment of primary sclerosing cholangitis (PSC). However, such markers are currently not available for PSC. Recent studies have implicated the significance of sugar molecules in modulating inflammatory responses. This study is directed towards investigating and determining the specificity of sugar molecules on immunoglobulin in patients with PSC. Specifically, we will apply exclusive, state-of-the-art technology using triple quadruple (QqQ) mass spectroscopy with subsequent multiple reaction monitoring (MRM) to elucidate the overall profile, composition and linkage of the sugar residues of plasma Ig glycans. This study will lay important groundwork for the discovery of new biomarkers in PSC by developing a specific Ig classifier panel and/or prognostic markers to better understand the natural history of the disease.

2016 Update

The contribution of sugar chain binding onto antibodies is gaining significant attention in autoimmune disorders. In this study, we examined the overall profile, composition and linkage of the sugar residues of plasma antibody sugar chain. Distinct sugar patterns were identified in plasma antibodies of patients with PSC when compared to PBC and healthy individuals. Next, we will evaluate sugar residue patterns in patients with IBD and other liver abnormalities. Our goal is to discover disease-specific sugar patterns of plasma antibody as biomarkers for early diagnosis and disease monitoring in PSC.

Awarded in 2014

In 2014, PSC Partners funded six research studies.

All of our lead investigators are of international renown and have proposed highly innovative studies to advance the diagnosis and therapy of primary sclerosing cholangitis. Each researcher has provided a lay summary for the PSC Partners community.

Unraveling genetics driving PSC sub-phenotypes: An IPSCSG Study

Rudi Alberts, PhD, Department of Gastroenterology and Hepatology, University of Groningen and University Medical Centre Groningen, The Netherlands

Amount Awarded: $60,000 over two years.

PSC is a severe and complex disease. Both environmental factors and genetics are involved in its development and course. Genetic studies have identified sixteen genetic loci on multiple chromosomes that are associated with PSC. The main contribution of these studies is increased insight into PSC pathogenesis. However, ever since the first human genome was sequenced in 2003, the clinical world has been waiting for translation of genetic knowledge into personalized care for individual patients. To which extent do genetics drive differences in PSC disease behavior? Do genetic mutations within PSC patients separate patients with a mild disease course from those suffering from severe progressive disease? Do genetic mutations within PSC patients separate patients that do and do not develop cholangiocarcinoma? To address these and related questions, within the International PSC Study Group (IPSCSG) we have performed the largest PSC clinical characterization effort to date, covering over 3000 patients from hospitals in 13 countries. The primary aim of this study is to identify genetic variants driving PSC clinical characteristics. The most important characteristics collected are date of birth and diagnosis, date and cause of death (PSC related, cholangiocarcinoma, hepatocellular carcinoma etc.), dates of liver transplantations and co-occurrence with IBD. Genetic mutations are identified using the Immunochip genotyping platform, interrogating ~200.000 genetic variants. By combining clinical characteristics with genetic variants we can discover genetic loci that are associated with these characteristics. Furthermore, information across all mutations will be combined to build genetic risk scores for these characteristics. These can potentially be used to predict, based on the DNA of the patient, whether he or she is likely to develop one of the aforementioned PSC characteristics or complications.

2015 Update

In the past year we have finished the collection of PSC sub-phenotypes. Also, for each of the 3402 PSC patients, we have coupled the sub-phenotypes to the genetic variants, i.e. genotypes.

We started our analyses with yes/no phenotypes, for example, comparing PSC patients that have developed cholangiocarcinoma with PSC patients that did not. This revealed several suggestive links between genetic variants and those phenotypes. Most interesting links were found by comparing PSC patients with and without Autoimmune Hepatitis.

Next we analyzed several phenotypes that are measured through time. For example, we have data indicating at which date patients were diagnosed with PSC and also, at which date they underwent a liver transplantation. By comparing this data with genetic data, we found a strong link between a genetic variant located on chromosome 6 and transplant-free survival. We are currently finalizing a manuscript reporting this finding.

Until now, no genetic variants are known that are linked to transplant-free survival. Now we have identified a protein that is possibly involved in transplant-free survival. Further research needs to be done to confirm this finding.

2016 Update

Previously, we have collected sub-phenotypes describing PSC disease severity and complications and genetic information for a large cohort 3402 PSC patients. We found a genetic variant on chromosome 6 that is significantly associated with the phenotype liver transplant-free survival. Also we identified the gene RSPO3 as most likely candidate for this association.

In the past year we investigated the gene expression of RSPO3 in disease-relevant cells. We found that in mouse cholangiocytes and cholestatic cholangiocytes indeed RSPO3 is highly expressed. We also found in human hepatic stellate cells, the cell type that is mainly involved in liver fibrosis, expression of this gene.

We have presented our results at two international congresses and are in the process of publishing them.

This project is partially funded by the generous support of Axel and Karen Rasmussen.

Distinguishing primary sclerosing cholangitis from IgG4-associated cholangitis: Specific IgG4 class-switched B-cells as diagnostic tool

Ulrich Beuers, MD, Department of Gastroenterology and Hepatology and Tygat Institute of Liver and Intestinal Research, Academic Medical Center, University of Amsterdam, The Netherlands.

Amount Awarded: $60,000 over a period of two years

Primary sclerosing cholangitis (PSC) and the more recently defined Immunoglobulin G4-associated cholangitis (IAC) are inflammatory diseases of the bile ducts and the liver of unknown cause which are difficult to distinguish with conventional biochemical and imaging studies. Patients with PSC and IAC are predominantly male, show a cholestatic serum enzyme pattern and similar stenoses and dilatations of the intra- and/or extrahepatic bile ducts. The serum immunoglobulin IgG4 may or may not be elevated both in IAC and PSC. An accurate diagnostic marker is lacking, but urgently needed as long-term course and treatment of PSC and IAC differ markedly. We have recently identified dominant IgG4+ B-cell receptor (BCR) clones in blood and affected tissue of patients with IAC, but not PSC and based on these findings have unravelled “blue collar” work with chronic exposure to environmental toxins such as solvents, industrial and metal dusts, pigments and oils used in the automotive industry as a potential risk factor for IAC, but not PSC. With our actual proposal, we aim to (i) provide a reliable, cost-effective and patient-friendly diagnostic marker to distinguish between PSC and IAC. We also want to provide a deeper molecular understanding of the role of IgG4 in IAC and PSC by (ii) trying to identify antigens responsible for the expansion of recently described dominant IgG4+ B-cell receptor (BCR) clones in IAC in comparison to PSC; (iii) study the role of T-cells by performing T-cell receptor (TCR) sequencing in patients with IAC in comparison to PSC who have previously undergone BCR sequencing; and (iv) further assess risk factors predisposing to IAC in comparison to PSC.

2015 Update

We have recently demonstrated with a novel genetic analytical method, the so-called next-generation sequencing (NGS) technique, with which the genetic information (RNA) for about 10.000 different proteins on the surface of B-lymphocytes can be determined that dominant IgG4+ B-cell receptor (BCR) clones accurately distinguish patients with IAC from those with PSC or cholangiocarcinoma. Based on these findings, we had unraveled “blue collar” work with chronic exposure to environmental toxins such as solvents, industrial and metal dusts, pigments and oils used in the automotive industry as a potential risk factor for IAC, but not PSC. We are meanwhile able to confirm our results in a total of 68 patients with IAC, PSC or carcinoma of the bile ducts or pancreas using this expensive and time-consuming NGS technique. Based on these results, we have now developed a novel, by far more affordable, widely applicable quantitative polymerase chain reaction (qPCR) protocol analyzing IgG4 RNA of B-cells in blood. In the 80 patients tested so far, the diagnostic accuracy of the simple new test was remarkable. Thus, IgG4+ B-cell receptor clones and IgG4 RNA may distinguish PSC (and cholangiocarcinoma) from IAC better than the tests so far available and may become a valuable diagnostic tool in the future. We continue our efforts to further validate the new diagnostic qPCR test in a large prospective study. In addition, we focus on unraveling the proteins against which the IgG4 are directed in IAC and PSC to better understand the differences in the inflammatory processes in these diseases.

2016 Update

With financial support of the PSC partners seeking a cure we recently unraveled that the IgG4-producing B cells in patients with active IgG4-RD of the biliary tract (IAC) or pancreas (autoimmune pancreatitis; AIP) are derived from a limited number of highly expanded IgG4+ B cell receptor (BCR) clones, by applying novel next generation sequencing (NGS) technology. These IgG4+ BCR clones were not found in healthy and diseased controls with PSC and hepatobiliary or pancreatic cancer. These findings indicated that development of an accurate (sensitive and specific) diagnostic marker for IAC was within reach. For comparison, conventional serum IgG4 did not reach an acceptable accuracy in distinguishing IgG4-RD from PSC and hepatobiliary/pancreatic malignancies. Still, NGS technology is work-intensive, expensive and unaffordable for routine clinical use. Based on our findings using NGS technology, we recently developed a widely affordable qPCR test (compared to NGS technology) to use blood IgG4/IgG RNA-ratio as marker for early diagnosis of IAC. We found that blood IgG4/IgG RNA-ratio as determined by a simple qPCR reached high accuracy for delineation from PSC and hepatobiliary and pancreatic malignancies comparable to that of IgG4+BCR clones identified by NGS.  IgG4/IgG RNA-ratio was also demonstrated to be potentially useful for monitoring of IAC disease activity during treatment . Use of this novel IgG4/IgG RNA-ratio was recommended as diagnostic test for patients evaluated for possible IgG4-related pancreatobiliary disease.

References

Hubers LM, Maillette de Buy Wenniger LJ, Doorenspleet ME, et al. IgG4-associated cholangitis: a comprehensive review. Clin Rev Allergy Immunol 2015;48:198-206.Maillette de Buy Wenniger LJ, Culver EL, Beuers U. Exposure to occupational antigens might predispose to IgG4-related disease. Hepatology. 2014;60:1453-4Maillette de Buy Wenniger LJ, Doorenspleet ME, Klarenbeek PL, et al. Immunoglobulin G4+ clones identified by next-generation sequencing dominate the B cell receptor repertoire in immunoglobulin G4 associated cholangitis. Hepatology 2013;57:2390-8.Doorenspleet ME, Hubers LM, Culver E, et al. IgG4+ B-cell receptor clones distinguish IgG4-related disease from primary sclerosing cholangitis and biliary/pancreatic malignancies. Hepatology 2016;64:501-7.

Tabibian JH, Lindor KD. Distinguishing IgG4-related disease from its pancreatobiliary mimics: Are we there now? Hepatology 2016;64:340-2.

Read MoreImmunoglobulin G41 B-Cell Receptor Clones Distinguish Immunoglobulin G4-Related Disease From Primary Sclerosing Cholangitis and Biliary/ Pancreatic MalignanciesDistinguishing Immunoglobulin G4–Related Disease From Its Pancreatobiliary Mimics: Are We There Now?This project is partially funded by the generous support of Don and Ricky Safer.

