Project title and Supervisors

Title: Investigation of the role of inhibition of NF-κB activity in mediating effects of hypercapnic acidosis in Ventilation induced Lung Injury
Supervisors:
Professor John Laffey, Professor and Head, Department of Anaesthesia, National University of Ireland, Galway
Dr Leo Kevin, Consultant Anaesthetist and Clinical lecturer, Department of Anaesthesia, National University of Ireland, Galway.

Project Summary

Acute Lung Injury (ALI) is the clinical syndrome of rapid onset bilateral pulmonary infiltrates and hypoxaemia of non-cardiac origin. When the hypoxaemia is severe, the condition is termed the acute respiratory distress syndrome. It is a significant public health problem, with an attributable mortality similar to breast cancer or HIV. Many survivors of ALI and ARDS are unable to return home at hospital discharge. Mechanical ventilation may worsen ALI/ARDS, a process termed Ventilator Induced Lung Injury (VILI). Hypercapnic Acidosis (HA) is a central component of lung ventilatory strategies to minimize VILI – this is termed “permissive hypercapnia”.
HA is a potent biologic state, exerting a myriad of effects on diverse biologic pathways. Deliberately induced HA is protective in multiple lung injury models. However, HA may inhibit the host response to bacterial sepsis, and also retard the repair process and slow recovery following ALI/ARDS. Thus HA may be a double edged sword, depending on the context.
An alternative approach is to manipulate a single key effector pathway that is central to the protective effects of HA. Hypercapnia attenuates NF-κB activation, and may exert its effects – both beneficial and deleterious – via this mechanism. NF-κB is a pivotal regulator of the pro-inflammatory response, but is also a key epithelial cytoprotectant. By selectively modulating the NF-κB pathway, at the pulmonary epithelial surface, it may be possible to accentuate the beneficial effects of HA during the injury process, while minimizing the potential for HA to delay lung tissue repair following injury.
We will investigate the role of NF-κB inhibition in mediating effects of HA – in both injury and repair – and characterize the direct effects of modulating NF-κB, in in vitro and in pre-clinical models of lung injury and repair. We will utilize gene therapy to inhibit pulmonary epithelial NF-kB activity, a technique which facilitates high intensity delivery of this therapy directly to the injury site, thereby maximizing the benefit.
These studies will provide novel insights into: key pathways contributing to lung injury and repair; the role of HA and NF-κB in these processes; the efficacy of strategies to modulate the NF-κB pathway in the presence and absence of hypercapnia; and the overall potential of pulmonary gene therapy in ALI/ARDS.

Project summary update (March 2010)

Acute Respiratory Distress Syndrome (ARDS) is a form of sudden and catastrophic failure of the respiratory system that can be rapidly fatal, and causes over 20,000 deaths annually in the US alone. There is no treatment for this devastating disease process. ARDS requires advanced medical care including mechanical support of lung function (Mechanical Ventilation) in an Intensive Care Unit. Mechanical Ventilation, the supportive therapy necessary to sustain life in most cases, may further damage the lung, termed Ventilator Induced Lung Injury (VILI). Our research is focused on lung repair after VILI. We have developed a unique rodent model of the disease process, and have already demonstrated an important mechanism by which mechanical ventilation of the lung may retard effective repair.
We wish to go on and develop novel therapeutic strategies for patients with ARDS and VILI. Stem cell therapies, particularly mesenchymal stem cells (MSC’s), have demonstrated considerable promise in relevant pre-clinical models of ARDS. We wish to determine the efficacy of MSC’s and find out more about how they work, in models of ARDS caused by VILI. We also wish to determine the best way of administering these stem cells, and establish if we can further increase their effectiveness by modifying the stem cells to produce specific therapeutic proteins. We will carry out these studies in our rodent VILI model. This work will yield important insights into the potential for stem cell therapies to treat patients with ARDS and enhance our understanding of how these cells produce their effects.

Project title and Supervisors

Title: Devising New Therapeutic Strategies for Multiple Myeloma: Cell Cycle Dysregulation
Supervisors:
Professor Michael O’Dwyer, Professor of Haematology, National University of Ireland, Galway; Consultant Haematologist, University Hospital, Galway
Professor Corrado Santocanale, Professor of Molecular Medicine, National University of Ireland, Galway
Dr Patrick Hayden, Consultant Haematologist, University Hospital, Galway

Project Summary

Multiple Myeloma remains an incurable disease. Recent advances in its treatment are still halted by eventual progression. In unravelling the biology of multiple myeloma, numerous potential therapeutic targets have emerged. Among these are the cell cycle, DNA repair and apoptotic proteins.Cell cycle dysregulation is both key to the development of a mutant clone as well as to eventual progression.The project will explore, expand and refine this understanding with an ultimate aim of translating this work into modern therapeutics.

Project summary update (March 2010)

Targeting Cdc7 kinase in Myeloma. Coyne MRE -Department of Molecular Medicine, National University of Ireland, Galway. -Department of Haematology, Galway University Hospitals, Galway.

	Cell Viability Curves. A. Cell lines B. Primary Cells treated with increasing doses of PHA-767491

Myeloma remains an incurable blood cancer and the development of new therapeutic approaches is of fundamental importance. Myeloma is characterised by altered control of the cell cycle, an internal clock-like process that allows cells to divide and replicate. Cdc7 is an essential protein required for initiation of DNA replication and subsequent cell cycle progression. In the last two years a number of compounds have been developed to target this protein as an anti-cancer strategy. This project utilises the prototype Cdc7 inhibitor or blocker, PHA-767491. The main question this project asks is whether inhibiting this process will translate into a new therapeutic in Myeloma? This underlines translational haematology, in particular, bringing progress from the bench to the bedside but equally from the bedside to the bench. To date we have showed that inhibiting this protein has a potent anti-cancer effect in myeloma models and cells directly harvested from patients resistant to current standards of care. This preliminary work was presented at the American Society of Hematology and has led to forging an International Collaboration with Prof Leif Bergsagel, Mayo Clinic Arizona, one of the leaders in validating new treatments for myeloma. Moreover, the progress of delivering this treatment to myeloma patients moves closer with extension of a Phase I Clinical Trial with a oral Cdc7 inhibitor, NMS-354, to blood cancers in Galway’s new Cancer Research Facility. From this initial work, the project has uncovered other exciting roles of Cdc7. This underlines the two-way traffic between the bed and bench of translational science. These are currently under active investigation. In conclusion, the project, almost at the half-way mark, has provided the initial preliminary pre-clinical data to support the use of Cdc7 inhibition in myeloma. Much work remains to cystallise this preliminary work and develop other leads arising out of this work.
Publications in 2009:
*Coyne M et al Targeting Cdc7 kinase in Multiple Myeloma. Blood. 114 (22), 1479. 2009.
Awards in 2009
Best oral poster presentation Hematology Association of Ireland, 2009.  

Project title and Supervisors

Title: Investigation of microRNA expression patterns as novel biomarkers for breast cancer
Supervisors:
Professor Michael J Kerin, Professor of Surgery, Department of Surgery Clinical Science Institute, National University of Ireland Galway
Dr Nicola Miller, Senior Scientist, Department of Surgery, Clinical Science Institute, National University of Ireland Galway

Project Summary

Breast cancer is the leading cause of cancer-related death in women in Ireland and despite recent reductions in mortality rates due to earlier diagnosis and improved therapeutics, over 600 Irish women die from their disease annually. The adverse effects of breast cancer have clear implications for Irish society in terms of disease burden and economic cost. Resultantly much research effort now focuses on the identification of biomarkers to ameliorate disease classification, prognostication and to monitor disease recurrence. However due to the highly heterogeneous nature of the disease, to date this task has proven insurmountable.
miRNAs are an abundant class of small, single stranded non-coding RNA molecules. They function to regulate gene expression and as a result, mediate of several key biological processes. Their association with carcinogenesis has also been established and they have been demonstrated to have both oncogenic and tumour suppressor properties. The discovery that certain miRNAs are dysregulated in breast cancer, and more recently their detection in the circulation of breast cancer patients, indicates their potential as novel diagnostic and prognostic biomarkers and possibly even therapeutically.
The principal objective of this study is to identify dysregulated miRNAs in breast tumour tissue and to correlate these expression patterns in the circulation of breast cancer patients so that any changes may be monitored following therapaeutic intervention. In vitro manipulation of candidate miRNA levels will help to define their cellular function so that their precise roles in carcinogenesis may be better understood. This study, it is hoped, will provide a deeper understanding of miRNA biology so that these small RNAs will fulfil their promise as valuable prognostic markers and therapeutic targets in cancer. 

