Er bezeichnete es u. Ivan Rebroff — Das Einsame Glöckchen https: 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.
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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 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.
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.
These findings indicated that development of an accurate sensitive and specific diagnostic marker for IAC was within reach. Still, NGS technology is work-intensive, expensive and unaffordable for routine clinical use. Distinguishing IgG4-related disease from its pancreatobiliary mimics: Are we there now?
Are We There Now? Genetically mutated strains of mice or other small animals sometimes exhibit features of human disease. 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.
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. 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. 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.
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. 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. In IBD pathophysiology, we have identified a key cellular and molecular module in mice that drives disease: 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.
We have found a key pathological cell type that can cause inflammation in both the liver and the intestine: Dendritic cells DCs are part of the immune system and serve two functions: 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.
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. And a second type of dendritic cell gives the signal to make a totally different inflammatory protein, IL 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 By getting ride of this dendritic cell we successfully reduced the amount of IL 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.
Inflammatory Th1 and Th17 in the intestine are each driven by functionally specialized dendritic cells with distinct requirements for MyD Cell Reports accepted, in press. This project is partially funded by the generous support of the Grover Family Foundation and Deborah Wente.
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. 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. 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.
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.
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. The biliary epithelium presents antigens to and activates natural killer T cells. Hepatology , Oct;62 4: Natural Killer T cells aggravate inflammation in a novel induced murine model of cholangitis. Primary sclerosing cholangitis PSC is an autoimmune disease that destroys the bile ducts in the liver and can lead to liver failure.
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. 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.
We believe that these studies have the potential to identify new therapeutic approaches to the treatment of PSC. 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.
These cells also exhibited immature phenotype and were more activated. Collectively, our data demonstrated that in patients with PSC, deficiency in maturation with aberrant enhanced cytotoxicity in the NK subset is intriguing and significant.
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. 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 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. Therefore, to identify further sources of heritability, e.
By using a next-generation sequencing-based approach, it is planned to sequence the exomes, i. 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.
Read a November progress update report on this grant. Read the final report for this grant. 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. 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. The pathogenesis of PSC remains enigmatic, making the development of targeted therapeutic strategies difficult. Elevated levels of bile acids can lead to hepatocyte injury, liver failure and the need for liver transplantation. 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. 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.
To date, we have found that mast cells infiltrate the liver during PSC and cholangiocarcinoma progression. 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. Read the final report and see the publications generated as a result of this grant. 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. In this regard, our recent data suggest that a subset of cultured i. We believe, and our data support, that this state is representative of a senescence-associated secretory phenotype SASP. 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. Read a July progress update report on this grant.
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. 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.
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.
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. 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. 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.
Read the November progress update report on this grant. Read the January progress update report on this grant. Also read the article Dr. Jochen Metzger;Professor Michael P. Manns and Professor Harald Mischak. Therefore, the aim of scientific contributions must be directed to early diagnosis of CC with high accuracy.
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. 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. 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.
Gert Van Assche and Dr. UC involves only the large bowel while CD may involve any part of the gastrointestinal tract. 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.
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.
Primary sclerosing cholangitis PSC is a multifactorial disease with genetic, microbial, and environmental components. 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.
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 ,s 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. A second cohort of cases and healthy controls is available for replication of observed signals. Identified variants will give important insights in disease pathogenesis. In addition we might identify mutations with predictive value for disease occurrence or disease behavior.
Read a September progress update report on this grant. Box , New Haven, CT Central themes of the research projects funded in this round, are: The following are the Project Summary and Specific Aims sections of the funded proposals. The etiology and pathophysiology of PSC remain unclear. 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. These studies will eventually provide a strong rationale for the development of new therapies. 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.. 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.
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.
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. 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. 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. 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.
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. 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.
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. 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:.
Does the expression of candidate genes identified through the previous aim differ between discordant MZ twins? 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.
We will take advantage of samples already available to the proponent from 6 MZ twin pairs discordant for PSC. We will take advantage of this unique cohort and of cutting-edge molecular methods to address two complementary and one ultimate issue.
What is the methylation pattern of single CpG sites within differently expressed 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. It is estimated that first-degree relatives of PSC patients have a 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. 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.
This approach has been used by Dr. 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. 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. The primary goal of this proposal is to better understand the relationship between the primary sclerosing cholangitis PSC and inflammatory bowel disease IBD.
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. Genetic contributions to both have been confirmed epidemiologic studies and by the identification of genetic associations for both IBD and PSC.
Interestingly, significant associations were not found for confirmed UC susceptibility loci. The unique relationship between PSC and IBD provides an ideal opportunity to leverage these comparative studies to provide insight into disease mechanisms and course. We propose to recruit patients and collect phenotype data and biospecimens from individuals with PSC-IBD using uniform protocols to enhance collaborative studies.
The Immunochip Consortium has developed a genotyping platform which includes all inflammatory disease loci, many of which are shared between different diseases. 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. Both genetic and environmental possible also dietary factors may be involved in disease development and progression.
Primary sclerosing cholangitis PSC is a chronic cholestatic disease of the liver and bile ducts that generally leads to progressive liver failure. 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.
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. 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. Should positive results be obtained in these studies, this combination therapy can be immediately tested in patients with PSC. We and others have demonstrated that orphan nuclear receptors like pregnane x receptor PXR abrogates intestinal inflammation induced by xenobiotic compounds e.
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. 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.
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. 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. 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.
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.
Mani will test this hypothesis. 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.
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.
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.
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 project will be divided in two phases: 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. Die wichtigsten Informationen zur Reformation in Zürich auf einen Blick. Skulpturen, Denkmäler und Installationen verschönern den öffentlichen Raum von Zürich, aber nicht nur!
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