Accelerated Identification of Preclinical Models for Primary Sclerosing Cholangitis (PSC) Using IBM Watson Cognitive Technology

Lisa B. Boyette, MD, PhD, SAVE JON, Inc., Pittsburgh, PA

Amount Awarded: $30,000 over a period of one year

Genetically mutated strains of mice or other small animals sometimes exhibit features of human disease.  These small animal “models” are tremendously useful for studying a disease and testing therapies for that disease.  Finding the right genetic mutation such that the mouse’s disease resembles a specific human disease is usually a matter of chance when the cause of the disease is not understood.  There are currently no good animal models for Primary Sclerosing Cholangitis (PSC), but identifying one or more would open new avenues for understanding the causes of PSC.  We propose to accelerate this process of identifying good animal models for PSC.  Using the cognitively enabled IBM Watson supercomputer, we will search all available published medical literature and animal model databases for small animal models exhibiting disease features of PSC.  We will use this information to formulate a comprehensive list of possible models for PSC from records of thousands of existing animal models currently in use to study other diseases.  We will publish a rank-ordered list of our findings, annotated with all of the information we were able to gather about each model, and make this data easily accessible to the entire PSC research community.  In the future tissues can be obtained from each of those animals to validate them as model organisms for PSC studies in the lab.

Update:

Laboratory animals mimicking human diseases are valuable resources that help us understand diseases and develop cures. However, as we do not have a complete understanding of what causes PSC, an animal model that mimics PSC perfectly has not yet been established through genetic engineering, and such a model has not spontaneously emerged for us to study. The animal model traditionally used for reference in PSC research is a mouse with a mutation in a gene called MDR2. This mouse has a liver disease that is very similar to the fibrosis (scarring) observed in the bile ducts and livers of PSC patients. However, mice with an MDR2 mutation do not display an increased frequency of bile duct cancer, which does occur in the human PSC population. Furthermore, PSC affects more men than women and can involve many symptoms outside of the liver, most notably IBD and symptoms that overlap with other autoimmune disorders. Therefore, we performed a large-­scale analysis of all mice with known gene modifications to determine if any other mice have features that mimic multiple aspects of human PSC. From our analysis, we produced a list of gene modifications that are known to affect the liver, digestive system, immune system and tumor development in mice. The features produced by these gene mutations were examined in detail, and the genes were then ranked for their ability to cause aspects of PSC in mice. However, the mouse symptoms recorded in association with gene changes often varied with the particular genetic background of each mouse and the environmental challenges presented to them. Furthermore, some mice that showed potential mimicking of PSC need to be analyzed in greater detail. Therefore, from our list we have created a database that will be made publically available to the PSC research community. Our database will guide researchers towards potential models of interest for PSC research that should be further examined in the context of different genetic and environmental conditions. As investigations ensue and more information is gathered, the database will be updated to refine the rankings and provide researchers with a reference tool detailing more specific mouse models for PSC..

This one year project is funded by the generous support of John and Chris Browner, David and Rosalind Parry.

Aberrant Dendritic cell functions that cause inflammatory bowel disease

Gianna E. Hammer, PhD, Duke University, Immunology Department, Durham, NC

Amount Awarded: $60,000 over a period of two years

Although patients with primary sclerosing cholangitis (PSC) often also have inflammatory bowel disease (IBD), next to nothing is known about the cell types and mechanisms that connect these two diseases. A major block to deciphering these connections is the lack of animal models that develop both PSC and IBD. In IBD pathophysiology, we have identified a key cellular and molecular module in mice that drives disease: dendritic cells that lack the intracellular enzyme A20 cause IBD by aberrantly activating inflammatory immune cells. How inflammatory responses of dendritic cells may cause PSC, and whether dendritic cells in the liver also require A20 to prevent aberrant immune activation, is unknown. The goal of this proposal is to define the role of A20 in dendritic cell subsets of the intestine and liver. Additionally, we will identify PSC-related pathologies in the livers of mice lacking A20 expression specifically in dendritic cells—we propose that these mice may be a novel animal model for the study of both IBD and PSC.

2015 Update

We have found a key pathological cell type that can cause inflammation in both the liver and the intestine: the dendritic cell. Dendritic cells (DCs) are part of the immune system and serve two functions: 1) Sense microbes and 2) Instruct the rest of the immune system to the presence of these microbes. We have been testing these DC functions in the intestine and determining how these functions are related to microbial commensals. We have identified DCs that interact with microbial commensals and determined that DCs that are hyper-active to microbial commensals incorrectly instruct pro-inflammatory immune responses in the intestine. These pro-inflammatory immune responses are largely the result of T cells that secrete dangerous molecules once they encounter a microbial target. We are further testing whether these same mechanisms are operating in the liver and whether T cells that cause inflammation in the intestine are similar to T cells that may cause inflammation in the liver.

2016 Update:

To understand the links between gut inflammation and liver inflammation we are studying dendritic cells. Dendritic cells are immune cells that live in both liver and gut. In these tissues dendritic cells interact with bacteria and other immune cells. We study dendritic cells because dendritic cells are like the volume button of the immune system. When dendritic cells have a very high response to gut bacteria, they give out signals to other immune cells. These signals tell other immune cells to make proteins that cause inflammation. These intense immune responses are responsible for inflammation. There are different types of dendritic cells and we found that each has a unique response to gut bacteria. Interestingly, when the response to gut bacteria is very high, each type of dendritic cell sends out a different kind of signal. Each type of signal tells immune cells to produce a different type of inflammatory protein. For example, we found one type of dendritic cell gives the signal to make the inflammatory protein IFN. And a second type of dendritic cell gives the signal to make a totally different inflammatory protein, IL-17. These inflammatory proteins cause gut inflammation.

Once we learned that certain types of dendritic cells were responsible for specific inflammatory proteins, we designed a type of therapy to block gut inflammation. Using mice, we eliminated the specific gut dendritic cell that gives out the signal for the inflammatory protein IL-17. By getting ride of this dendritic cell we successfully reduced the amount of IL-17 in the gut and reduced the level of gut inflammation. Now that we understand how dendritic cells cause gut inflammation, we will determine how these dendritic cells impact liver inflammation. We will also test whether liver dendritic cells behave similar to gut dendritic cells. Once we answer these questions our research could lead to new therapies for PSC.

Publications:

Liang J, Huang H, Benzatti F, Karlsson AB, Zhang JJ, Youssef N, Ma A, Hale LP and Hammer GE. (2016). Inflammatory Th1 and Th17 in the intestine are each driven by functionally specialized dendritic cells with distinct requirements for MyD88. Cell Reports (accepted, in press)

This project is partially funded by the generous support of the Grover Family Foundation and Deborah Wente.

The Role of CD1D In Regulating Inflammation in PSC

Espen Melum, MD, PhD, Norwegian PSC Research Center, Oslo University Hospital, Rikshospitalet, Oslo, Norway

Amount Awarded: $60,000 over two years

Primary sclerosing cholangitis (PSC) is a chronic inflammatory biliary disease. At present little is known about the pathogenesis of PSC. Findings in genetic studies clearly suggest that inflammatory mechanisms are involved. Natural killer T (NKT) cells are activated by lipid antigens, and represent a major lymphocyte population in the liver with potent regulatory properties that are likely to regulate immune driven liver diseases.This project aims for in-depth exploration of the role of NKT cells in the bile ducts and PSC by using human samples and an induced mouse model of cholangitis, resembling PSC. The level of CD1D, the molecule that activates NKT cells will be examined in liver from patients with PSC and compared to controls. We have previously demonstrated that the cells lining the bile ducts (cholangiocytes) can activate NKT cells and now we plan to develop a novel mouse model where we can examine this. This model will increase our understanding of NKT cells in the bile ducts and how their activation is affecting the inflammation. The establishment of a novel induced murine model can also be used to test other interventions and potential treatments in future studies.

2015 Update

During the first year of funding from PSC Partners we have closely examined certain molecules involved in activating an important subset of lymphocytes called NKT cells. In these studies we have demonstrated that these activating molecules are decreased in PSC patients, implying a potential of less ability to activate these regulatory cells.

It is absolutely necessary to have proper animal models to move the field of PSC research further. In an attempt to establish such a model, we have developed a novel surgical technique where we are able to inject substances into the bile ducts of mice under anesthesia. This model can be useful for testing compounds directly injected into the bile ducts of diseased livers and to elicit inflammation in healthy livers.

Our first experiments using this novel model have been to inject irritants that can activate the lymphocyte subsets mentioned in the first paragraph. To aid these studies we have used genetically modified mice that lack these NKT cells and thus been able to demonstrate the activation of these cells in the bile ducts of normal wild type animals.

During the next year we will substantially expand on the murine models and try further to dissect the mechanisms that govern this induced inflammation in the bile ducts. We are extremely grateful for the funding received from PSC Partners Seeking a Cure that has enabled these studies. PSC Partners Seeking a Cure will be acknowledged in all publications based on these results.

2016 Update

As it is absolutely necessary to have proper animal models to move the field of PSC research further, we aimed at establishing a new mouse model to study bile duct inflammation and the role of bile duct cells in disease. During the first year of funding we began the work of developing a novel surgical technique that would allow injection of substances into the bile ducts of mice under anesthesia. This model can be useful for testing compounds directly injected into the bile ducts of diseased livers and to elicit inflammation in healthy livers.

After much work with optimizing the surgical technique we finally succeeded in the establishment of the surgical technique. In the past year we have applied this technique to inject irritants into the biliary tree, which allows us to study the role of the above-mentioned NKT cells and their activating molecule in bile duct inflammation. We have run multiple experiments in normal wild type mice and genetically modified mice that lack these NKT cells. We have now successfully established a bile duct inflammation model that is driven by NKT cells. While normal wild type mice develop disease when injecting an irritant into the bile ducts, the genetically modified animals that lack NKT cells are somewhat protected from disease. Furthermore, we have been able to inhibit development of disease in normal wild type mice by treating them with blocking of the NKT activating molecule before surgery. We are presently working with preparation of manuscripts for both the establishment of the new surgical technique and the bile duct inflammation model.

We are extremely grateful for the funding received from PSC Partners Seeking a Cure that has enabled these studies. PSC Partners Seeking a Cure will be acknowledged in all publications based on these results.

Published results (Note: link for publication included below) The biliary epithelium presents antigens to and activates natural killer T cells.

Schrumpf E, Tan C, Karlsen TH, Sponheim J, Björkström NK, Sundnes O, Alfsnes K, Kaser A, Jefferson DM, Ueno Y, Eide TJ, Haraldsen G, Zeissig S, Exley MA, Blumberg RS, Melum E. Hepatology, 2015 Oct;62(4):1249-59. doi: 10.1002/hep.27840.

Natural Killer T cells aggravate inflammation in a novel induced murine model of cholangitis. Berntsen NL, Fosby B, Tan C, Schrumpf E, Bjornetro T, Foss A, Line PD, Karlsen TH, Blumberg RS, Melum E. AASLD 2015 in San Francisco.

This project is partially funded by the generous support of Don and Ricky Safer.

Role of Natural Killer Cells in the Pathogenesis of Primary Sclerosing Cholangitis

Guoxiang Yang, MD, PhD, Department of Internal Medicine, Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, CA

Amount Awarded: $60,000 over two years

Primary sclerosing cholangitis (PSC) is an autoimmune disease that destroys the bile ducts in the liver and can lead to liver failure. Most people with PSC also have ulcerative colitis or Crohn’s disease, two type of inflammation that affect the large intestine. Current evidence suggests that the immune cells that become active in the intestine lead to the inflammation and damage caused by PSC in the liver. One type of immune cells which might cause this damage are called natural killer (NK) cells. These cells have been identified as central to several other autoimmune diseases and recent genetic data implicate them in PSC as well. Our pilot data has shown that NK cells are activated in the blood of PSC patients. We believe that these NK cells originate in the intestine and subsequently migrate to the liver where they cause PSC. Our project will determine if the NK cells of PSC reflect a unique type of immune cells and will provide evidence of the potential role of these cells in the targeted destruction of bile ducts. Altogether, this application will provide important insights into the link between PSC and IBD. We believe that these studies have the potential to identify new therapeutic approaches to the treatment of PSC.