Project summary update (March 2010)

Breast cancer is a complex heterogeneous disease affecting over 1700 Irish women annually. Recent molecular advances have resulted in the reclassification of breast cancer into 5 distinct subtypes, each differing in terms of patient outcome, response to therapy and prognosis. Mi(cro)RNAs are a class of endogenous RNA molecules whose dysregulated expression in multiple disease processes is becoming increasingly well defined. Furthermore, dysregulated miRNA expression has also been identified in breast cancer. Recent work from our institution identified certain miRNAs to be elevated in the circulation of breast cancer patients with a subsequent decrease in the perioperative period. The purpose of this study is to identify clinically useful miRNA biomarkers to aid in the diagnosis, classification and prognostication of breast cancer patients.
In order to accurately and reliably quantify miRNA expression in the circulation, a comprehensive evaluation of candidate genes with which to normalise real time quantitative PCR data was required. A panel of candidates has been evaluated in miRNA derived from a cohort of 20 breast cancer patients and similarly matched control individuals. Validation of dysregulated miRNA expression is being undertaken in an expanded cohort of 69 breast cancer patients. A parallel study on miRNA gain-of-function analysis has been initiated in breast cancer cell lines. Thusfar, this has involved the maintenance and extraction of RNA from cell lines representative of the intrinsic subtypes of breast cancer to identify those cell lines with the lowest endogenous levels of candidate miRNAs.

Project title and Supervisors

Title: The developmental protein HOXc11 mediates endocrine resistance in breast
cancer
Supervisors:
Dr. Leonie Young, Leader Of Surgical Research, Royal College of Surgeons in Ireland
Prof. Arnold Hill, Professor of Surgery, Beaumont Hospital, Royal College of Surgeons in Ireland

Project Summary

Breast cancer will affect one in every twelve women in Ireland today. Despite recent advances in diagnosis and treatment it continues to be the leading cause of female cancer deaths in Ireland.
Along with surgery, hormone therapy has been a cornerstone in the treatment of breast cancer since the drug tamoxifen gained widespread use. This hormone therapy with tamoxifen and the recently developed aromatase inhibitors aim to remove hormones or block their action to stop cancer cells from growing and thus prevent the disease from recurring. While many tumours initially respond to these drugs, many will eventually relapse. This project is aimed at examining the mechanisms involved in enabling the breast cancer to escape from hormone therapy and grow again.
Our group has been particularly interested in a protein called SRC-1. Through previous work, we have shown that breast cancer patients who express this protein do not respond as well to hormone therapy as those who do not express SRC-1. Recently we have identified another protein called HOXc11 that interacts with SRC-1. HOXc11 is one of a class of proteins that control cell differentiation and it is well established that the development of cancer cells is a result of aberrant cell differentiation. We propose that the protein HOXc11 plays a central role in the development of resistance to hormonal therapy in breast cancer. Our aim is to establish exactly how HOXc11 contributes to the development of this resistance. We have identified one protein so far, S100ß, that is produced by the actions of HOXc11. This protein is secreted into the bloodstream and it is currently measured in patients with skin cancer, malignant melanoma, where it is useful in predicting response to treatment. Our initial work into S100ß in the setting of breast cancer has proved promising and we have started measuring levels in the bloods of patients with breast cancer. Our hope would be that this protein could be developed whereby it can be used a simple blood test to predict recurrence of breast cancer before the disease becomes clinically apparent.To further investigate the mechanisms of action of HOXc11 we plan to use a new experimental technique called ChIP-on-ChIP, to identify what other genes are affected by the actions of HOXc11 and what the functional significance of these genes is. By eliciting these mechanisms we can hope to develop new biomarkers of breast cancer and potential therapeutic targets for this devastating disease.

Project summary update (March 2010)

The calcium binding protein S100β as a prognostic indicator in breast cancer
SRC-1 is a protein known to play an important role in disease progression in breast cancer.  Through an interaction with a developmental protein called HOXc11, we have identified that SRC-1 helps regulate the production of another protein, S100β, in breast cancer cells that do not respond to traditional endocrine treatments.  The current aim of this work is to investigate the implications of S100β expression in breast cancer patients and secondly, to identify new SRC-1 target genes.
On a large group of human breast cancer specimens, we found that patients whose tumours expressed S100β protein were at greater risk of experiencing breast cancer progression.  Given that S100β is secreted into the bloodstream, we hypothesised that levels could be measured in patients with a simple blood test prior to any treatment.  We found elevated levels of S100β in 10% of patients and similar to our findings in the tumour specimen, patients who had elevated blood levels of S100β at time of diagnosis were more likely to experience disease progression.
As the range of therapeutics available for the management of breast cancer continues to expand, the ability to define those patients at greatest risk of disease progression will be central to the development of personalised medicine.  As a secreted protein S100β offers exciting potential as a biomarker in breast cancer.  Through a series of molecular and translational studies we aim to validate recently identified SRC-1 target genes to elucidate their putative role in SRC-1 mediated breast cancer progression.
Publication 2009
Interaction of Developmental Transcription Factor HOXc11 with Steroid Receptor Coactivator SRC-1 Mediates Resistance to Endocrine Therapy in Breast Cancer.
McIlroy M, McCartan D, Early S, Gaora PO, Pennington S, Hill AD, Young LS.
Cancer Res. 2010 Feb 9 (Epub ahead of print) PMID: 20145129

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Project title and Supervisors

Title: The Neurobiology of Psychogenic Non-Epileptic Seizures
Supervisors:
Professor Kieran Murphy, Chairman, Department of Psychiatry, RCSI
Professor Hugh Garavan, Director of Functional Imaging, Trinity College Institute of Neuroscience, Dublin

Project Summary

Epilepsy, a chronic disabling disorder affecting 1-2% of the population, is associated with significant levels of healthcare and carer burden. Several studies report that 20-25% of people with epilepsy have psychogenic non-epileptic seizures (PNES), seizures that occur without discernibly abnormal electrical activity in the brain. Several studies have suggested that people with PNES have a history of exposure to adverse environmental stressors. In addition, some studies suggest that people with PNES have specific difficulties in recognising and verbalising emotions.
Recent evidence suggests that abnormal alterations of the structure of the brain can occur following exposure to stress and may result in emotional dysregulation. Despite this, no study has investigated brain structure and function in people with PNES. Thus, we propose to examine brain structure and function of 30 people with PNES and 30 matched controls using structural and functional MRI. This is a truly translational medicine project where improved understanding of the neurobiological substrate of PNES will have significant implications for the development of novel diagnostic and therapeutic opportunities.
I will test the following hypotheses that:
People with PNES have significant differences in attention and cognitive processing on neuropsychological testing compared to age, IQ and gender matched controls.
People with PNES show abnormalities of brain structure, particularly affecting the amygdala-hippocampal complex, compared to age, IQ and gender matched controls.
People with PNES have functional abnormalities in implicit processing of emotion using functional magnetic resonance imaging. 

