Research



Drug discovery and development of new interventions

The PBRU is engaged in a drug discovery programme and in development of new interventions. This is to find new agents/interventions, which may eventually be used for the treatment of pancreatitis and pancreatic cancer. At present no drugs exist for the treatment of acute pancreatitis, and the prognosis of pancreas cancer patients following conventional radiotherapy/chemotherapy is poor.

1. Calcium Signalling and Drug Discovery

A major focus of research is the role that cytosolic calcium overload plays in all forms of pancreatic acinar cell injury (due to alcohol, gallstones and other aetiologies. 

Actimod

Work in the Department of Physiology, University of Liverpool and by others, has elucidated the physiological basis of the secretion of digestive enzymes from the pancreatic acinar cell, a process which occurs through the generation of spatiotemporally-defined rises in cytosolic calcium levels ([Ca2+]c). At Liverpool, advanced confocal microscopy and electrophysiological studies have shown that these rises are transient and generally confined to the apical pole of the cell. Furthermore, we have demonstrated that under pathophysiological conditions, such as hyperstimulation with cholecystokinin, non-oxidative alcohol metabolites and bile acids, normal Ca2+ signalling is disrupted and sustained, global rises of [Ca2+]c are generated that impair mitochondrial function and ultimately lead to necrosis. Normal secretory function is compromised under these conditions and inappropriate activation of digestive enzymes occurs within the cell, leading to autodigestion of the organ commonly associated with acute pancreatitis. The basis for the generation of these Ca2+ signals has been well characterised, with the inositol trisphosphate receptor (IP3R) playing an important role. In addition, detailed confocal imaging studies of mitochondrial function have resulted in our proposing that formation of the mitochondrial permeability transition pore (MPTP), induced by Ca2+ overload of mitochondria, may be a critical event which leads to necrosis of the pancreatic acinar cell, a major predictor of the severity of acute pancreatitis.

M_Cell_colour

We have demonstrated that several compounds are able to pharmacologically inhibit IP3R and MPTP preventing necrosis in models of pancreatitis. We are working with the Department of Chemistry and other collaborators to optimise further compounds to test against these targets using low through put technology employing Zeiss LSM 710 confocal scanning microscopy.

  

2. Models of Disease

Pancreatitis
Researchers at the PBRU have established that non-oxidative ethanol metabolites are important in the pancreatic acinar cell damage seen in pancreatitis. Fatty acid ethyl esters such as palmitoleic acid ethyl ester (formed from  intake of ethanol)  have been shown to cause acinar cell damage following the release of calcium. We have recently developed a model where  acute pancreatitis is induced following administration of ethanol and fatty acids.

Xenograft Models of Pancreatic Cancer
Xenograft models have been used to establish the potential effectiveness of therapeutic interventions in pancreas cancer, using sub-cutaneously injected pancreatic cells (e.g. MIAPACA2). 

 

Application of new diagnostic and imaging strategies

1. Confocal endoscopy, laparomicoscopy

The PBRU has established a specialised microscopy facility for use in translational clinical studies.

Inestinal_Villi In collaboration with the Liverpool BRC we are examining the use of confocal microscopy in various clinically applicable approaches. We are undertaking proof of principle/early phase studies of confocal endomicroscopy, laparomicroscopy and pancreatomicroscopy in acute pancreatitis, chronic pancreatitis  and pancreatic cancer. We are also developing protocols to carry out confocal studies of the small/large intestine (during endoscopy) and pancreas (during ERCP or necrosectomy) to assess pancreatic function, gut permeability, endotoxic and bacterial translocation. This work will be linked to other prognostic marker evaluations.
 
   
 
Red_Blobs 

To undertake this work we have purchased a Pentax confocal endomicroscopy system  and an Optiscan  confocal laparoscope.

 

 

 

 

2. PET/CT imaging

The PBRU has access to a Positron Emission Tomography combined with CT (PET/CT) scanner, which has been installed at the Royal Liverpool University Hospital. This has allowed us to take part in a study sponsored by MERCK (MERCK PN144) to evaluate PET/CT for monitoring chemotherapy response in pancreatic cancer. This is a multicentre study involving Manchester, Cambridge, London and Liverpool. MERCK has appointed the Liverpool GCLP facility as the central laboratory for processing samples from all centres participating in the trial. Also, Liverpool will become a site for the clinical arm of the study. We are also setting up a further multi-centre study PET-Panc (DoH/NIHR) to examine the diagnostic accuracy and clinical value of PET/CT in pancreatic cancer.