2015 Update

Primary sclerosing cholangitis (PSC) is a chronic autoimmune disorder targeted to intra and extra-hepatic bile ducts. Although cellular and genetic studies have shown that nature killer (NK) cells are involved in the pathogenesis of PSC, the immunological mechanism is unclear. Herein, we dissected the circulating NK cells and compared the phenotypic and functional characteristics of different subsets of NK cells from patients with PSC to that from healthy controls. We utilized special technique, called single cell staining, to evaluate the specific molecules expressed on the cell surface. We found that the number of NK cells is decreased in peripheral blood in patients with PSC. These cells also exhibited immature phenotype and were more activated. Furthermore, the NK cells from patients with PSC have a great capacity to kill the target cells. Collectively, our data demonstrated that in patients with PSC, deficiency in maturation with aberrant enhanced cytotoxicity in the NK subset is intriguing and significant. This distinct population of NK cells could be functionally involved in the pathogenesis of PSC. Of particular importance in our next step of study is to determine 1) if these aberrant NK cells are present in the liver; and 2) whether these NK cells have biliary epithelial cell cytotoxic activity.

This project is partially funded by the generous support of Mohammed Aslam and Fred Sabernick.

Awarded in 2013

In 2013, PSC Partners funded seven PSC research studies.

Whole exomes sequencing of primary sclerosing cholangitis patients with severe disease

Eva Ellinghaus, MD and Andre Franke, MD, Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Germany.

Amount Awarded= $60,000 over two years

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the intra- and extra hepatic bile ducts. There is scarce knowledge on etiology and pathogenesis of PSC, and due to lack of effective therapy, PSC has become one of the leading indications for liver transplantation in Northern Europe and the US. The strong genetic component to PSC risk is highlighted by an approximately 9-39 fold increase in sibling risk relative to the general population and recent studies have identified 16 susceptibility regions in the human genome that increase the risk for developing PSC. These, however, explain only 7.3% of variance in PSC liability. Therefore, to identify further sources of heritability, e.g. rare susceptibility variants with potentially high penetrance, systematic resequencing studies need to be performed.

By using a next-generation sequencing-based approach, it is planned to sequence the exomes, i.e. the protein-coding part of the human genome, of 48 unrelated PSC patients with severe disease to dissect the contribution of rare and low frequency coding variants to the complex phenotype of PSC. Newly identified mutations with potentially damaging effect will be validated and followed up in a large case-control sample. Functional analysis of these mutations is aimed for and the ultimate goal is to identify potential targets for therapeutic intervention.

Progress Update: Read a November 2014 progress update report on this grant.

Read the final report for this grant.

Role of epithelial toll like receptor dependent innate immune responses in the pathogenesis and treatment of primary sclerosing cholangitis

Romina Fiorotto, PhD, Mario Strazzabosco, MD, PhD, Department of Internal Medicine, Section of Digestive Diseases, Yale University and Tom H. Karlsen, MD, PhD, Norwegian PSC Research Center, Oslo, Norway.

Amount Awarded: $60,000 over two years

The etiology and pathophysiology of PSC remain still unclear. Two hypotheses have been proposed. The association with inflammatory bowel disease (IBD) suggests an involvement of innate immune mechanisms as a possible trigger. On the other hand, the retention of toxic bile acids able to damage the biliary epithelium is proposed as an alternative hypothesis (‘toxic bile’ hypothesis). However a common pathogenetic mechanism that explains the progression of the disease is still lacking. PSC has several common features with cystic fibrosis (CF), a genetic disease caused by mutation in CFTR, a chloride channel involved in biliary secretory function. Using a mouse model of CF we have recently described that lack of CFTR alters the innate immune responses in the biliary epithelium to endotoxins. In addition, we found that experimental bile acid toxicity causes more severe liver damage in CF mice and induces the expression of High Mobility Group Box-1 (HMGB-1), a molecule known to activate TLRs 4 and 2 mediated innate immune responses. Furthermore, we have preliminary evidence that cholangiocytes isolated from PSC patients present enhanced innate immune responsiveness to endotoxins.

Based on our recent findings, we propose that different types of biliary damage may be responsible for aberrant activation of the innate immunity in CF as well as in PSC and that activation of innate immune pathways represent a common event in the pathogenesis of sclerosing cholangitis. To pursue our hypothesis, we aim to study in vivo the effect of bacterial components and toxic bile acids on the innate immune response of the biliary epithelium in the CF mouse model. We expect to find the release of specific molecules that trigger innate immune responses after liver injury. We will then use these molecules to challenge in vitro CFTR-defective mouse cholangiocytes and human cholangiocytes isolated from PSC patients and show the activation of specific immune pathways. If our hypothesis is correct, it would also suggest that targeting the biliary innate immunity responses would represent the correct approach to treat sclerosing cholangitis. Therefore, in line with the recent reported anti-inflammatory and immune modulator role of vitamin D nuclear receptor (VDR) we will then test the effect of vitamin D treatment as a novel therapeutic approach to modulate the biliary epithelium innate immunity in vivo in the CF mouse model and in vitro in CFTR-defective mouse cholangiocytes and in human cholangiocytes isolated from PSC patients. These studies will have a strong translational potential for the treatment of PSC.

The first year of this project was funded by a generous donation from Abe and Rachel Gomel.

Read the final report for this Grant including the article published in the AASLD Journal of Hepatology.

Bile acid activated receptors TGR5 and FXR as therapeutic targets for the treatment of PSC

Stefano Fiorucci, MD and Barbara Renga, PhD, Department of Experimental and Clinical Medicine, University of Perugia, Italy.

Amount Awarded: $60,000 over two years

Primary sclerosing cholangitis (PSC) is a rare chronic cholestatic disorder of the liver and bile ducts that is characterized by fibrosing inflammation of the intra- and/or extrahepatic biliary tree generally leading to progressive bile duct obstruction, fibrosis, cirrhosis and end-stage liver disease. The pathogenesis of PSC remains enigmatic, making the development of targeted therapeutic strategies difficult. Primary sclerosing cholangitis, like other chronic cholestatic liver diseases, is characterized by hepatic retention of potentially toxic substances normally excreted into bile, in particular hydrophobic bile acids, as a consequence of impaired hepatobiliary secretion and/or obstruction of bile flow. Elevated levels of bile acids can lead to hepatocyte injury, liver failure and the need for liver transplantation.  Bile acids are endogenous ligands for the nuclear receptor farnesoid X Receptor (FXR) and the G-protein coupled receptor TGR5. FXR agonists are not the first candidate for long-term treatment of PSC. On the contrary, it has been recently demonstrated that FXR antagonism could be useful in the treatment of liver cholestasis and that theonellasterol, a natural highly selective FXR antagonist, exerts beneficial effects in the treatment of obstructive cholestasis. TGR5 has been recently identified as an important candidate gene for several inflammatory and metabolic conditions. Indeed, mutations of TGR5 gene were detected in both ulcerative colitis (UC) and PSC. The specific aims of this study are to investigate the effect of an FXR antagonist, such as the marine steroid theonellasterol, alone or in combination with UDCA in animal models of PSC and to evaluate the role of TGR5 in the pathogenesis of PSC and the effect of TGR5 agonists in the treatment of PSC.

This project will be entirely funded by generous donations from David and Ros Parry and ITW. Progress Update: Read a November 2014 progress update report on this grant.

Bile acid-regulation of mast cells in models of primary sclerosing cholangitis and cholangiocarcinoma

Heather Francis, PhD, Assistant Professor and Sharon DeMorrow, PhD, Associate Professor, Texas A&M Health Science Center.

Amount Awarded: $60,000 over two years

Primary Sclerosing Cholangitis and the primary liver cancer, cholangiocarcinoma, are devastating liver diseases that target cells within the liver called cholangiocytes.  These diseases have limited treatment options.  Patients that present with Primary Sclerosing Cholangitis have a higher incidence of developing cholangiocarcinoma. When cholangiocytes (that line bile ducts where bile flows out of the body) become damaged and ducts become blocked, cholangiocytes will increase in number and this disrupts the normal state of the liver and bile flow. Bile acids are synthesized by the liver to aide in the digestion of foods and the secretion of bile. Patients with liver disease are sometimes treated with bile acid therapy to attempt to improve liver function.  Ursodeoxycholate is a secondary bile acid that is used in patient therapy for Primary Sclerosing Cholangitis and has been found to have beneficial effects in patients suffering from these diseases.  Besides cholangiocytes, mast cells (inflammatory cells that release histamine in the body) are found in the liver and increase in number during liver disease.  When mast cells are treated with bile acids there is a release or blockage of histamine release (depending on the type of bile acid). Our proposal will aim to determine if mast cells and cholangiocytes interact with each other during disease progression and if bile acid treatment can alter their interactions. We will use animal models of both Primary Sclerosing Cholangitis and cholangiocarcinoma as well as cell culture lines to evaluate our specific aims. We believe that by altering mast cell histamine release, cholangiocytes will be repaired and disease progression may be halted.

The first year of this project will be funded by a generous donation from Ken and Patty Shepherd in addition to the proceeds from their annual fundraiser.

Progress Update: Read a November 2014 progress update report on this grant.

Progress Update: December 2015

To date, we have found that mast cells infiltrate the liver during PSC and cholangiocarcinoma progression. Once these cells migrate to the liver they release numerous mediators that can negatively impact a patient’s prognosis. One of the main factors released is histamine that can contribute to the progression of liver injury or tumor growth.  Since we have found a large number of mast cells in the liver following injury, we recently developed a technique to isolate or extract these cells from rodent liver giving us a tool to better examine their effects on PSC and cholangiocarcinoma models. Further, following treatment with cromolyn sodium, a compound that blocks the release of histamine from mast cells, we have demonstrated that there is a decrease in liver fibrosis (which is a consequence of diseases like PSC) and tumor growth.  We have found that treatment with ursodeoxycholate decreases the release of histamine from mast cells, which may also be an important finding in the treatment fibrosis or cholangiocarcinoma.  Our results demonstrate that targeting mast cells may be a potential therapy for patients suffering from PSC and cholangiocarcinoma.  Using human patient samples we have demonstrated that in PSC patients (advanced and late stages) there is an infiltration of mast cells compared to normal liver samples and histamine levels increase significantly in these patients.

Progress Update: May 2016

Read the final report and see the publications generated as a result of this grant.

The cholangiocyte senescence associated secretory phenotype as a therapeutic target in primary sclerosing cholangitis

Nicholas F. LaRusso, MD, Steven P. O’Hara, PhD, and James H. Tabibian, MD, Mayo Clinic Center for Cell Signaling in Gastroenterology.

Amount Awarded: $30,000 for one year.  This is a follow up from previous funding.