Project summary update (March 2010)

Psychogenic non-epileptic seizures (PNES) are episodes of altered movement, sensation or experience resembling epileptic seizures, but associated with patho-physiological processes and not with ictal discharges in the brain. This condition is classified as a conversion disorder and is present in 20-25% of patients who present to neurology services with epilepsy. People with PNES have a history of high rates of psychopathology, emotional dysregulation and neurocognitive impairment. Although such impairments are associated with brain structural and functional abnormalities, no previous study has examined quantitative brain structure or function in this patient group.
We recruited 20 patients with PNES without co-morbid epilepsy or other neurological condition and 20 healthy controls matched for age, IQ, gender and handedness.
Each participant underwent psychometric & neuropsychological assessment and brain scanning using structural MRI, diffusion tensor imaging and functional MRI. The results of these investigations will be presented and implications for future research and treatment discussed.

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Project title and Supervisors

Title: Modulation of ion transport and human airway epithelial physiology by two classes of anti-inflammatory molecules, lipoxins and glucocorticoids
Supervisors:
Professor Brian Harvey, Director of Research, Professor of Molecular Medicine, Royal College of Surgeons in Ireland
Professor Richard Costello, Associate Professor of Medicine and Consultant Chest Physician,
RCSI Education and Research Centre, Smurfit Building, Beaumont Hospital

Project Summary

Glucocorticoids are anti-inflammatory molecules used in the treatment of airway inflammatory diseases such as asthma. Lipoxins are biologically active eicosanoids and potent anti-inflammatory agents; however, their use in treatment of asthma or cystic fibrosis has not yet been reported in the literature. The precise molecular mechanism of action of glucocorticoids on epithelial inflammation, tight junction formation/repair and fluid secretion in the lung are largely unknown.
My research programme aims to test the hypothesis that glucocorticoids and lipoxins (LXA4) act synergistically on ion transport, tight-junction repair and eosinophil penetration in human airway epithelium. Using fluorescence confocal microscopy and short-circuit current measurements in human bronchial epithelial cells and primary bronchial epithelial cell cultures, we will test the effects of dexamethasone and lipoxins on cell signalling, ion transport and air-surface liquid volume (as a measure of the physiological function of the epithelium). Tight-junction breakdown occurs in a number of inflammatory diseases such as severe asthma and cystic fibrosis. Epithelial tight junctions can also provide a vital route for electrolyte and fluid transport across airway epithelia and provide a barrier to the migration of toxic substances.
We aim to compare dexamethasone and LXA4 effects on tight-junction (TJ) protein expression, TJ complex assembly and on transepithelial electrical resistance. Eosinophil infiltration into lung tissue is an important pathological feature found in the inflammatory process associated with asthma. We will test the hypothesis that one of the consequences of the tight-junction regulation by LXA4 is the limitation of the transmigration of eosinophils through human airway epithelium.
The translational aspect of this project aims to investigate the delivery of LXA4 stable analogues and third generation epithelial sodium channel blockers and their effect on epithelial surface hydration in a phase II clinical trial.

Project summary update (March 2010)

Lipoxin A4 increases Airway Surface Liquid height in Cystic Fibrosis bronchial epithelia
Mazen Al-Alawi, Valia Verriere, Olive McCabe, Raphael Chiron, Richard W. Costello, Valerie Urbach and Brian J. Harvey
Lipoxin A4 (LXA4) is an endogenous anti-inflammatory lipid mediator which is reduced in Cystic Fibrosis (CF) airways1. The altered Cl- secretion and Na+ hyperabsorption in CF affects the airway surface liquid (ASL) homeostasis and leads to a defective mucociliary clearance, chronic infection, inflammation and progressive lung destruction. In this study, the role of LXA4 in modulating ion transport and ASL height in CF and non-CF airway epithelia was investigated. CF (CuFi-1) and non-CF (NuLi-1) bronchial epithelial cell lines were grown into well-differentiated epithelia. LXA4 effects were explored using laser confocal fluorescence microscopy to measure ASL height, and short-circuit current and whole-cell patch clamp to investigate ion transporter activity. The spontaneous steady-state ASL height was significantly lower in CF than in non-CF epithelium. LXA4 (1nM) treatment for 15 minutes, increased ASL by a third in Nuli-1 epithelia (n=18) and doubled ASL height in CuFi-1 epithelia (n=19). This effect was sustained over 24 hours in the CF epithelia. The increase in ASL height by LXA4 was inhibited by Boc-2 (FPR1 antagonist), bumetanide (Cl- transport inhibitor), amiloride (Na+ channel inhibitor), reactive blue (P2Y2 receptor antagonist) and extracellular hexokinase (ATP hydrolysis). In addition, LXA4 stimulated Cl- secretion and inhibited Na+ absorption in CF bronchial epithelium. Our results provide evidence for a novel effect of LXA4 involving the FPR1 receptor, ATP secretion and purinoreceptor activation, inhibition of Na+ absorption and stimulation of Cl- secretion in CF and non-CF epithelia to finally increase ASL height. These novel effects of LXA4 open up a new therapeutic avenue in the treatment of CF.

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Project title and Supervisors

Title: Investigating the role of estrogen in cystic fibrosis
Supervisors:
Prof. Noel G. McElvaney, Dept. Medicine, Respiratory Research Division, RCSI
Dr. Catherine M. Greene, Dept. Medicine, Respiratory Research Division, RCSI
Prof Brian Harvey, Dept. Molecular Medicine, RCSI

Project Summary

Cystic fibrosis (CF) is a disorder characterised by chronic neutrophil-dominated airway inflammation and colonisation of the lungs with bacteria. Dysfunctional innate immune responses in the lung are known to play a key role in the pathogenesis of the pulmonary manifestations of CF. Females with CF exhibit a more aggressive course with a median survival of 1 year less than males and are particularly prone to deterioration of lung function with the onset of puberty.
This project will investigate the role of estrogen on airway inflammation in CF by evaluating the inflammatory consequences of microbial agonists on initiating and promoting inflammation in airway epithelial cells in response to estrogen stimulation in vitro. This project will also investigate the effect of estrogen on chloride ion secretion and KCNQ1 channel function in CF airway epithelial cells. Clinical studies using biological samples from females and males with CF will also be performed. These studies may have important implications for inflammatory gene expression and compensatory chloride secretion mechanisms in CF airway epithelial cells in females with cystic fibrosis.

Project summary update (March 2010)

Oestrogen Effects in the Lung may Answers to the Gender Difference in Cystic Fibrosis
Sanjay H. Chotirmall, Catherine M. Greene, Irene K. Oglesby, Warren Thomas, Shane J. O’Neill, Brian J. Harvey, Noel G. McElvaney
Cystic Fibrosis (CF) is a genetic disorder affecting many organs with the leading cause of mortality being recurrent infection and airway destruction. Ireland has the highest incidence and carrier rate of CF worldwide.  Females with CF have more aggressive disease particularly with the onset of puberty. They have worse lung function, increased mortality and earlier bacterial colonization compared to male CF patients. An explanation for this “gender gap” in CF remains elusive. We investigated the role of the female sex hormone oestrogen as a potential cause of the CF gender gap. W

SLPI protein microscopy was performed to confirm sub-cellular localization at baseline and following 10nM oestrogen (E2) treatment +/- cystic fibrosis bronchoalveolar lavage fluid (CF BALF). The images show that SLPI protein localizes to the nucleus upon oestrogen exposure

e have found that exposing the cells which line the airways of the lungs  (epithelial cells) to oestrogen produces an  inhibition of an important inflammatory protein (interleukin-8) in CF. This IL-8 protein is normally released by the epithelial cells in response to bacterial and viral infection in the CF lung. Oestrogen acts on cells by binding to two types of receptors, oestrogen receptor alpha (ERα) and oestrogen receptor beta (ERβ) and we measured the expression of both of these receptors in CF epithelial cells grown in the laboratory and also from samples obtained from the lungs of CF patients.  Both types of oestrogen receptors were detected in CF cells although ERβ receptor expression was greater than the expression of the ERα type.. Using drugs which activate or inhibits these different oestrogen receptors, we were able to show that oestrogen acting through the ERβ receptor caused the inhibition of IL-8 release from CF cells in response to an inflammatory stimulus. We also showed that amounts of another protein called secretory leucoprotease inhibitor (SLPI) in the cell increased after exposure to E2 and that this protein moves into the nucleus of CF cells after exposure to oestrogen.  The SLPI protein acts to prevent the release of IL-8 from the CF epithelial cells. In summary, E2 inhibits IL-8 release through the ERβ receptor in CF cells by increasing the amounts of SLPI protein in the cell. This research shows a novel anti-inflammatory mechanism for E2 in females with CF, which may predispose to infection and colonization. These data may at least in part explain the gender differences observed in CF. 