Validation of new biomarkers and screening protocols

1. PBRU Biobank
The PBRU has obtained ethical approval to collect and store samples of urine, serum, plasma, RNA and white cell preparations from patients with acute pancreatitis. These samples are stored in the PBRU biobank, which is part of the University of Liverpool biobank located in the GCLP laboratory. Patient information and clinical data is also collected. These data are securely and ethically stored on computer in password protected, secure backed up files. We are also in the process of storing samples from patients with chronic pancreatitis and pancreatic cancer. Stored RNA from a selected cohort of acute pancreatitis patients at admission will be examined by microarray using Affimetrix GeneChips.

To permit reliable approaches including genome-wide association and epidemiological, studies we are working towards creating one of the largest collections of samples in Europe from patients with chronic pancreatitis, acute pancreatitis and pancreatic cancer. This has required us to widen our collaborations both Nationally and Internationally. This has been endorsed enthusiastically by several leading European pancreatic groups based in Spain, Germany, Italy and Sweden. The PBRU is also expanding its biobanking activities with new clinical trials/studies starting in 2010.

2. Identification of Biomarkers
The PBRU is pursuing a biomarker discovery programme for pancreatitis and pancreatic cancer, to improve diagnosis, treatment and prognosis. The PBRU has experience in the identification of blood-borne protein biomarkers for pancreatic disease. Whilst blood is recognised as a highly important source of disease-related biomarkers, the complexity and dynamic protein concentration range of plasma/serum complicates the analysis process. Consequently, a number of strategies for simplification of blood prior to protein analysis have been developed. We recently published a technically detailed protocol for iTRAQ (Isobaric Tags for Relative and Absolute Quantification)-based quantification of serum proteins following immunodepletion of high abundance protein. This mass spectrometry-based approach enabled the identification of 217 proteins (5773 peptides) with a false discovery rate of 1 % or 254 proteins with 95% confidence, respectively. A number of proteins that were specifically over-expressed in patients with chronic pancreatitis or pancreatic cancer compared to disease- and healthy controls are currently undergoing validation. The PBRU  has access to a QTRAP 5500 hybrid triple quadrupole/linear ion trap mass spectrometer. This mass spectrometer can be used for biomarker discovery, and is also an excellent tool for the quantification of known protein biomarkers using Multiple Reaction Monitoring.

In addition, biomarker discovery work using Luminex assays for the detection of cytokines, chemokines and growth factors are being performed on samples including diagnosed pancreatic cancer cases, benign-disease controls and healthy sera.

We are also examining the effect of the cellular microenvironment on the progression of pancreas cancer to identify new biomarkers.
 
3. Bioinformatics
The PBRU has invested in bioinformatics, since systems biology provides a powerful approach to examine the transcriptome and proteome for diagnostic and prognostic marker discovery. We are employing Microarrays and LC MS/MS (e.g. by iTRAQ relative quantification) platforms using RNA/DNA serum and plasma from large numbers of patients with acute and chronic pancreatitis and pancreatic cancer to provide genetic/protein patient profiles. The large data sets generated in microarray experiments are highly parallel and highly variable and so require specialist bioinformatics analysis for robust interpretation. Bioinformatics support is available in PBRU to plan, analyse and interpret the outcome of microarray experiments. Appropriate experiments are designed to maximise statistical robustness and minimise costs, where good quality hybridisations on Affymetrix, Agilent or bespoke arrays are performed locally thanks to close collaboration with the Advanced Genomics Facility. The pipeline of statistical analysis is chosen to suite the particular platform and study type and performed using the most appropriate data models, which are implemented in R/Bioconductor or proprietary software such as Partek or GeneSpring. Data mining and functional interpretation in functional studies is performed using Ingenuity software, generating molecular hypotheses that can be tested in the lab. Microarray data will be stored on dedicated servers and, in common with other research activities in PBRU, the aim is to implement GCLP standards to data handling and computation.
 

4. EUROPAC and secondary screening

The European Registry of Familial Pancreatic Cancer and Hereditary Pancreatitis (EUROPAC) is the world’s largest registry of its kind for inherited pancreatic diseases. The registry orchestrates clinical trials for the alleviation of symptoms of pancreatitis and secondary screening for pancreatic cancer as well as genetic studies to identify the mutations and polymorphisms that predispose to pancreatic disease. EUROPAC has been running since 1997 and has 249 families with pancreatitis (723 affected individuals). It has been shown that patients with hereditary pancreatitis have a 40% lifetime risk of developing pancreatic cancer. Also, EUROPAC has registered 168 families with more than 1 case of pancreatic cancer. We are analysing epidemiological and genetic factors which may contribute towards the development of pancreatic cancer in high risk families, which may help explain the aetiology of pancreatic cancer and provide new approaches for diagnosis and treatment of the disease.

For further information on the EUROPAC screening programme please click here for EUROPAC website.