Primary sclerosing cholangitis (PSC) is a multifactorial disease believed to have genetic, immunologic, microbial, and other environmental components.  Emerging evidence suggests that cholangiocytes, i.e. the cells that line the bile ducts, may not only be cellular targets of PSC, but participate in driving the disease.  In this regard, our recent data suggest that a subset of cultured (i.e. in vitro) cholangiocytes, in response to certain injurious stimuli, undergo transition to a cellular state in which they are no longer able to replicate but remain capable of secreting pro-inflammatory and pro-fibrotic mediators—and do so in excess amounts.  We believe, and our data support, that this state is representative of a senescence-associated secretory phenotype (SASP).  SASP cholangiocytes, as newly presented by us at the 2013 AASLD Presidential Plenary Session, “Advances in Hepatology”, are not only induced experimentally, but are also present in PSC liver specimens as well as an animal model of PSC.  Therefore, based on our ongoing work in this area, we propose that chronic exposure to injurious, SASP-derived molecules promotes biliary injury, thereby contributing to the development and progression of PSC.  In this proposal, we will examine the mechanisms, consequences and pathologic outcomes of inhibiting cholangiocyte SASP through different techniques in both cholangiocyte culture and an animal model of PSC.  We believe that in doing so, we may identify novel therapies for further study, with the hope that they will ultimately be sufficiently promising for clinical trials in patients suffering from this disorder.

Progress Update: Read a July 2014 progress update report on this grant.

Linking gene susceptibility and environmental factors to biliary damage in primary sclerosing cholangitis: the CD28/vitamin D axis

Evaggelia Liaskou, PhD, Post-Doctoral Research Fellow and Gideon M. Hirschfield, NIHR Biomedical Research Unit in Liver Disease & Centre for Liver Research. Institute of Biomedical Research School of Immunity and Infection, University of Birmingham, England.

Amount Awarded: $60,000 over two years

Primary sclerosing cholangitis (PSC) is a chronic autoimmune liver disease that is seen across all ages. Over time damage to the bile ducts can cause bile duct obstruction, secondary liver scarring and also a risk of cancer. A combination of environmental and genetic factors are important in why someone gets PSC, therefore understanding the interaction between these two factors will help us understand better the causes of disease, something critical to helping develop new drug treatments.

Many genes and environmental factors have now been linked to PSC. We are interested in vitamin D levels in PSC, because low levels are associated with other autoimmune diseases. We are also interested in one particular gene associated with PSC, called CD28, because this gene is very important in controlling how cells respond to injury. Some white blood cells that lose the ability to make CD28 protein are more inflammatory and such cells are more frequently found in autoimmune diseases.

In our project we are going to study how in PSC the cells that lose CD28 work, whether they cause bile duct inflammation, and if there is an association between a person’s gene signature, and CD28 function. We’ll also look to see if vitamin D is important in this particular pathway, particularly as we know if it is possible to supplement vitamin D in patients.

Work to Date

Primary sclerosing cholangitis (PSC) is a devastating and progressive disorder in which persistent inflammation of the bile ducts (the vessels that collect the bile made by hepatocytes and drain into the gall bladder for storage) results in scarring with blockage of the bile flow and subsequent risk of liver failure and/or bile duct cancer. PSC is considered an autoimmune disease where the patient’s own immune cells are attacking parts of the body. Currently, patients with PSC have no effective medical treatment and frequently need liver transplantation, with the chances of getting the disease again in their new liver being quite high. A combination of environmental and genetic factors are important in why someone gets PSC, therefore understanding the interaction between these two factors will help us understand better the causes of disease, something critical to helping develop new drug treatments. To achieve this patient support in funding our research has been very helpful.

Many genes and environmental factors have been linked to PSC. In our study we were interested in one particular gene called CD28, because this gene is very important in controlling how cells respond to injury. Some white blood cells that lose the ability to make CD28 protein are more inflammatory and such cells are more frequently found in autoimmune diseases. Thus we studied how in PSC the cells that lose CD28 work, whether they cause bile duct inflammation and whether the environmental factor vitamin D is important in this particular pathway.

Our data have shown that a significant proportion of white blood cells, called CD4 and CD8 T cells, found in the liver of PSC patients, have lost the expression of CD28 protein. We found that these CD28 null T cells are highly active cells that express molecules on their surface that can lead them around bile ducts. Moreover, we found that these cells are able to activate and kill the bile duct cells. In the liver tissue of PSC patients we detected high levels of TNFα, and in the laboratory we could show that this molecule could reduce the expression of CD28 by T cells, an effect that was prevented by the administration of vitamin D. We have now extended our study to investigate whether there is an association between a person’s gene signature and CD28 function using samples from healthy volunteers.

Regulation of hepatic lymphocyte responses in pediatric sclerosing cholangitis

Alexander G. Miethke, MD, Assistant Professor of Pediatrics, Pediatric Gastroenterology, Hepatology, and Nutrition, Cincinnati Children’s Hospital Medical Center.

Amount Awarded: $60,000 over two years

It is currently unknown whether PSC in children is different from PSC in adults. Additionally, mechanisms that drive initiation and disease progression in pediatric PSC are not well described. In a mouse model of sclerosing cholangitis we found that T- and NK-lymphocytes infiltrated the liver during the early stages of disease. The surge of these lymphocytes which are capable of injuring bile duct epithelial cells was accompanied by an influx of regulatory T cells. Regulatory T cells represent a small proportion of T helper lymphocytes and are critical for constraining immune responses and preventing autoimmune disorders. Therefore, regulatory T cells seem to be an attractive cellular target for immunotherapy. Here, we propose experiments that manipulate number and activation of regulatory T cells in mice during the early stage of sclerosing cholangitis to better understand how these cells interact with other immune cells in the liver and whether they suppress immune-mediated bile duct injury.  In complementary studies on tissue samples from children affected by PSC we will examine which types of cells important for control of immune activation are present in the liver at the time of diagnosis. Collectively, this data may provide novel insights into mechanisms of hepatic immune activation in sclerosing cholangitis which might lead to new avenues of pharmacotherapy in pediatric PSC.

A significant portion of this project is funded by generous donations from the John and Chris Browner Family.

Progress Updates:

Read the November 2014 progress update report on this grant.

Read the January 2016 progress update report on this grant.  Also read the article Dr. Miethke et. al. published in the AASLD Hepatology Journal about this grant.

Awarded in 2012

In 2012, PSC Partners funded three PSC research studies.

A Combined Bile and Urine Proteomic Test for the Detection of Cholangiocarcinoma in Patients with Primary Sclerosing Cholangitis

Dr. Tim O. Lankisch, MD,Associate Professor of Medicine, Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; Dr. Jochen Metzger;Professor Michael P. Manns and Professor Harald Mischak.

Amount Awarded: $60,000 over two years.

The detection of cholangiocarcinoma (CC) remains a diagnostic challenge particularly in patients suffering from primary sclerosing cholangitis (PSC) who are known to develop CC in approximately 15% of cases. Therefore, the aim of scientific contributions must be directed to early diagnosis of CC with high accuracy. Two case/control studies performed by our group, the one using bile, and the other urine as sample matrix suggested that proteomic analysis is a feasible method to achieve this aim since this has led to peptide multi-marker models with classification accuracies above 80 %. PSC Partners has generously provided us with the resources required to analyze additional peptide marker candidates in bile and urine. This helps us to select those peptides which upon inclusion into our established bile and urine peptide marker sets will further improve classification performance. Particular emphasis will be placed on enhanced sensitivity of CC diagnosis since this will allow the combination of bile and urine proteomic analysis to a unique classification scheme. The later will include both local and systemic changes of CC development and progression into the diagnostic decision of CC during surveillance of PSC patients.

Read the final report for this grant.

Metagenomic Analysis of Bile Microbial Diversity in Primary Sclerosing Cholangitis

Dr. Patrick S.C. Leung, PhD, Adjunct Professor of Immunology, Division of Rheumatology/Allergy, University of California Davis School of Medicine; Jonathan Eisen, PhD and Hoi Shan Kwan, PhD, The Chinese University of Hong Kong.

Amount awarded: $60,000 over two years.

The frequent association of primary sclerosing cholangitis (PSC) and inflammatory bowel diseases strongly implicates that translocation (movement) of bacteria/bacterial antigens into the liver can trigger immunological responses in the biliary tract. Such immunological responses that arise from cross-recognition between microbial antigens and self might lead to chronic liver disorders in PSC. We believe evidence of such microbial translocation could be found in the bile from patients with PSC. This study is directed to define the bacterial populations in bile samples obtained from patients with PSC and controls. We will use the state of the art “in low cost high throughput ultra sequencing” (next generation of gene sequencing) to determine the nucleotide sequence of bacterial ribosomal RNA gene in bile samples. Sequences will then be analyzed against DNA data bank to find out what the candidate microorganisms are and how they are related to the gut microbes in these samples. Data obtained could shed light on future work in defining the microorganisms responsible for biliary pathology (behavior) in PSC.

Read the final report for this grant.

Characterization of the Intestinal Microbiome in UC Patients With and Without PSC

Dr. Mark S. Silverberg, MD, PhD, Associate Professor, University of Toronto, Canada; Dr. Gert Van Assche and Dr. David Kevans.

Amount awarded=$10,000.

The Inflammatory Bowel Diseases (IBD), Ulcerative Colitis (UC) and Crohn’s Disease (CD) affect about 0.5% of North Americans. UC involves only the large bowel while CD may involve any part of the gastrointestinal tract. Primary Sclerosing Cholangitis (PSC) is a liver condition that affects approximately 2-5% of patients with IBD. Patients with IBD affecting their colon are at increased risk of developing colon cancer (CRC), a risk which is even further elevated in patients with colonic IBD and PSC. Why patients with IBD involving the colon are at increased risk of colorectal cancer and why this risk is even further elevated in patients with concurrent PSC is not known. Additionally, the causes of PSC are unknown and there are few effective therapies. Eventually, individuals affected by PSC may require liver transplantation.

The human intestine contains huge numbers of bacteria and it has been shown that these bacteria may trigger UC and CD. Emerging evidence has also suggested that bacteria may play a role in determining disease behaviour and cancer development. It is speculated that the particular composition of the bacteria in the large intestine may be an important factor causing PSC. New techniques now allow the characterisation of intestinal bacteria using tissue and stool samples. The aim of this study is to assess the amount and type of intestinal bacteria in UC patients with and without PSC to evaluate whether there are differences between these groups which may explain their contrasting disease behaviour. We will also assess whether the composition of intestinal bacteria within these groups is influenced by genetic factors and immune system responses. Our findings may provide insights into the processes underlying disease behavior, cancer development and PSC evolution in UC and potentially lead to new diagnostic and treatment strategies.

Awarded in 2011

The recipients of the 2011 awards include Geoff Baldwin, PhD, Imperial College, London, UK; Nicholas F. La Russo, MD, The Charles H. Weinman Professor of Medicine at the Mayo Clinic in Rochester, MN; and Rinse W. Weersma, MD, PhD, Associate Professor, The University of Groningen, The Netherlands.

A Nano-Device for the Early Stage Detection of Cholangiocarcinoma

Dr Geoff Baldwin, Reader in Biochemistry, Imperial College London; Professor Richard Kitney; Professor Paul Freemont and Mr. James Field.

Amount Awarded: $60,000 over two years.Final Lay Report, September 2015

As many PSC sufferers will be aware, cholangiocarcinoma (CCA) is a bile duct cancer and PSC predisposes these individuals to developing this cancer so that it may occur in up to 40% of sufferers. Of great concern is that it is ultimately a leading cause of death for PSC patients. If identified at an early stage, CCA can be treated by resection of the liver. Unfortunately for individuals with PSC, the inadequacy of current screening technologies means that CCA is frequently diagnosed at a late stage when palliative care is the only remaining option.

We obtained funding from PSC Partners to try and change this situation. Our approach has been to develop protein nanocages as a platform for early stage diagnosis of CCA. Protein nanocages (PNs) are naturally occurring spherical structures with large internal cavities. We have developed methods to fluorescently label these PNs so that they can be seen with high sensitivity. In addition we have conjugated them to antibodies. Since antibodies are known that target specific cancer markers on cells, this enables the specific targeting of the PNs to cancer cells. We have shown that the fluorescent PNs can thus be targeted to CCA cells as well as other cancer cell types.