Research Highlights for 2009
• Awarded “International Trainee Travel Award (ITTA)” by the American Thoracic Society Assembly on Allergy, Immunology and Inflammation (AII), San Diego 2009.
• Presentation at the “International Scholars Poster Colloquium” at the American Thoracic Society Congress, San Diego 2009.
• Poster Presentations
-23rd North American Cystic Fibrosis Conference, Minneapolis 2009
-Irish Thoracic Society Annual Meeting, Galway 2009
-Beaumont Hospital Sheppard Prize 2009
-Royal College of Surgeons in Ireland Research Day 2009

 

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Dr David Prichard, TCD

Project title and Supervisors

Title: Ursodeoxycholic Acid – a Molecular Modulator of the Inflammation-Cancer Sequence in the Oesophagus?
Supervisors:
Professor Dermot Kelleher, Department of Clinical Medicine, Trinity College Dublin
Dr Aideen Long, Department of Clinical Medicine, Trinity College Dublin

Project Summary

Each of the previous few decades has seen a 10% increase in the incidence of oesophageal adenocarcinoma (OAC) making OAC the carcinoma that is growing at the fastest rate in terms of incidence in the developing world. This cancer has a poor prognosis regardless of treatment. OAC commonly arises from a pre-malignant metaplastic transformation of oesophagus cells known as Barrett’s Oesophagus (BO). Over time, BO may progress through dysplasia to OAC. Our current understanding is that BO most commonly occurs in the setting of Gastro-Oesophageal Reflux Disease (GORD). Acid in the refluxate has been identified in epidemiological studies as a major risk factor for OAC; however, the use of effective acid suppression therapies (proton pump inhibitors) has yet to impact on either disease pathology or epidemiology. This suggests that factors other than acid in the refluxate contribute to development and progression of oesophageal metaplastic lesions.
The presence of abnormal concentrations of bile acids (in particular deoxycholic acid (DCA) in refluxate from GORD patients suggests that they may be involved in the promotion of oesophageal injury and metaplasticity.
Ursodeoxycholic acid (UDCA) has been shown to inhibit the effects of DCA in oesophageal and other cells. In animal models, UDCA has also been shown to inhibit the development of upper GI malignant disease. UDCA has been shown to activate the glucocorticoid receptor and as such its effects may be mediated through this pathway. It may also act as an anti-inflammatory through this pathway.
Thus, research into UDCA’s gene activation profile and UDCA’s effect on modulating genes activated by low pH and bile acids in oesophageal cells may identify new therapeutic strategies for preventing BO and the progression of BO to OAC. Investigations regarding the role of the GR these processes will not only develop our understanding of the protective role of UDCA in the oesophagus but will also further our knowledge of UDCA as an anti-inflammatory molecule.

Project summary update (March 2010)

Hydrophobic bile acids present in oesophageal refluxate have been identified as pro-inflammatory mediators and as carcinogens. Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is established as a therapeutic agent in liver disease in which exposure to high concentrations of hydrophobic bile acids such as deoxycholic acid (DCA) results in chronic inflammation. UDCA exerts its effects through anti-apoptotic and anti-inflammatory properties. Both have been shown to be partly mediated by UDCA activation of the glucocorticoid receptor (GR) rather than simply by displacement of more toxic bile acids. As such UDCA may be of benefit in reducing chronic inflammation in the oesophagus and the progression to OAC.
Our research using HET-1A cells, representing normal oesophageal mucosa, has demonstrated that UDCA at doses of 300μMol inhibits cellular proliferation but does not significantly increase apoptosis or necrosis. DCA treatment results in a significant reduction in cell viability in a dose and time dependent fashion. This appears to be mediated through both apoptosis and necrosis. Pre-treatment of HET-1A cells with UDCA does not inhibit the overall level of DCA induced cell death, but appears to reduce DCA induced apoptosis.
UDCA can induce translocation of the GR in a variety of oesophageal cell lines in a time and concentration dependent manner and can activate GR dependent transcription. Finally, we have demonstrated that UDCA-can to inhibit DCA induced transcription of pro-inflammatory cytokines such as IL-8.
Our research suggests that UDCA does not propagate DCA induced cell death and may have benefit in reducing the inflammatory response to hydrophobic bile acids.

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Project title and Supervisors

Title: Generation and Characterisation of Activated Protein C variants with Altered Functional Properties and Enhanced Therapeutic potential
Supervisors:
Professor James O'Donnell
Dr Roger Preston

Project Summary

The only effective therapy currently available for patients admitted to Intensive Care Units with severe sepsis is a recombinant anticoagulant protein called Activated Protein C (APC). Interestingly, studies have shown that the beneficial effects of APC in such patients are not entirely attributable to the anticoagulant effects of APC. Rather, APC can also directly modulate the inflammatory response by initiating complex intracellular signalling. The aims of my project are to generate and characterize novel APC variants with enhanced functional properties that could be utilized for potential therapeutic gain. Furthermore, these variants will provide further insight into the molecular and cellular basis through which the anticoagulant and anti-inflammatory effects of APC are mediated. 

Project summary update (March 2010)

Generation and Characterisation of Activated Protein C Variants with Altered Functional Properties and Enhanced Therapeutic Potentia. The only effective therapy currently available for patients admitted to intensive care units with severe sepsis

Activated protein C – an anticoagulant and anti-inflammatory enzyme Activated protein C (APC) uses the enzymatic activity in its serine protease domain (blue) to break down coagulation factors and initiate anti-inflammatory signaling in the vaculature. These properties have lead to the utilization of APC for the treatment of severe sepsis and other inflammatory diseases. Image generated from Gla domainless APC crystal structure (1AUT 31), using PYMOL molecular visualization software.

is a recombinant anticoagulant protein, Activated Protein C (APC). Interestingly, studies have shown that the beneficial effects of APC in such patients are not entirely attributable to the anticoagulant effects of APC. Rather, APC also has a separate “cytoprotective” function and can directly modulate the inflammatory response by initiating complex intracellular signalling. The aims of my project are to generate and characterize novel APC variants with enhanced functional properties that could be utilized for potential therapeutic gain. Furthermore, these variants will provide further insight into the molecular and cellular basis through which the anticoagulant and anti-inflammatory effects of APC are mediated. 
Major findings to date include generation of a novel APC variant which lacks anticoagulant function but exhibits enhanced cytoprotective properties. This variant has potential as an improved therapy in sepsis because administration of APC in this setting is frequently limited by bleeding.
An important interaction between APC and the heparin-reversing agent protamine sulphate was also characterized. This work led to the elucidation of the molecular mechanism through which protamine can cause bleeding, an unwanted side effect often occurring immediately after cardiothoracic surgery.
Ongoing work is directed towards discovering novel approaches to enhance the therapeutic potential of APC and ultimately to improve its efficacy in sepsis treatment.

Peer-reviewed publications since commencement of MMI clinician scientist fellowship programme:
*Ní Áinle F, Preston RJ, Jenkins PV, Nel HJ, Johnson JA, Smith OP, White B, Fallon PG, O'Donnell JS. Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation. Blood 2009;114(8):1658-65.
*Preston RJ, Tran S, Johnson JA, Ní Áinle F, Harmon S, White B, Smith OP, Jenkins PV, Dahlbäck B, O'Donnell JS. Platelet factor 4 impairs the anticoagulant activity of activated protein C. J Biol Chem. 2009;284(9):5869-75.
*Harmon S, Preston RJ, Ní Áinle F, Johnson JA, Cunningham MS, Smith OP, White B, O'Donnell JS. Dissociation of activated protein C functions by elimination of protein S cofactor enhancement. J Biol Chem. 2008;283(45):30531-9.