In principle these results demonstrate that PNs can be used as a platform for diagnosis of cancer. However, there is still a long way to go before we can use this in the clinic. We are really focused on keeping the target of clinical application in our sights and so we are now working with clinicians at Hammersmith hospital in London to establish whether we can target cancer in liver tissue from patients.

We are continuing to work with PNs to develop them for diagnostic and therapeutic applications by simultaneously delivering drugs within the cage. Through this we now have interest from pharmaceutical companies and we look forward to exploring the exciting opportunities for protein nanocages.

Potential Role of Cholangiocyte Senescence in PSC.

Nicholas F. LaRusso, MD, Mayo Clinic Center for Cell Signaling in Gastroenterology, Mayo Clinic.

Amount Awarded: $60,000 over two years.

Primary sclerosing cholangitis (PSC) is a multifactorial disease with genetic, microbial, and environmental components. Emerging evidence suggests that cholangiocytes, i.e. the cells of the bile ducts, may not only be affected in PSC, but may actually participate in driving disease progression. Our recent data suggest that cholangiocytes, in response to biologically-relevant injurious stimuli, transition from a proliferative to a senescent phenotype, a metabolically active cellular state in which the cell is no longer capable of cell division. Furthermore, these cells secrete excess amounts of inflammatory mediators. Based on our recent work, we propose that chronic exposure to injurious molecules promotes cholangiocyte senescence and secretion of inflammatory molecules, thereby contributing to the development and progression of PSC. The proposed experiments will establish cholangiocyte senescence as a fundamental cholangiocyte response to persistent injury, address the mechanisms by which relevant injurious stimuli induce cellular senescence, and interrogate whether PSC-associated senescent cells are mediators of disease. This is a novel approach to understanding the development and progression of PSC that may have important implications for understanding disease initiation/progression and provide insights for the development of novel therapies.

The Exome in PSC

Rinse K. Weersma MD, PhD, Associate Professor, Department of Gastroenterology and Hepatology, University of Groningen and University Medical Centre Groningen, The Netherlands; Prof. Dr. rer. nat. Andre Franke, Institute of Clinical Molecular Biology; Christian-Albrechts-University of Kiel, Universitätsklinikum Schleswig Holstein, Campus Kiel Schittenhelmstr.

Amount awarded: $60,000 over two years.

PSC is a complex disease, with both environmental and genetic factors involved in its development and disease course. It is thought that changes in multiple genes in the human DNA explain part of the etiology of the disease. In recent years it became possible to screen the human genome by using 100,000s or even millions of markers spread around the genome. Several regions on the human genome have now been identified that contain genes that are associated to disease. Since PSC is a rare disease and large numbers of patients are required, this work has been performed by multiple centers throughout the world that have collaborated. It is expected that additional genetic regions will be identified in the near future

By using these large numbers of genetic markers, we now know which broad regions on the human genome are associated with the disease, but we do not know which specific genetic variants within these regions are causing protein changes that lead to disease. Most of the associated variants are not causal, but merely markers for other functionally important, causative variants. Variants that reside in the so-called exons of the genes have direct implication in protein function and are therefore very interesting. Part of the heritability is thought to reside in these rare exomic variants with greater effect-sizes. Therefore, the primary aim of the study is the identification and confirmation of rare exomic variants involved in PSC pathogenesis.

This will be done by using a custom-made exome genotyping array including ≈250.000 variants, to genotype a cohort of 1000 PSC patients and 6000 healthy controls. A second cohort of 1000 cases and 1000 healthy controls is available for replication of observed signals. Identified variants will give important insights in disease pathogenesis. The variants on the exome array will have direct functional implications and will therefore enable additional functional studies and hopefully targets for novel therapeutic interventions. In addition we might identify mutations with predictive value for disease occurrence or disease behavior.

Progress Update: Read a September 2014 progress update report on this grant.

Awarded in 2010

In 2010, the Scientific/Medical Advisory Committee selected five proposals for two years of funding:

Pathogenesis of PSC: Role of TGR5 in the regulation of the innate immune response in the biliary epithelium

Mario Strazzabosco, M.D., Ph.D, Department of Internal Medicine Section of Digestive Diseases, Yale University, 333 Cedar Street-1080 LMP, P.O. Box 208019, New Haven, CT 06520-8019, and Michael Trauner, M.D., Ph.D, Department of Internal Medicine III, Divison of Gastroenterology and Hepatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

Amount Awarded: $40,000 over years.

Epigenetics associated with primary sclerosing cholangitis in monozygotic twins discordant for the disease

Carlo Selmi, MD, PhD, Assistant Professor of Internal Medicine, University of Milan, Physician Scientist, IRCCS Istituto Clinico Humanitas, Rozzano, Milan, Assistant Professor of Medicine, University of California, Davis, Division of Rheumatology, Allergy, and Clinical Immunology, GBSF suite 6515, 451 E Healthy Sciences Drive, Davis, CA 95616.

Amount Awarded: $40,000 over 2 years.

Identification of genetic profiles unique to PSC-IBD

Judy Cho, M.D., Associate Professor of Medicine and Genetics, Section of Digestive Diseases, Director, Inflammatory Bowel Disease Center, Yale University, Internal Medicine, 333 Cedar Street, Room LMP-1072, P.O. Box 208056, New Haven, CT 06520-8056.

Amount Awarded: $40,000 over 2 years.

Combination treatment with ursodeoxycholic acid and all-trans retinoic acid for primary sclerosing cholangitis (PSC)

Shi-Ying Cai, Ph.D., Research Scientist, Section of Digestive Diseases, Department of Internal Medicine, Yale Liver Center, Yale University School of Medicine, 1080 LMP, 333 Cedar Street, New Haven, CT 06520, and James L. Boyer, M.D., Ensign Professor of Medicine, Section of Digestive Diseases, Department of Internal Medicine, Yale Liver Center, Yale University School of Medicine, 1080 LMP, 333 Cedar Street, New Haven, CT 06520.

Amount Awarded: $37,000 over 2 years.

Establishing the role and molecular mechanisms for pregnane X receptor in progressive sclerosing cholangitis

Sridhar Mani, M.D., Professor, Medicine, Oncology and Genetics, Miriam Mandel Faculty Scholar in Cancer Research, Albert Einstein College of Medicine, 1300 Morris Park Ave, Chanin 302D-1, Bronx, NY 10461.

Amount Awarded: $40,000 over 2 years.

Central themes of the research projects funded in this round, are: genetics and “epigenetics” of PSC, and the use of mouse models of sclerosing cholangitis to probe the pathogenesis mechanisms and accelerate development of novel therapies.

The following are the Project Summary and Specific Aims sections of the funded proposals. Each Project Summary/Specific Aims description of the project (provided by the investigators) is followed by a brief “Interpretation for the layperson” (prepared by David Rhodes). David Rhodes accepts full responsibility for any errors/omissions that may occur in these “layperson” interpretations.

Pathogenesis of PSC: Role of TGR5 in the regulation of the innate immune response in the biliary epithelium.

Mario Strazzabosco, M.D., Ph.D, Department of Internal Medicine Section of Digestive Diseases, Yale University, 333 Cedar Street-1080 LMP, P.O. Box 208019, New Haven, CT 06520-8019, and Michael Trauner, M.D., Ph.D, Department of Internal Medicine III, Divison of Gastroenterology and Hepatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria.

Amount Awarded: $40,000 over 2 years.Summary

The etiology and pathophysiology of PSC remain unclear. The strong association with IBD suggests a dysregulation of the innate immunity system and among the different hypothesis, an increased response to bacterial products released by a ”leaky gut” has been suggested. This hypothesis has not been adequately tested because of the lack of adequate animal models. PSC has several features in common with Cystic Fibrosis, an autosomic recessive disease caused by mutation in CFTR, a cAMP-stimulated chloride channel that is involved in the secretory function of the biliary epithelium. We have recently found that CFTR deficiency alters the innate immunity of the biliary epithelium and generates a strong TLR4-mediated inflammatory response when the epithelium is exposed to endotoxins. Recent data suggest that TGR5, a Gprotein-coupled receptor for bile acids, that increases cellular cAMP, co-localizes with CFTR and regulates CFTR targeting and function in response to bile acids. The recent finding that the TGR5 gene is a likely disease gene in PSC let us to hypothesize that, similarly to CFTR, TGR5 may be a regulator of TLR4-mediated innate immunity and that a defective function of TGR5 may lead to an excessive inflammatory response to LPS. In this project, we plan to study the liver inflammatory response to DSS-colitis in TGR5-knockout mice, the effects of LPS on cytokine secretion in cultured TGR5-KO cholangiocytes, and the role of TGR5 on the regulation of the TLR4 pathway. These studies will eventually provide a strong rationale for the development of new therapies.

Specific Aims

Sclerosing cholangitis is a spectrum of progressive cholestatic liver diseases characterized by obliterative fibrosis and inflammation of the intrahepatic and/or extrahepatic biliary system. Primary Sclerosing Cholangitis (PSC) is associated with inflammatory Bowel Diseases (IBD), but while IBD is present in 75-80% of PSC cases, only 5-8 % of patients with IBD develop PSC. The etiology and pathophysiology of PSC, as well as the nature of the relationship with IBD remain unclear. Besides the “autoimmune” and the “toxic bile” hypothesis, the “leaky gut” hypothesis suggests that PSC is caused, in susceptible individuals, by exposure of the biliary tree to bacterial endotoxins. The lack of experimental models has hampered progresses in the study of PSC pathogenesis. Recent data generated by our lab indicate that cystic fibrosis may represent an interesting model disease. Cystic Fibrosis (CF) is a genetic disorder of secretory epithelia, caused by the defective function of a Cl- channel essential for bile secretion, called CFTR.. Several patients with CF present a cholangiopathy that shares many similarities with PSC. Both conditions manifest as a slowly progressive fibrosing cholangitis, affecting any tract of the biliary tree, with similar histologic and radiographic findings and a common evolution in biliary cirrhosis. Similar to PSC, that affects only a minority of IBD patients, liver disease affects a minority of CF patients. CFTR-knockout (CTR-KO) mice do not show spontaneous liver pathology, but develop a severe cholangitis after induction of colitis with dextran sodium sulfate (DSS). DSS-treatment had no effects on wildtype animals, suggesting that development of biliary inflammation and liver disease required the interaction between genetic predisposition and acquired factors. We have shown that, independently from the secretory defect, the biliary tree of CF-KO mice is more susceptible to endotoxins that enter the portal circulation because of the increased intestinal permeability. In fact, CFTR-defective cholangiocytes show an altered posttranslational regulation of TLR4 activity, and exhibit a stronger Src-dependent TLR4/NF-kB-mediated inflammatory response to an endotoxin challenge. Thus, CF-cholangiopathy, rather than being the consequence of ductal cholestasis, results from altered innate immune responses of CFTR-KO cholangiocytes. Similar mechanisms may apply to the primary form of sclerosing cholangitis. Recent genome wide association data provided circumstantial evidence that the G-protein-coupled bile acid receptor-1 (gpr1 or TGR5), is a potential disease gene in PSC. TGR5 is a bile acid receptor that generates cAMP upon binding of bile acids. TGR5 was shown to colocalize with CFTR and to be able to activate and stimulate the translocation of CFTR to the apical membrane of gallbladder cells. Moreover, TGR5 was shown to mediate bile-acid-induced suppression of LPS-induced cytokine secretion in macrophages. Thus, we hypothesize that, similar to the mechanism we have shown for CFTR, TGR5 modulates the innate immune response in cholangiocytes, and that defective function of TGR5 my lead to an excessive inflammatory response to LPS.