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Project title and Supervisors

Title: Brain structure, function and connectivity in autism
Supervisors:
Dr Louise Gallagher, PhD, MRCPsych, Consultant Child and Adolescent Psychiatrist, Clinical Senior Lecturer, School of Medicine, Department of Psychiatry, Trinity College Dublin
Professor Hugh Garavan, PhD, Associate Professor, Trinity College Institute of Neuroscience, Department of Psychology, Trinity College, Dublin.
Dr Katherine Johnson, PhD, Research Fellow, Trinity College Institute of Neuroscience, Department of Psychology, Trinity College, Dublin.

Project Summary

Background: People with high functioning autism (HFA) have difficulties in social interaction, communication and behaviour, but normal intelligence. In spite of the enormous impact associated with autism, no effective treatment has yet been developed. To devise a therapy for any disorder, knowledge of the basic underlying problems is essential. Unfortunately, in autism, these are not yet fully understood. It has been suggested that a multidisciplinary approach is essential in furthering our understanding of the cause of autism (Dawson et al, 2002). This study is strongly interdisciplinary, seeking to investigate links between neuropsychological and neuroimaging abnormalities in autism. Scientific research shows that individuals with HFA have trouble changing thought-processes, inhibiting inappropriate responses and perceiving faces. Abnormalities in brain structure and function and unusual connections between brain regions may underpin these problems but are not yet fully understood. Such an understanding is essential for the development of rational treatments for this disorder.
Theory: That people with HFA have difficulties changing thought-processes, inhibiting responses and perceiving faces and that these problems are associated with underlying structural and functional brain abnormalities.
Objectives
• To investigate brain activity and connections between brain regions in HFA during performance of psychological tasks.
• To relate brain activity and connectivity to brain structure.
Methods: Computer-based psychological tasks will be administered to a group with HFA and controls. Neuroimaging techniques will detect differences in brain activation and brain structure between the groups during these tasks.
Likely outcomes: During the psychological tasks, we expect children with HFA to have performance difficulties and abnormal brain activity. We anticipate that abnormal brain activity will occur in areas of the brain that are structurally abnormal.

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Project title and Supervisors

Title: The association of variations in the immune response with the development of idiopathic bronchiectasis
Supervisors:
Dr. John Jackson FRCPath, Chief Scientist, Department of Immunology, St. James’s Hospital Dublin
Dr. Mary Therese Keogan, MD. FRCPI, FRCPath, FFPath(RCPI), Consultant Immunologist, Beaumont Hospital, Medical Director of the National Histocompatibility and Immunogenetics Service for Solid Organ Transplantation, Hon. Senior Lecturer, Royal College of Surgeons in Ireland
Prof. Con Feighery FRCPI, Consultant Immunologist, St. James’s Hospital, Director of Department of Clinical Immunology
Supporting Collaboration: Prof. Luke O’ Neill, School of Biochemistry and Immunology
Trinity College Dublin

Project Summary

Bronchiectasis is a chronic suppurative lung disease associated with significant mortality and morbidity. Dilated airways act as reservoirs for infecting organisms resulting in a vicious cycle of infection, inflammation with airway damage, impairment of local defences and further infection. Bronchiectasis complicates many immunodeficiencies. Over recent years our expanding knowledge of immunology has shown that subtle immune variations normally compatible with health can, in some individuals, result in a predisposition to infection. Our hypothesis is that subtle variations in the immune response in combination may constitute an immune deficiency.
Many of the key pathogens associated with bronchiectasis are coated in polysaccharide capsules. Opsonisation of these organisms is essential for effective phagocytosis. A functional polymorphisms of CD32, the only human receptor for IgG2 leaves some individuals unable to bind IgG2, and therefore unable to effectively phagocytose organisms opsonised with IgG2. While this variation is compatible with good health, in those people who make a predominantly IgG2 response to polysaccharide antigens, the combination of these two factors is likely to be significant. Further subgroups of people fail to mount any response on vaccine challenge, or make antibody responses that wane quickly.
We wish to assess whether these impaired antibody responses are associated with a reduction in the memory B cell population, and whether poor antibody production is more clinically relevant in combination with other opsonic defects. Polymorphisms in mannose binding lectin resulting in reduced protein levels may also result in reduced opsonisation. Additionally recent developments in the role of pattern recognition molecules, the toll-like receptors and their adaptor molecules have established a role for functional polymorphisms in the modulation of the immune response.
This study will investigate the role of CD32 polymorphisms, MBL polymorphisms, TLR polymorphisms, responses to anti-polysaccharide vaccine challenges and alterations in memory B cell population, in the development and severity of idiopathic bronchiectasis.
Our ultimate aim is to define the clinical significance of immune variation in susceptibility to infection and to translate the research protocols established for assays found to be clinically relevant to a diagnostic immunology laborator.

Project summary update (March 2010)

This project aims to examine the impact of subtle variations in the immune system on the development of the chronic suppurative lung disease bronchiectasis. Bronchiectasis is characterized by structural damage to the lungs caused by recurrent chest infections. We have examined factors thought to predispose to recurrent infection in a well characterized population of over 100 patients with bronchiectasis.
We first examined the prevalence of mutant versions of the gene coding for the important complement protein mannose binding lectin. We determined that polymorphic versions of the gene occur in about 30% of the healthy Irish population but are not increased in the bronchiectasis cohort. We also examined the prevalence of a polymorphism (H131R) of the gene coding for the IgG receptor CD32. We found that the prevalence of the homozygous R131 variant was doubled in our bronchiectasis group compared with the control group. The presence of this variant is thought to result in defective phagocytosis of encapsulated bacteria.
To investigate the impact of this variant further we have established flow cytometric assays to measure phagocytosis. We have used fluorescent latex beads coated with bacterial sugars and fluorescently labeled bacteria as models for phagocytosis. We are currently examining the functional impact of these polymorphism on phagocytosis of infective targets, generation of oxidative burst and expression of markers of neutrophil activation. We are also examining variations of our phagocytosis assays using small volume whole blood samples which are versatile and suited to paediatric applications and using high content screening applications which allow measurement of morphological data.
These investigations will give us a better understanding of the impact of CD32 polymorphisms and may provide evidence of a direct link to the pathology of bronchiectasis.

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Project title and Supervisors

Title: Cellular mechanisms of insulin resistance due to the R482W mutation of the LMNA gene in Familial Partial Lipodystrophy, Dunnigan Variety (FPLD)
Supervisors:
Rosemary O'Connor, Ph.D., Professor of Cell Biology, Department of Biochemistry, BioSciences Institute, University College Cork
Dr. Domhnall J. O’Halloran, Consultant in Endocrinology & Diabetes Mellitus, Cork University Hospital

Project Summary

In this study we aim to further elucidate the molecular mechanisms involved in producing the marked clinical phenotype of insulin resistance (IR) / metabolic syndrome (MetS) that occurs in FPLD. Our working hypothesis is that the LMNA mutation (R482W) causes a disruption in gene expression as well as signalling responses to insulin and sex hormones.
This leads to altered adipocyte differentiation and function giving rise to lipodystrophy and in turn to the severe metabolic phenotype seen in FPLD. We will do this by investigating differentiation, physiology and cell signalling via insulin and IGF–1 receptors in adipocyte cell lines generated from 3T3-L1 preadipocytes that have been transfected to over-express wild type and R482W mutant LMNA.
We will also investigate the role of estrogens and androgens in adipocyte differentiation as the onset of this disease is triggered by puberty, and females tend to have worse phenotypes and outcomes. In achieving these goals we hope to gain valuable insights into the pathogenesis of IR and Type 2 Diabetes Mellitus (T2DM) in FPLD. This in turn will further our understanding of adipocyte biochemistry and function. 

Project summary update (March 2010)

Mutations in the LMNA gene, which encodes key proteins in the wall of a cell nucleus (lamins A & C), cause several diseases including Dunnigan-type familial partial lipodystrophy (FPLD). This disease causes subjects to develop severely deranged fat tissue distribution at puberty, leading to a striking physical appearance including enlarged neck fat pads and thin & muscular limbs. Patients develop insulin resistance, fatty liver disease, hypertension, raised cholesterol levels and tend to die in their fifth and sixth decades of life from sudden cardiac death.
We used two established fat cell precursor cell-lines to investigate the mechanisms underlying of abnormal fat distribution in FPLD (3T3-L1 and LMNA WT/KO cells). When the mutant LMNA gene (R482W) was transfected into the cells, a key step in fat cell differentiation was inhibited (PPAR gamma gene expression). Our investigation of cell lines derived from patients’ B cell lymphocytes show that these cells express the mutant LMNA gene and there is an abnormal build-up of the precursor pre-lamin A, which has been implicated in altered cell differentiation in other cell types.
Annual turnover of fat cells in humans has recently been estimated to be as high as 10%. This is the first study to show alterations in fat cell differentiation of animal cell models as a result of over-expressing the R482W mutant as compared to over-expressing wild type LMNA. Site-specific fat cell expression of mutant LMNA may underlie the changes seen in FPLD - populations of fat cells expressing more R482W mutated LMNA may have impaired ability to re-generate. Our next step is to look at fat cells from our patients and devise a mechanism for reducing pre-lamin A build-up in these cells in order to normalize fat tissue distribution.
Findings presented at ENDO 2009 (Annual Scientific Meeting of The Endocrine Society in Washington D.C. June 2009) – Nominated for President’s Prize.
Also presented at the annual meeting of the Irish Endocrine Society in November 2009.

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Project title and Supervisors

Title: Paracrine mediators of progenitor cell function and myocardial repair post ischaemia
Supervisors:
Professor Noel M Caplice MD PhD, Director, Centre for Research in Vascular Biology, Biosciences Institute, UCC; Professor of Cardiovascular Sciences UCC; Consultant Physician/ Cardiologist Cork University Hospital

Project Summary

We anticipate a substantial contribution to the understanding of myocardial infarction (MI). MI is obviously a very common and serious condition: cardiovascular disease is the leading cause of mortality in the Western world. The treatment of MI is constantly evolving, and is at a very interesting stage currently.
The use of fibrinolytic agents such as streptokinase, second-generation tissue plasminogen activator (t-PA) alteplase, and newer third-generation agents such as reteplase and tenecteplase have been the first line of treatment for acutely occluded coronary arteries over the last three decades. However, with current fibrinolytic drugs, about 40% of patients do not achieve epicardial (vessel) patency, and 20% to 50% of those who achieve vessel patency do not achieve myocardial patency.
Coronary-artery bypass grafting (CABG) and percutaneous coronary intervention (PCI) have been the mainstay of mechanical coronary revascularization for the last few decades. These techniques have been very effective in reducing the symptoms associated with coronary disease, but in the long-term the disease process continues despite adjunctive therapy, ultimately meaning the majority of patients require repeat procedures. Adjunctive therapy refers to pharmacologic therapy with medications such as statins (HMG CoA reductase inhibitors), antiplatalet agents such as aspirin and plavix, beta-blockers, and angiotensin-receptor blockers which aim to slow the atherosclerotic disease process and decrease the hazardous complications of the healing process which results in unwanted complications such as myocardial fibrosisThe most recent developments in PCI technology involving the use of medications delivered via polymer coating of (bare) metal stents have decreased the rate of restenosis in diseased coronary arteries, but have introduced many more challenges most often associated with the prolonged dual antiplatalet therapy required with their usage. Their initial superiority over bare-metal stents has not been as substantial with their increased usage as was initially postulated six years ago. Technological developments have provided the ability to stent more lesions, but the mortality benefit has not been realised.
The limitations of the mechanical modality to improve mortality highlight the need to continue to investigate alternative therapeutic means to improve outcome. Perhaps we need to look more closely at earlier stages of the disease process: certainly we need to understand it more thoroughly. The discovery of endothelial and smooth muscle progenitor cells in the circulation after MI revealed a completely new aspect to the development of coronary artery disease. These are precursor cells discovered in the peripheral circulation after MI, and by various imaging modalities have been shown to target injured myocardium and stimulate its repair. In an experimental model the injured myocardium has been shown to regain viability and function.
There has been much debate recently regarding the benefit of injecting progenitor cells into patients’ coronary arteries in terms of improved clinical outcome, as results have not been as encouraging as was initially hoped. We believe this is missing the point entirely- there is no question that these cells are released post MI, that they target the injured myocardium, and stimulate recovery and repair- we cannot expect to transfer this benefit to improve long-term clinical outcome when we barely understand the mechanism involved, and the numerous different cytokinetic and physiologic messenger pathways implicated.
Much more research needs to be undertaken, and we especially need to understand the mediators of progenitor cell release, proliferation, and homing to injured myocardium, and how the myocardium is subsequently repaired. When we have reached this point we will be in a better position to manipulate the disease process for potential therapeutic benefit.

Project summary update (March 2010)

Rescuing Ischaemic Myocardium: Novel Therapies and Imaging Modalities
Cardiovascular disease remains the leading cause of death in the Western World. I am investigating new mechanisms of heart tissue repair and regeneration following a “heart attack” or myocardial infarction (MI), and new methods of imaging the damaged heart tissue in this context.
We are demonstrating promising cardioprotective effects of both Insulin-like Growth Factor-1 (IGF-1) and Nitrite. We are using the latest generation 64-slice CTPET to image myocardial function, remodelling, and tracking of therapies to the infarct zone.
At 24 hours we found a significant reduction in cell death in damaged heart tissue, and an improvement in heart function with IGF-1 treatment. This benefit was sustained long term, since at two months, with IGF-1, we found a significant reduction in infarct size, heart wall thinning, wall motion defects, and a significantly reduced impairment of heart contractility.
Using Nitrite, which is the precursor to nitric oxide used by angina patients, we demonstrated via myocardial perfusion on CT, that there is a significant cardioprotective effect independent of blood flow. Using microscopic fluorescent microspheres which can travel down the smallest heart vessels, we have demonstrated that nitrite increases flow into the core of the heart attack area, known as the “microvascular obstruction” area. This has significant implications for future therapeutic delivery, as it literally “opens up” this area for drug delivery.
Publications in 2009:
1. Kenneth Martin, Sharon Weiss, Pat Metharom, Jeffrey S Schmeckpeper, Brian G Hynes, John F O’Sullivan, Noel M Caplice.
‘Thrombin Stimulates Smooth Muscle Cell Differentiation from Peripheral Blood Mononuclear Cells via Protease-Activated Receptor-1, RhoA, and Myocardin.
Circulation Research 2009. Jul 31;105(3):214-8.
Pubmed: 19574550
2. Leblond AL, O’Sullivan J, Caplice NM.
Bone marrow mononuclear stem cells: potential in the treatment of myocardial infarction.
Stem Cells and Cloning: Advances and Applications.
3. O’Sullivan JF, McFadden E
‘Ostial left main stenosis in a frequent flyer’
Int J Cardiol 2009 May 15;134(2):e66-7. Epub 2008 Apr 2.
Pubmed: 18378023
4. Book Chapter
John F O’Sullivan, Anne-Laure Leblond, Noel M Caplice.
Chapter 12 ‘A key role of angiogenesis in recovery from ischaemic heart disease’.
Therapeutic Angiogenesis. Editor: M Slevin. (In Press)

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Project title and Supervisors

Title: An investigation of the therapeutic potential of lipoxins and lipoxin analogues in diabetic nephropathy
Supervisors:
Professor Catherine Godson, Diabetes Research Centre, Conway Institute, School of Medicine and Medical Sciences, UCD
Dr. Denise Sadlier, Consultant Nephrologist and Senior Lecturer, Mater Misericordiae University Hospital and Clinician Investigator , Diabetes Research Centre, Conway Institute, School of Medicine and Medical Sciences, UCD.