The hypothesis will be addressed through the following specific aims:

Aim 1: to study the effects of DSS-induced colitis on biliary inflammation in TGR5-KO mice. Aim 2: to study the effects of LPS on cytokine secretion in cultured TGR5-KO and WT cholangiocytes. Aim 3: to study the role of TGR5 on TLR4 pathway regulation in cultured TGR5-KO and WT cholangiocytes.

These studies will generate a better understanding of the innate immune response mechanisms in cholangiocytes and of the pathogenesis of PSC. Furthermore, the results of our study are likely to have a strong translational potential because several therapeutic molecules able to target these mechanisms are being developed.

Interpretation for the Layperson

One of the explanations for the cause of PSC (primary sclerosing cholangitis) in association with inflammatory bowel disease (IBD) is that an immune dysregulation in the gut leads to a “leaky gut” which allows toxic bacterial products to be transported to the liver, causing inflammation around the bile ducts. Both genetic and environmental factors may be involved in disease development and progression. It is essential that we understand the precise causes of PSC, so that we can ultimately learn how to slow or halt disease progression. Significant progress has been made in recent years concerning the identification of genes that may be associated with PSC. An important research approach is to take information obtained from these human PSC genetic studies to develop mouse models that mimic human PSC. Such models will be critical in understanding the pathogenic mechanisms, and accelerating the development of novel therapies. One of the exisiting mouse models of PSC is the cystic fibrosis mouse model. When given colitis, the cystic fibrosis transmembrane conductance regulator (CFTR) deficient mouse develops bile duct injury closely resembling human PSC. The CFTR gene encodes a protein that functions in bicarbonate secretion into bile. This mouse model has already been used to show that docosahexaenoic acid (DHA; a component of fish oil) protects against bile duct injury, and has led to clinical trials of DHA in the treatment of PSC. The relevance of this mouse model to human PSC is that variants of the cystic fibrosis gene have been associated with PSC, and pediatric PSC patients have been shown to have a dysfunction of the cystic fibrosis protein.

The TGR5 gene has been recently identified as a strong candidate for a susceptibility gene in PSC by the Norwegian PSC research center. The TGR5 gene encodes a protein that is a “bile acid sensor” and a regulator of the cystic fibrosis protein. To learn more about what TGR5 is doing, the researchers will use mice in which TGR5 has been knocked-out, and study whether the mice develop sclerosing cholangitis. Drs. Strazzabosco and Trauner will test whether deleting the TGR5 gene in mice will result in an abnormal inflammatory response to the bacterial product lipopolysaccharide (LPS; also known as endotoxin). A characteristic feature of inflammation caused by endotoxin is the activation of the pro-inflammatory complex, nuclear factor kappa-B (NF-kB). Endotoxin binds to the Toll-like receptor 4 (TLR4) and activates NF-kB. Thus, the assessment of the effect of deletion of TGR5 on TLR4-NF-kB pathway regulation in biliary cells (cholangiocytes) will provide important information about the inflammatory mechanisms controlled by TGR5. Drugs that target TGR5 are already in development, and so this study will pave the way for testing whether these drugs may inhibit liver inflammation. Drs. Strazzabosco and Trauner have extensive experience working with mouse models of sclerosing cholangitis, and have been instrumental, respectively, in clarifying the disease mechanisms in biliary tract diseases, and in demonstrating that nor-ursodeoxycholic acid (nor-UDCA) is superior to ursodeoxycholic acid (UDCA) in preventing liver injury, and bringing nor-UDCA to human clinical trials.

The first year of this project will be funded by a generous donation from Craig and Ali Wiele.

Epigenetics associated with primary sclerosing cholangitis in monozygotic twins discordant for the disease

Carlo Selmi, MD, PhD, Assistant Professor of Internal Medicine, University of Milan, Physician Scientist, IRCCS Istituto Clinico Humanitas, Rozzano, Milan, Assistant Professor of Medicine, University of California, Davis, Division of Rheumatology, Allergy, and Clinical Immunology, GBSF suite 6515, 451 E Healthy Sciences Drive, Davis, CA 95616.

Amount Awarded: $40,000 over 2 years.Summary

Primary sclerosing cholangitis (PSC) is characterized by the presence of a chronic and relapsing inflammation of the biliary tract. As illustrated by recent association studies, genomic factors cause individual susceptibility in a subgroup of patients. Monozygotic (MZ) twins are a powerful tool to estimate the role of genetic and environmental factors, yet the concordance rate for PSC in such twins is unknown. Epigenetic changes are ideal candidates to provide a link between genomic susceptibility and environmental stimuli, particularly in the unique model of MZ twins discordant for the disease. We will take advantage of MZ twins discordant for PSC to utilize cutting-edge technologies which allow a large-scale analysis of epigenomics to address two specific issues:

Issue #1. Do MZ twins discordant for PSC manifest consistent patterns in terms of DNA methylation changes within repetitive and non-repetitive elements? We will utilize custom-designed MeDIP multiplex arrays for non repetitive and the most represented repetitive elements (LINE-1, Alu). Issue #2. Does the expression of candidate genes identified through the previous aim differ between discordant MZ twins? We will utilize RTPCR on mRNA samples and then pinpoint methylation differences through bisulfite sequencing. Issue #3. Is there an in silica disease model that can be summoned from the identified putative genes? The results obtained are expected to provide insights into putative epigenetic marks implicated in PSC onset that may well be integrated with the recently identified genetic loci. These results ultimately provide the bases for new epigenetic treatments in inflammatory conditions.

Specific Aims

We will take advantage of samples already available to the proponent from 6 MZ twin pairs discordant for PSC. Samples include DNA, mRNA, and mononuclear cells from peripheral blood and were previously obtained by the proponent through a worldwide effort supported by PSC Partners Seeking a Cure. We will take advantage of this unique cohort and of cutting-edge molecular methods to address two complementary and one ultimate issue.

Issue #1. Do MZ twins discordant for PSC manifest consistent patterns in terms of DNA methylation changes within repetitive and non-repetitive elements? Issue #2. Does the expression of candidate genes identified through the previous aim differ between discordant MZ twins? What is the methylation pattern of single CpG sites within differently expressed genes? Issue #3. Is there an in silica disease model that can be summoned from the identified putative genes? This analytical step will include data from the recently concluded genome-wide genetic association study as well as from the present proposal. Such findings will be utilized with the appropriate bioinformatics tools to determine what molecular pathways are involved in PSC pathogenesis or whether a genetic signature can be extrapolated.

Interpretation for the Layperson

It is estimated that first-degree relatives of PSC patients have a 40-fold higher risk of developing PSC than the general population. Therefore there is a strong genetic component to PSC. The genetic basis of PSC is likely to be complex involving more than one gene and may well be insufficient to explain disease onset. There may also be as yet unknown environmental factors contributing to disease initiation and progression. At the interface between genetics and environment is a relatively new field of research called “epigenetics”. Here, certain gene modifications can take place, such as methylation of cytosine residues of DNA, converting cytosine to 5-methylcytosine, mostly at sites called “CpG sites”. When some areas of the genome are methylated more heavily than others this can alter gene expression and thus the production of the corresponding protein. A key approach to discovering whether epigenetic changes are involved in disease development/progression is to carefully examine the DNA methylation patterns of monozygotic twins (i.e. twins that are genetically identical) but differ with respect to disease (i.e. only one twin is affected, and the pair is thus discordant for disease). This approach has been used by Dr. Selmi and colleagues to investigate the role of “epigenetics” and DNA methylation in particular, in primary biliary cirrhosis (PBC), a liver disease that mostly affects women. These pioneering studies have recently shown that monozygotic twins that are discordant for PBC have distinct methylation patterns of certain genes on the X chromosome. Dr. Selmi and colleagues will now extend these studies to investigate DNA methylation patterns in 6 pairs of monozygotic twins discordant for PSC, recruited with the assistance of PSC Partners Seeking a Cure. The results of this project are expected to provide insights into putative epigenetic markers implicated in PSC onset, painting a more complete picture of the genetic landscape of PSC and its interface with environmental exposures. These results may ultimately provide the basis for novel epigenetic treatments.

Identification of genetic profiles unique to PSC-IBD

Judy Cho, M.D., Associate Professor of Medicine and Genetics, Section of Digestive Diseases, Director, Inflammatory Bowel Disease Center, Yale University, Internal Medicine, 333 Cedar Street, Room LMP-1072, P.O. Box 208056, New Haven, CT 06520-8056.

Amount awarded: $40,000 over 2 years.Project Summary

The primary goal of this proposal is to better understand the relationship between the primary sclerosing cholangitis (PSC) and inflammatory bowel disease (IBD). We propose to accomplish this by identifying the genetic and genomic factors which contribute to the cause of PSC-IBD, and comparing this to identified genetic and genomic factors associated with Crohn’s disease (CD) and ulcerative colitis (UC). Identification of these factors will ultimately contribute to better screening for disease risk, improve predictions of disease severity, influence treatment regimens and advance therapies for treatment and quality of life care. PSC-IBD is poorly characterized, clinically and genetically. A strong association has been observed with IBD and PSC, some analyses identified IBD in 80-90% of PSC patients. Conversely, about 5% of UC patients will have associated PSC compared to 3.4% of CD patients. Genetic contributions to both have been confirmed epidemiologic studies and by the identification of genetic associations for both IBD and PSC. A recent GWAS found the strongest PSC association for the HLA-B region at chromosome 6p21, and some evidence for associations at 2q35, 3p21 and 13q31. Interestingly, significant associations were not found for confirmed UC susceptibility loci. These observations underlie PSC and IBD as complex genetic traits, and support the hypothesis that PSC-UC is a distinct sub-phenotype of IBD. We plan to advance the genetic and genomic knowledge in PSC-IBD by expanding the collection of PSC-IBD cases and biospecimen collections (Specific Aim #1). We propose to compare and contrast both the genetic loci (Specific Aim #2) and peripheral blood serum miRNA patterns (Specific Aim #3) between PSC and IBD. The unique relationship between PSC and IBD provides an ideal opportunity to leverage these comparative studies to provide insight into disease mechanisms and course.

Specific Aims

Specific Aim #1: Development and expansion of PSC-IBD resources for collaborative genetic and genomic studies. We propose to recruit patients and collect phenotype data and biospecimens from individuals with PSC-IBD using uniform protocols to enhance collaborative studies. Identification and a better understanding of the genetic factors contributing to PSC-IBD will greatly increase the potential for identifying disease predictors/markers, novel therapies for disease management and/or the prevention of invasive treatments.

Specific Aim #2: To compare and contrast genetic loci between PSC and IBD. The Immunochip Consortium has developed a genotyping platform which includes all inflammatory disease loci, many of which are shared between different diseases. We propose studies to define the genetic overlap between PSC and IBD, with a particular focus on combining PSC data with two IBD subtypes, namely,extensive colitis and fibrostenosing CD.