Project Summary

Diabetes mellitus is a disease that is characterised by high blood sugar and the consequences of which affect many organs. Although the effects of high glucose help explain some of the ways in which diabetes affects patients there are many other mechanisms at play which have recently come to light. Currently, available treatments may slow the development of complications of the disease but cannot reverse these devastating conditions. Diabetes remains a major public health problem that is a common cause of heart disease, blindness, limb loss and kidney disease (The commonest cause for end-stage renal disease in Ireland). In order to devise better treatments for diabetic kidney disease it is important that we understand how it is initiated and progresses with a view to reversing the relentless progression to renal failure.
My research is dedicated towards finding better markers that indicate diabetic kidney disease progression and novel medications that will prevent or reverse this disease. In particular I seek to explore whether a novel class of potential therapeutics i.e. lipoxins may play a role in reversing or limiting scarring in diabetic kidney disease. Recent research has shown that inflammation plays an important role in diabetic kidney disease and that unresolved inflammation leads to scarring in the kidney which has been shown to be one of the key steps to progression to end stage renal failure and requiring replacement for native renal function, in the form of transplantation or dialysis. Lipoxins are substances that have been shown to have anti-inflammatory properties and effects that inhibit some of the key factors involved in scarring in general and in particular those cells of the kidney vulnerable to damage in diabetes. These observations form the basis of my hypothesis: That lipoxins may be useful agents in the treatment of diabetic kidney disease. I will test this hypothesis in cell and animal models of the disease.
In summary I believe that diabetic kidney disease is an important public health issue, in which inflammation and scarring have been shown to be key factors in disease progression. Lipoxins are a novel group of potential therapeutic agents that have been shown to have some effects on the latter processes and our research proposal, which is outlined in more detail forthwith, seeks to determine what affects these agents will have on diabetic kidney disease.

Project summary update (March 2010)

Tubulo-interstital fibrosis is the final common pathway in chronic kidney disease (CKD) leading to end-stage kidney disease (ESKD). This process results in replacement of the functional unit of the kidney with scar tissue resulting in a requirement for either dialysis or transplantation. At present there is a limited of availabe effective therapies that limit this process. The aim of this research project is to explore the therapeutic potential of lipoxin A4 (LXA4) as a novel suppressant of renal

LXA4 modulation of TGF-β1 induced Smad and ERK activation

fibrosis.
Renal fibrosis may reflect recruitment of circulating fibrocytes, activation and proliferation of resident renal fibroblasts and transition of epithelial cells to a mesenchymal phenotype [EMT]. We have previously reported that the novel anti-inflammatory and proresolution eicosanoid LXA4 can attenuate EMT in models of fibrotic injury (Rodgers et al., Am J Pathol  167(3): 683-94; 2005).  Here we have explored whether LXA4 might also impact on aldosterone and TGF-β1- driven renal fibroblast proliferation, activation and extracellular matrix (ECM) production.
Recent research suggests a growing appreciation of the role of aldosterone as a key mediator in renal fibrosis however the exact molecular mechanisms involved remain unknown. We provide novel evidence of the key role of TGF-β1 in aldosterone induced fibroblast activation and CTGF induction. In summary this research indicates the potential of LXA4 as a novel suppressant of renal fibrosis, which we will further explore using a relevant in vivo model.
Publications 2009:     
Animal models of glomerulonephritis  by Ryan A , Sadlier D, Godson C  published by Cambridge University Press (Invited chapter to a textbook entitled Fundamentals of Inflammation, edited by Serhan C, Ward P and Gilroy D,  submission  date Febuary  2009).
Diabetes mellitus and apoptosis: inflammatory cells.  Ryan A, Murphy M, Godson C, Hickey FB.  Apoptosis. 2009 Dec;14(12):1435-50. Epub 2009 Apr 10. 
Lipoxins: regulators of resolution. Ryan A, Godson C.  Current opinion in Pharmacology ( Submitted Dec 2009, Accepted for publication Feb 2010).                        

Project title and Supervisors

Title: Human and in vitro studies examining the early effects of antiretroviral drugs on mitochondrial DNA and genes regulating lipid metabolism
Supervisors:
Dr Patrick Mallon, MB BCh FRACP PhD; Lecturer in Medicine and Consultant Infectious Diseases Physician, UCD School of Medicine and Medical Sciences, Catherine McAuley Education and Research Centre, Mater Misericordiae University Hospital, Dublin
Dr Peter Doran, BSc PhD, Scientific Director, UCD Clinical Research Centre, Genome Resource Unit, UCD School of Medicine and Medical Sciences, Mater Misericordiae Hospital, Dublin

Project Summary

Highly active antiretroviral therapy (HAART) comprising combinations of nucleoside analogue reverse transcriptase inhibitors (NRTI), non-nucleoside analogue reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI) has markedly decreased morbidity and mortality for HIV infected individuals. However, as therapy is lifelong, concerns have arisen over the long term side effects of HAART.
The syndrome of HIV associated lipodystrophy is the most common long term side effect of antiretroviral therapy. This is a heterogenous syndrome associated with central adiposity, subcutaneous fat wasting (lipoatrophy) as well as dyslipidemia and type 2 diabetes. As well as being stigmatising and painful, this syndrome can affect drug adherence and the dyslipidaemia and diabetes can increase the risk of cardiovascular disease.
Although there is a definite association between the use of particular NRTIs and PIs and the development of HIV lipoatrophy, trhe underlying mechanisms remain unclear. NRTIs are known to cause mitochondrial toxicity while PIs inhibit cellular factors important in lipid and glucose metabolism such as peroxisome proliferator-activated receptor-gamma (PPARgamma). However this data is derived from studies on patients with advanced lipoatrophy who have often received both PIs and NRTIs for prolonged periods. Little work has been done on the early effects of drug exposure on changes in genes affecting lipid metabolism either as a potential marker for the development of lipoatrophy or as a target for therapy for HIV lipoatrophy.
Our study will look at the expression of genes associated with mitochondrial function, lipid metabolism and insulin metabolism in adipose tissue obtained from subjects in four multicenter trials looking at the effects of antiretrovirals in HIV infected and uninfected individuals both before and after therapy. These can be correlated with clinical and biochemical findings from these trials. We will also examine mitochondrial DNA (mtDNA) and gene expression in human hepatocyte and preadipocyte cell lines exposed to antiretrovirals in vitro in an attempt to find a reliable in vitro model which can be used to intensely study underlying pathogenic mechanisms. We hope that this will allow the development of both safer antiretroviral medications as well as potential therapies for this condition.