Specific Aim #3: To compare and contrast peripheral blood serum miRNA patterns between PSC and IBD. We propose a pilot study to explore the feasibility of using micro-RNAs as biomarkers for PSC-IBD. The analysis of miRNAs in peripheral blood may provide an important and novel source for biomarkers. At present the extent to which small RNAs can be specifically mapped to and regulate, protein-coding mRNA expression is unknown. However, given the plethora of SNP associations in 3’UTR regions, specifically for IBD, the concept that disease-associated variation may modulate mRNA expression through variable miRNA regulation of genetic variation will be explored. We propose an initial analysis of circulating miRNA from serum of CD, UC, PSC-IBD cases and healthy controls.

Interpretation for the Layperson

PSC (primary sclerosing cholangitis) is strongly associated with inflammatory bowel disease (IBD), particularly ulcerative colitis (UC). Both genetic and environmental (possible also dietary) factors may be involved in disease development and progression. Much work during the last decade by the NIDDK Inflammatory Bowel Disease Genetics Consortium (led by Dr. Judy Cho [http://medicine.yale.edu/intmed/ibdgc/]) has identified a multitude of genes associated with UC and Crohn’s disease. The identification of these genes is leading to a more complete understanding of the mechanisms of UC and Crohn’s disease initiation and progression, and identification of new targets for intervention, and novel therapies for disease management. Parallel studies on the genetics of PSC are beginning to reveal that the IBD associated with PSC may have a different genetic signature from classic UC and Crohn’s disease. Dr. Judy Cho will rigorously investigate this important question by recruiting patients and collecting phenotype data and biospecimens from individuals with PSC-IBD for comparison with classic UC and Crohn’s disease patients, and healthy controls. Dr. Cho will use the “Immunochip” [which includes all known inflammatory disease genes] to investigate the precise genetic overlap between PSC and the various forms of IBD.

There is recent interest in whether microRNAs (small RNA molecules with potent gene regulatory functions) play a role in IBD and PSC [Dr. Invernizzi at UC Davis is currently funded by PSC Partners Seeking a Cure to investigate whether specific microRNAs are associated with PSC and cholangiocarcinoma]. Dr. Cho will extend this work to determine if circulating microRNAs in the serum of Crohn’s disease, UC, and PSC-IBD patients differ from one another, and from healthy controls. This work may lead to new diagnostic tools, perhaps enabling early identification of UC and Crohn’s disease patients who may be at risk for developing PSC.

This project will be entirely funded by our anonymous donor who generously donated $100,000 in our Itching for a Cure: Road to Connecticut fundraiser of 2009/2010 to help support PSC research.

Combination treatment with ursodeoxycholic acid and all-trans retinoic acid for primary sclerosing cholangitis (PSC)

Shi-Ying Cai, Ph.D., Research Scientist, Section of Digestive Diseases, Department of Internal Medicine, Yale Liver Center, Yale University School of Medicine, 1080 LMP, 333 Cedar Street, New Haven, CT 06520, and James L. Boyer, M.D., Ensign Professor of Medicine, Section of Digestive Diseases, Department of Internal Medicine, Yale Liver Center, Yale University School of Medicine, 1080 LMP, 333 Cedar Street, New Haven, CT 06520.

Amount Awarded: $37,000 over 2 years.Project Summary

Primary sclerosing cholangitis (PSC) is a chronic cholestatic disease of the liver and bile ducts that generally leads to progressive liver failure. The pathogenesis of PSC remains enigmatic, but malfunction of hepatic immunity has been proposed to play a role in the development/progression of the disease. There is no FDA approved medical treatment for PSC. Although norUDCA has shown beneficial effects in Mdr2 (Abcb4) knockout mice (an animal model for PSC), there is an urgent need to develop novel treatment strategies and to test them in animal models and in PSC patients. Very recently, we have found that combination treatment with UDCA and retinoic acid substantially improved animal growth rate and significantly reduced bile salt pool size, liver fibrosis, necrosis, inflammation, and bile duct proliferation in an animal model of cholestasis, the common bile duct ligated rat. (UDCA and retinoic acid are FDA-approved medications for treating primary biliary cirrhosis and acute promyelocytic leukemia and inflammatory disorders such as psoriasis, acne, and rheumatoid arthritis, respectively). Parts of the molecular mechanisms of this beneficial effect of UDCA and retinoic acid have also been verified in primary human liver cells, including hepatocytes and hepatic stellate cells (manuscript submitted for publication). Therefore, we propose to test if RA alone or in combination with UDCA will improve liver pathology in Mdr2-/- mice. Completion of the proposed study will provide critical information for determining whether RA alone or in combination with UDCA might be potentially beneficial for patients with PSC. If this project demonstrates beneficial effects in this animal model, we plan to test this therapy in patients with PSC.

Specific Aims

The specific aim of this study is to determine whether RA alone or in combination with UDCA has beneficial effects on liver fibrosis, necrosis and or inflammation in Mdr2-/- mice, an animal model for PSC.

Interpretation for the Layperson

It is urgent that novel therapies/treatment strategies be developed for PSC, and the use of mouse models of sclerosing cholangitis will be critical in accelerating the discovery and pre-clinical testing of these new approaches. It is well recognized that vitamin A deficiencies are common in PSC patients, and that the deficiencies become more pronounced as the disease progresses. The liver is a major storage organ for vitamin A, and as cholestatic liver diseases progress, this vitamin A can be released from the vitamin A storing cells of the liver (hepatic stellate cells) as they transition away from vitamin A storage towards a state that promotes collagen biosynthesis and liver fibrosis. Vitamin A (retinol) is a precursor of all-trans retinoic acid (RA), and so loss of vitamin A during advancing liver disease may lead to retinoic acid deficiencies, which may in turn contribute to a vicious cycle of inflammation, necrosis and fibrosis. In this proposal, Drs. Cai and Boyer will test whether the combination of ursodeoxycholic acid (UDCA) with retinoic acid (RA) will be superior to the individual drugs alone in delaying liver fibrosis, necrosis and inflammation in a mouse model of sclerosing cholangitis, the Mdr2 (-/-) mouse model. Should positive results be obtained in these studies, this combination therapy can be immediately tested in patients with PSC.

This project will be entirely funded by our anonymous donor who generously donated $100,000 in our Itching for a Cure: Road to Connecticut fundraiser of 2009/2010 to help support PSC research.

Establishing the role and molecular mechanisms for pregnane X receptor in progressive sclerosing cholangitis

Sridhar Mani, M.D., Professor, Medicine, Oncology and Genetics, Miriam Mandel Faculty Scholar in Cancer Research, Albert Einstein College of Medicine, 1300 Morris Park Ave, Chanin 302D-1, Bronx, NY 10461.

Amount Awarded: $40,000 over 2 years.Project Summary

We and others have demonstrated that orphan nuclear receptors like pregnane x receptor (PXR) abrogates intestinal inflammation induced by xenobiotic compounds (e.g., DSS). These experiments serve as a proof-of-concept that PXR plays a significant role in pathogenic diseases of the gut that result from unregulated inflammatory responses (e.g., IBD). Fish oils have beneficial effects on inflammation and are relatively non-toxic xenobiotics. Since the pathogenesis of progressive sclerosing cholangitis is parallel to that observed in arteriosclerotic inflammation (where fish oils show clear benefit), our 2-year project will focus on determining the significance of PXR activation in the pathogenesis and maintenance of PSC. Specifically, we will use the DDC-induced mouse model of cholangiopathy in our mouse models of PXR activation [PXR wt type, PXR-/- and humanized (h)PXR] to determine if PXR activation by non-hepatotoxic agonists (e.g., hyperforin or rifampicin) abrogates biliary inflammation. In our second aim, we will determine whether PXR mediates the actions of fish oils in abrogating biliary inflammation. Specifically, we hypothesize that fish oils will transactivate PXR and inhibit inflammation.

Specific AimsTo determine the effect of PXR activation on biliary inflammation in the DDC-induced mouse model of cholangiopathy in PXR+/+, PXR-/- and humanized PXR mice. DDC induces a cholangiopathy in mice at 8 weeks of treatment that resembles early inflammatory changes seen in PSC. (a) To determine the effect of PXR on early inflammation in the bile duct tract, PXR +/+ will be treated with DDC (+ PCN, a potent mPXR agonist). At 4, 6 and 8 weeks, cohorts of mice from each genotype, will undergo histopathologic and immunohistochemistry assessment of the entire bile duct tract (intra- and extrahepatic) as previously described. (b) The same experiments will be repeated with PXR-/- and hPXR mice (+ hyperforin or rifampicin, potent hPXR agonists), with accurate sample size estimates obtained from data from (a). These experiments will determine the clinical impact of PXR in biliary inflammation as typified in PSC.To determine the therapeutic efficacy of fish oils alone or in combination with classical PXR ligands on DDC-induced mouse models of cholangiopathy. Fish oils (DHA, EPA) are commercially available and serve as weak ligands to several nuclear receptors – RXRalpha and PPARalpha. Since PXR dimerizes with RXRalpha and is a direct target gene of PPARalpha, we hypothesize that fish oils will transactivate PXR and inhibit inflammation. Furthermore, we surmise that multiple ligands acting on PXR, given its promiscuous ligand-binding pocket, will synergistically activate PXR and inhibit inflammation. To test this concept, we will treat PXR+/+, PXR-/- and humanized PXR mice with vehicle, fish oils and/or PCN (as mPXR ligand) or rifampicin (as hPXR ligand).Interpretation for the Layperson:

It is becoming clear that there is a complex network of nuclear receptors in the gut and liver that are “master” controllers of gene expression, regulating all aspects of metabolism, ranging from bile acid transport and detoxification, to energy metabolism, to lipid metabolism, and circadian rhythms (day-night cycles). An important “master” contoller is the receptor called pregnane X receptor (PXR), also known as the steroid and xenobiotic receptor (SXR). PXR is known to be down-regulated in ulcerative colitis, and the PXR gene itself may be an ulcerative colitis susceptibility gene, and a gene that affects the rate of progression of PSC. This receptor plays an important role in detoxification of lithocholic acid, a toxic bile acid produced from ursodeoxycholic acid (UDCA) by gut bacteria. Recent studies indicate that activation of PXR by drugs such as rifampin (rifampicin) and rifaximin results in reduction of inflammation and fibrosis.

Because the adverse effects of high-dose UDCA in PSC may be associated with conversion of UDCA to lithocholic acid, there is also particular urgency in investigating whether drugs that target PXR may help prevent these adverse effects of UDCA.

In this proposal, Dr. Mani will use humanized mice in which the mouse PXR gene has been replaced with the human PXR gene so that it responds to rifampin (rifampicin), and will then test whether activation of PXR by rifampin will result in a reduction of biliary inflammation.

Another “master” controller of gene expression in the liver and gut is the receptor called retinoid X receptor (RXR), which partners with PXR (and many other nuclear receptors) to regulate metabolism. A known activator of RXR is a component of fish oil, docosahexaenoic acid (DHA). It is plausible that the combination of DHA and rifampin, as activators of both RXR and PXR, respectively, will be superior to either drug alone in reducing inflammation. Dr. Mani will test this hypothesis. If positive results are obtained, this may lead to novel therapies in PSC, perhaps combining UDCA with rifampin and DHA. It should be noted that rifampin is already used by many PSC patients for the control of pruritus, and that DHA has shown some early positive results in reducing alkaline phosphatase levels in PSC patients. Some mouse models suggest that DHA may be protective against colitis and colorectal cancer, and there is growing evidence that human ulcerative colitis may be associated with low intake of omega-3 fatty acids such as those found in fish oils.

Awarded in 2009

Eight awards were selected for funding for two years each.