Project summary update (March 2010)

Mitochondrial toxicity (MtT) is a common side effect of highly active anti-retroviral therapy (HAART) for HIV infection.  We are examining the risk factors and mechanisms of MtT with HAART.
Lactic acidosis (LA) and symptomatic hyperlactatemia (SHL) are life-threatening complications of severe MtT.  Through a multicenter international collaboration with samples from the INITIO trial we are examining clinical and molecular risk factors for LA and SHL. In particular we are examining whether mitochondrial changes in peripheral blood mononuclear cells (PBMCs) predict MtT in other tissues. This is the largest randomized study to date of LA and SHL, and is important for predicting this severe toxicity which is being increasingly reported with HAART in resource-limited settings.
HIV lipodystrophy (HIVLD) occurs due to MtT in adipose tissue in individuals on HAART. Through collaboration within the European AIDS Treatment Network (NEAT) we are investigating how different components of HAART alter adipose tissue function, with tissue samples from 3 international trials. We are examining changes in mitochondrial DNA and gene expression, and the expression of genes involved in adipose tissue differentiation, inflammation and glucose and lipid metabolism. Through our NEAT collaborators we are correlating molecular data with clinical and histologic findings from subjects enrolled in these trials.  We are validating these findings through adipose cell cultures established from samples obtained from elective abdominal surgery in the Mater Misericordiae University Hospital.
This research will allow a better understanding of mitochondrial and adipose tissue physiology, as well as the mechanisms of toxicity of HAART.
Publications 2009:
Peripheral Blood Mononuclear Cell Mitochondrial DNA (mtDNA) and Mitochondrial RNA (mtRNA) Does Not Predict Lactic Acidosis or Symptomatic Hyperlactatemia for HIV-infected patients on Antiretroviral Therapy : a Sub-study of the INITIO Trial.
Abstract 730, 17th Conference on Retroviruses and Opportunistic Infections, San Francisco, February 15-19th 2010.
Eoin Feeney*1, C Chazallon2, N O’Brien1, V Meiffredy2, R Goodall3, D Cooper4, J-P Aboulker2, P Yeni5, P Mallon1,6, and on behalf of the INITIO Trial Intl Coordinating Committee
1Univ Coll Dublin, Ireland; 2INSERM SC10, Villejuif, France; 3Med Res Council Clin Trials Unit, London, UK; 4Natl Ctr in HIV Epi and Clin Res, Univ of New South Wales, Sydney, Australia; 5Univ Diderot, Paris 7, France; and 6Mater Misericordiae Univ Hosp, Dublin, Ireland

Project title and Supervisors

Title: The role of Endothelial to Mesenchymal Transition in Cardiac Fibrosis
Supervisors:
Dr. John Baugh, Principal Investigator & College Lecturer, UCD School of Medicine & Medical Science
Dr. Ken McDonald, Consultant Cardiologist, St. Vincent’s University Hospital

Project Summary

Heart Failure is an emerging epidemic due in part to the increasing age of our population and also to the prolonged survival of individuals with cardiovascular disease. Diastolic Heart Failure (DHF) accounts for over half of these cases and it is estimated that the total cost of DHF care in Ireland likely exceeds 100 million per annum or just less than 1% of the entire HSE budget. Here, in parallel with the lack of evidence-based treatments, mortality rates remain unacceptably high. DHF is characterised by reduced cardiac compliance due to increased stiffness of the cardiac tissue that occurs as a result of exaggerated fibrosis or scarring.
This project investigates the reversible ability of endothelial cells within the heart to develop a fibroblast-like phenotype via endothelial to mesenchymal transition (EMT) and contribute to the process of exaggerated fibrosis in DHF. This translational research builds upon an established collaboration between Dr. Ken McDonald and Dr. John Baugh in the UCD Conway Institute of Biomolecular and Biomedical Research.
It will test the hypothesis that EMT occurs as a consequence of vascular injury in hypertensive patients, predisposing them to exaggerated myocardial fibrosis, diastolic dysfunction and diastolic heart failure. By developing an in-vitro model using primary human coronary artery endothelial cells, potential promoters [pro-fibrotic agonists, stretch, pathological extra-cellular matrix, coronary sinus serum from hypertensive patients with differing degrees of diastolic dysfunction, primary cardiac fibroblasts from patients with macroscopic evidence of cardiac fibrosis] and inhibitors [BNP and statins] of EMT will be investigated.
Ultimately, by targeting this reversible pathway, we may be able to prevent adverse cardiovascular remodeling and thus make inroads into reducing the significant disease burden caused by diastolic heart failure.

Project summary update (March 2010)

The emergence of cardiac fibrosis as an important adverse predictor of risk in heart disease is not surprising given that it is largely responsible for cardiac stiffness, provides a substrate for rhythm disturbances and promotes tissue hypoxia and development of heart failure. Yet, this diagnosis remains difficult, treatment options remain limited and underlying mechanisms remain elusive.
I have completed an assessment of how levels of fibro-inflammatory markers vary throughout the natural history of hypertension and having obtained ethics committee approval to procure cardiac tissue biopsies from patients undergoing cardiac surgery, I am currently investigating how markers of collagen turnover, advanced as potential biomarkers, relate to tissue fibrosis. Our group and others have identified biochemical elevations in collagen 3 synthesis to be of particular significance with regard to heart failure prognosis and I have begun a detailed study of how collagen 3 relates to the major fibrillar cardiac collagen type 1 at a tissue level using histological and immunohistochemical techniques.
The natriuretic peptide system represents an antagonising pathway in fibrosis and I am also investigating whether brain natriuretic peptide (BNP) may represent a novel biomarker for cardiac fibrosis and whether alterations in BNP signalling may predispose to increased cardiac fibrosis in human tissue.
A new collaboration with the UCD Conway Nanoscale Function Group has enabled me to image tissue structures in high resolution and in a novel way, to correlate clinical, biochemical and immunohistochemical findings with tissue stiffness as measured using atomic force microscopy.

Project title and Supervisors

Title: Do the dynamics of quasispecies complexity and IP-10 concentration in chronic hepatitis C provide an opportunity to individualise treatment strategies?
Supervisors:
Dr Liam Fanning, Director, Molecular Virology Diagnostic & Research Laboratory, Senior Lecturer and Research Scientist, Department of Medicine, Cork University Hospital
Dr Orla Crosbie, Consultant Hepatologist, Department of Medicine, Cork University Hospital

Project Summary

Hepatitis C virus [HCV] is an RNA virus and affects up to 170 million people worldwide Pegylated interferon and ribavarin are the current standard of care and are likely to remain so for the foreseeable future. Not all patients respond to these with response rates varying from 40-50% in those infected with genotype 1 to 70-80% in those infected with genotype 2 and 3. Current strategies for identifying likely non responders are based on population data and give no indication of likely response in individual cases.
HCV is a virus which is prone to mutation. This results in the development of numerous populations of slightly different viruses known as quasispecies. Recent data suggests that quasispecies complexity is associated with likely response to treatment. Little is known of variations in quasispecies complexity [QSC] over time and we hope to measure these. This may identify windows of improved sensitivity to treatment.
A growing body of evidence suggests that levels of interferon gamma inducible protein [IP-10] could provide a large dividend in classifying likely responders and non responders. Current research has not identified whether IP-10 varies with time or whether it could be used as a surrogate marker for QSC. We plan to measure changes in IP-10 and compare this with QSC over time.
Finally changes in viral load (the amount of virus detectable in a patient’s blood) in the early treatment phase have also shown potential in predicting those who will respond to therapy. We propose to prospectively recruit patients who are starting treatment and to measure QSC, IP-10 over four months before commencing treatment and viral kinetics in the first 4 weeks following treatment. We plan to use them to create a mathematical model to identify responders and non responders at an earlier stage than has previously been possible in order to minimise potentially serious side effects and optimise cost effectiveness in treatment programs. 

Project summary update (March 2010)

Hepatitis C virus [HCV] is an RNA virus and affects up to 170 million people worldwide. Not all patients respond to the current therapies with response rates varying from 40-50% in those infected with genotype 1 to 70-80% in those infected with genotype 2 and 3. Current strategies for identifying likely non responders are based on population data and give no indication of likely response in individual cases.
HCV is prone to mutation. This results in the development of numerous populations of slightly different viruses known as quasispecies. Recent data suggests that quasispecies complexity is associated with likely response to treatment. Little is known of variations in quasispecies complexity [QSC] over time and we hope to measure these. This may identify windows of improved sensitivity to treatment.
We are also investigating whether a protein called IP-10 which has been shown to predict likely response to treatment can be used as a marker indicating QSC.
We plan to use this data to create a mathematical model to identify responders and non responders at an earlier stage than has previously been possible in order to minimise potentially serious side effects and optimise cost effectiveness in treatment programs. The data generated will also yield insights into short term temporal variation in quasispecies complexity in an immunocompetent host.
Recruitment and data collection has commenced. Preliminary data representing the dominant genetic sequences are being generated and analysed. Preliminary results are encouraging with genetic variance between viral isolates evident.