Examining the Disease Impact of Genetic Variation in Logical Candidate Genes for PSC: a PROGRESS Study

Konstantinos N. Lazaridis, M.D., Assistant Professor of Medicine, Center for Basic Research in Digestive Disease, Mayo Clinic College of Medicine 200 First Street SW, Rochester, MN 55905.

Project Summary: Genetic predisposition is thought to play a key role in the susceptibility to primary sclerosing cholangitis (PSC). However, the rarity of PSC has rendered the collection of sufficient multiply-affected families to perform traditional genetic analyses impossible, hampering efforts to identify the associated genetic variants. To this end, we established the PSC Resource Of Genetic Risk, Environment and Synergy Studies (PROGRESS) registry and biorepository, a national research resource aimed at elucidating the genetic and environmental contributors to PSC by combining association-based study designs with cutting-edge and emerging approaches to their analysis. We are now in the position to utilize PROGRESS for genetic studies and have elected to focus on highly selected, biologically plausible candidate genes. This logical candidate gene approach remains important in the era of genome-wide studies as it provides for superior gene coverage and a more subtle appreciation of disease affects for these highly suspect genes than afforded by the more comprehensive studies. Moreover, this genetic information may prove valuable to future gene-gene and geneenvironment interaction studies, even when no primary association is identified.

Our seminal effort has identified a strong association between PSC and a promoter polymorphism in the tumor necrosis factor alpha (TNFalpha) gene. Here we propose to build upon this finding by genotyping haplotype tagging single nucleotide polymorphisms (SNPs) in a number of PSC candidate genes, performing association analysis for disease status as well as subphenotypes of disease, and exploring potential gene-gene and gene-environment interactions by utilizing the unique resource available to us in the PROGRESS registry and biorepository.

Cholangiocarcinoma-associated serum microRNAs in primary sclerosing cholangitis: Identification and prognostic potential

Pietro Invernizzi, M.D., Ph.D., Assistant Professor of Medicine, Division of Rheumatology, Allergy, and Clinical Immunology, University of California, Davis GBSF suite 6515, Davis, CA 95616.

Project Summary: Primary sclerosing cholangitis (PSC) is associated with a high risk of cholangiocarcinoma (CCA), a rare but often fatal malignancy of the bile duct epithelium. Several tumour markers have been used to aid diagnosis, but no useful markers specific for CCA are currently available. Because of the ease of obtaining blood samples, there is an obvious need for accurate serum markers for screening of CCA in PSC patients at an early stage of this cancer or, even better, for pre-cancer biomarkers. MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs involved in the control of cell differentiation processes, and consistently with this they have been demonstrated to be useful cancer biomarkers. Recently, this emerging field of study has discovered that miRNAs can also be efficiently evaluated in serum. The aim of the project is to identify specific serum miRNAs with a role as pre- or early-cancer biomarkers for human CCA in patients with PSC. The project will be divided in two phases: (i) In the first phase (first year) we will investigate the miRNAs expression profile (1) in available serum from 30 patients with CCA, 30 with PSC, and 30 healthy subjects, matched by sex and age to CCA patients, (2) in five human CCA immortalized cell lines and one normal immortalized biliary epithelial cells (BECs) line, and (3) in available human primary BECs cultures from CCA, PSC and normal subjects (ii) In the second phase (second year) we will take advantage of an extraordinary unique multi-center series of PSC sera (n=400) collected in tertiary referral liver centers in USA, Italy, and Norway in order to evaluate with a longitudinal (retrospective/prospective) study the role as pre- or early-cancer biomarkers of the CCA-specific miRNAs identified in the phase 1 of the study.

Aberrant homing of lymphocytes to the liver in patients with primary sclerosing cholangitis; the missing link between colon and liver

Cyriel Y. Ponsioen, MD, PhD, Department of Gastroenterology & Hepatology, Academic Medical Center, C2-112, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands.

Project Summary: Primary sclerosing cholangitis (PSC) is a rare chronic inflammatory disease of the biliary tree leading to bile duct strictures, to fibrosis and cirrhosis of the liver and eventually to liver failure. The etiopathogenesis is unknown. There is no medical therapy of proven benefit for the long-term prognosis so far. Liver transplantation is the treatment of choice in late stage disease. PSC is highly associated with inflammatory bowel disease (IBD). It was proposed that T lymphocytes primed to home to gut tissue, by mistake instead home to the liver in PSC, thereby keeping the chronic inflammation ongoing. It was hypothesized that erroneous homing of gut-primed lymphocytes in PSC is caused by aberrant over-expression or functioning of the chemokines, integrins, or addressins involved in transendothelial migration of gut-homing T-cells. We hypothesize that this may occur through functional gene mutations.A cohort of greater than 400 PSC patients with detailed phenotyping and appropriate controls (primary biliary cirrhosis, IBD-patients and healthy partners) is being accrued from 42 hospitals in central-Netherlands. The expression of CCL25, CCR9, alpha4Beta7, and MAdCAM-1 will be studied as candidate chemotactic factors in cases and controls by immunohistochemical expression in PSC liver and colon tissue, as compared to control tissues. Peripheral blood and colonic lamina propria lymphocytes (PBL and LPL) will be harvested and analyzed by FACS to examine the expression of CCR9 and alpha4Beta7.

Potential relevance of our research will be 1. to ascertain the gut-homing lymphocyte paradigm in the IBD-PSC association; 2. to assess the presence of PSC associated gut-homing chemokines, addressins and integrins in colonic tissue; 3. to pinpoint the role of CCL25, CCR9, alpha4Beta7, and/or MAdCAM-1 in the pathogenesis and/or sustaining of inflammation in PSC. This will lend support to initiate trials in PSC with specific inhibitors of gut-homing chemotaxis such as natalizumab and Traficet; 4, the building of a large population-based PSC cohort including biobanking for future studies.

The topic of our research project is to study location-specific expression of these chemotactic molecules in PSC, in particular to prove that there is colonic expression of CCL25.

alpha4Beta7-Integrin Ligand Development for the Treatment of PSC

Christopher Bowlus, M.D., Associate Professor, Division of Gastroenterology, UC Davis Medical Center, 4150 V Street, PSSB 3500, Sacramento, CA 95817

Amount Awarded: $40,000 over 2 years.

This project will be entirely funded by a generous donation from Abe and Rachel Gomel.

Project Summary: Primary sclerosing cholangitis (PSC) is due in part to gut-derived lymphocytes trafficking to the liver in response to specific adhesion molecules and chemokines normally expressed in the gut. Specifically, alpha4Beta7-integrin expressing lymphocytes home to the PSC liver where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is aberrantly expressed. Blocking alpha4 integrins has been successfully targeted by monoclonal antibodies for the treatment of Crohn’s disease. However, the use of this therapy in PSC is hindered by the risk of progressive multifocal leukoencephalopathy (PML), believed to be due to the lack of specificity for alpha4Beta7. Vedolizumab (formerly known as MLN002 and MLN02), is a monoclonal antibody with specificity for alpha4beta7 that has shown efficacy in phase 2 clinical trials of moderately active ulcerative colitis and Crohn’s disease. However, monoclonal antibodies have limitations. The antibody molecule is relatively large with a molecular weight of 160,000 kD requiring infusion or subcutaneous injection. In addition, even with humanized forms of these monoclonal antibodies, human anti-chimera antibodies (HACA) are frequently generated and are sometimes associated with a lower rate of response. Peptides are considerably smaller than monoclonal antibodies and generally do not illicit a humoral immune response. They are chemically stable and relatively easy to derivatize. Although peptides are susceptible to proteolytic degradation in vivo, this can be inhibited with blocking of their N- and C-termini, making them cyclized, or incorporating D-amino acids. 
In this proposal, we will take advantage of our expertise in One Bead-One Compound (OBOC) combinatorial chemistry, integrin biology, immunology and PSC to identify novel lead compounds for the treatment of PSC. We have previously identified a high affinity, high specificity peptidomimetic ligand for alpha4Beta1-integrin that is currently under pre-clinical development. In this project we will use the same technology to identify lead compounds for alpha4Beta7 ligands. In Aim 1 we will design and synthesize a diverse combinatorial chemistry library based upon the known alpha4Beta7 binding motif. In Aim 2 we will use a whole cell method to screen this library with alpha4Beta7+ T cells from PSC patients. The specificity and binding affinity of these lead compounds will be assessed in Aim 3. Upon completion of this project we will have identified lead compounds that will be ready to proceed to functional testing. In addition, the knowledge gained will be used to design focused libraries which will be screened for additional ligands.

Non-invasive Assessment of Disease Progression in Primary Sclerosing Cholangitis. 

Gregory T. Everson, M.D., Transplant Center and Hepatology, Clinic Mail stop B, 154 Anschutz Outpatient Pavilion, 1635 N. Ursula Street, Room 7085, Aurora, CO 80045. 

Amount Awarded: $40,000 over 2 years. 

Project Summary: Current methods for monitoring primary sclerosing cholangitis (PSC) disease progression are insensitive, invasive, and nonspecific. We have developed a novel noninvasive technique to accurately measure liver function by quantifying portal-systemic shunting, the cholate shunt test. Our overriding hypothesis is that our liver function test will accurately monitor PSC disease progression.

The Role of Abcb11 and Fibroblast Growth Factor 15/19 in the Pathogenesis of Primary Sclerosing Cholangitis

Richard M. Green, M.D., Associate Professor of Medicine, Division of Hepatology, Northwestern University Feinberg School of Medicine.

Amount Awarded: $40,000 over 2 years.

This project will be entirely funded by a generous donation from David and Ros Parry.

Project Summary: Primary sclerosing cholangitis (PSC) is a hepatic disease of unknown etiology that can lead to cirrhosis, cholangiocarcinoma and the need for liver transplantation. It is characterized by inflammation and fibrosis of cholangiocytes, the cells that compose the liver bile ducts. Unfortunately, no effective therapy has been shown to slow disease progression, in part because the pathogenesis of primary sclerosing cholangitis remains poorly understood. By enhancing our understanding of the pathogenesis of PSC, one can better design rational therapies for this potentially devastating form of chronic liver disease. 
The cholangiocytes that form the bile ducts are exposed to extremely high concentrations of bile salts, which induce signaling changes in cholangiocytes. The high bile salt concentrations in bile are created by the ATP-dependent secretion of bile salts from the liver into the biliary system. Our laboratory has a long-standing interest in identifying the mechanisms by which hepatocytes (liver cells) secrete bile salts into the biliary system. We, along with other investigators, have determined that bile salts are secreted by the liver canalicular membrane transporter Abcb11. Furthermore, we have cloned Abcb11 and developed a transgenic mouse that over-expresses Abcb11 in the liver. In addition, the level of expression of Abcb11 in humans is highly variable (by a factor of over 10) and therefore it is likely important in the manifestation of many cholestatic liver diseases. However, the impact of high levels of Abcb11 expression on primary sclerosing cholangitis is poorly understood. 
A recently identified gut-derived hormone named fibroblast growth factor 15/19 (FGF15/19) is secreted by the ileum in response to bile salt stimulation and subsequently interacts with hepatocytes and cholangiocytes to regulate their function and maintain normal cellular homeostasis. Although FGF15/19 has been shown to be an important physiologic regulator of ductular cells in the gallbladder and for gallstone formation, the function of FGF 15/19 in other biliary tract diseases remains unknown. In the proposed studies, we will explore the role of Abcb11 and FGF 15/19 in biliary tract disease; since our enhanced understanding of these physiologic processes will allow for the design of rational, novel therapeutic targets for treating patients with PSC

A Pilot study of Vancomycin or Metronidazole in patients with Primary Sclerosing Cholangitis.