Research Pillar: Biomedical

The genetic basis of immune escape and its clinical relevance in lymphoid cancers

Lymphomas are a group of cancers derived from white blood cells. This project focuses on how some lymphomas carry mutations that render the immune system unable to recognize and destroy them.

We have recently described a gene named CIITA that is mutated in certain lymphomas. CIITA plays a major role in regulating the production of proteins on the surface of cells that allow cells of the immune system to recognize them. Mutations in CIITA can lead to a reduction in these proteins so the cancerous cells are not controlled by the immune system.

We will use DNA sequencing to explore genetic changes within CIITA in a large number of tumour samples from lymphoma patients. We will compare these genetic findings with the clinical data from the patients and look for survival trends. We will also use cell lines and mouse models to investigate the impact of CIITA mutations on tumour biology.

Ultimately, we hope to unveil novel mechanisms that will help build the foundation for the development of new diagnostic tests and/or therapeutic strategies.

Multimodal characterization of airway remodeling with label-free nonlinear optical imaging

Michael Smith Foundation for Health Research/AllerGen Post-Doctoral Fellowship Award

The number of Canadians who will die from asthma is estimated at 200 per year and over 3 million suffer daily with the disease. A better understanding of the disease could give rise to more effective treatments.

Within the normal lung, collagen and elastin fibers provide the structural components of the airways. In asthmatic airways, the collagen and elastin fibers are disorganized and more collagen accumulates within the airway, making the airway more narrow and harder to breath through.

We will use nonlinear optical microscopy (NLOM) to understand the changes in the three-dimentional structure of the airways' elastin and collagen fibres that occur within asthma. Additionally, we will observe changes that occur inside these fibers by transmission electron microscopy (TEM).

Thus, if collagen were a "rope", we would be taking images of this rope with NLOM, and then images of each thread that composes the rope with TEM. To study the spatial distribution of the "ropes" and the ratio of collagen to elastin in asthmatic airway tissue, we will use textural analysis.

This study will give rise to results that could aid researchers in developing better asthma therapies, this improving the quality of life for millions of asthma patients.

Role of allergen-experienced Group 2 Innate Lymphoid Cells in allergic lung inflammation

Allergic asthma is an incurable respiratory disease that affects more than 300 million people worldwide. Asthmatic patients first become sensitized by inhaled substances that trigger an allergic reaction (allergens). Repeated exposures to the same allergens cause allergic inflammation in the lung because allergen-specific cells of the immune system called lymphocytes acquire memory: they react when they re-encounter the same allergen.

Another set of cells, the recently identified Group 2 Innate Lymphoid Cells (ILC2s), can also trigger allergic lung inflammation. Unlike regular lymphocytes, ILC2s do not recognize specific allergens. However, experienced ILC2s can vigorously react to new allergens, causing a stronger allergic lung inflammation than they did on their first exposure to unrelated allergens. Therefore, ILC2s can acquire memory that is not specific to particular antigens.

We believe that upon allergen encounter, different subsets of memory ILC2s are generated. The goal of this research is to characterize memory ILC2s in the chronic phase of allergic lung inflammation.

The results obtained in this research would explain why sometimes the causative allergen is not identified and why asthma vaccines are not always effective. This research may lead to the development of novel therapies for chronic asthma.

Are there indicators of Alzheimer’s disease in the eye?: New computational imaging and analysis algorithms

Michael Smith Foundation for Health Research/The Pacific Alzheimer Research Foundation Post-Doctoral Fellowship Award

 

Alzheimer’s disease (AD) is progressive degeneration of the brain that results in loss of memory and cognitive abilities. The prevalence of the disease presents a daunting challenge — as of 2015, 46.8 million people in the world live with AD, with the number expected to double by 2035. In Canada, 14.9 percent of those 65 and older have the disease. The global health-care cost for dementia has exceeded 1 percent of the global gross domestic product (GDP).

 

AD significantly affects the quality of life for the patients and caregivers, and this makes early detection critical. However, the brain imaging required for the diagnosis is costly, and AD is often discovered only after it has progressed considerably.

 

The overarching theme of this project is finding the connection between the eye and AD, by investigating it for potential biomarkers of the disease. The eye is an extension of the brain, with the optic nerve forming a direct physical connection between the retina and the brain’s visual cortex. Recent advances in ophthalmic imaging techniques, such as optical coherence tomography (OCT), provides high-resolution 3D visualization of the inner structures of the eye, including the retina, nerve fibres, and blood vessels, in a noninvasive manner. OCT and other imaging techniques give us a comprehensive picture of the eye’s health and function.

 

We will develop image processing and analysis tools to examine chemical biomarkers, structural degradation, and functional loss in the eye that may be associated with AD. The project will potentially lead to discovery of novel AD biomarkers in the eye, and a cost-effective and accessible diagnostic tool for early detection of AD.

 

End of Award Update

Source: CLEAR Foundation

 

What did we learn?

We know that amyloid beta, a hallmark of Alzheimer’s disease, is present not only in the brain but also in the retina of the patients, and its deposition can vary by location and comorbidity such as cerebral amyloid atrophy.

 

Why is this knowledge important?

Retina can be readily imaged in high detail, and it contains rich information about the person’s neuronal health. Retinal imaging has potential as an early and accessible screening tool for neurodegenerative diseases. Studying the mechanisms of Alzheimer’s disease pathology in the retina also gives us insight into those in the brain.

 

What are the next steps?

Professor Joanne A. Matsubara’s group at the University of British Columbia and I are continuing to collaborate to study retinal biomarkers of Alzheimer’s disease. We are currently looking into how amyloid beta affects glial cells and blood vessels in the retina.

 

Publications

Optimizing anti-cancer peptides for use as novel cancer therapies

Anticancer peptides (ACPs) are small peptides (short chains of amino acids) that kill cancer cells by puncturing them or by triggering programmed cell suicide. In the lab, certain ACPs kill slow-growing and multidrug-resistant cancer cells, enhance action of anticancer drugs, and trigger the immune system to attack tumours. However, many also kill normal cells.

The goal of this work is to understand how the amino acid sequence contributes to selective cancer cell killing. This project will:

  1. Screen large numbers of ACPs to identify amino acids that maximize cancer cell selectivity
  2. Explore mechanisms of anti-tumour activity of our lead peptide Mastoparan

Peptide arrays allow hundreds of ACPs to be tested inexpensively. We will create many variant forms of the highly active ACP Mastoparan by substituting every amino acid in its chain with every other naturally occurring amino acid. A peptide array will be used to test which amino acids are vital for cancer cell killing and which ones harm normal cells.

Computer analysis will generate new ACP sequences with better predicted selectivity for cancer cells. The ACPs predicted to be most selective for cancer cells will be synthesized and screened, and the most promising ones will be tested for toxicity against human cancer cells and normal cells.

In addition, the immune system component of the anti-tumour activity of Mastoparan L will be explored further in vivo.

Ultimately, this project could give rise to peptides that selectively target cancer cells and induce an anti-tumour immune response.

Development of novel indolmycin derivatives for the treatment of MRSA

Since the discovery of antibiotics over 80 years ago, bacterial infections have been relatively straightforward to treat. However, the improper use of antibiotics has caused bacteria to develop antibiotic resistance, posing a serious global threat to preventing and treating common bacterial infections.

This project seeks to combat multi-drug resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) by improving on an antimicrobial compound that naturally occurs in the environment. Indolmycin is naturally produced by Streptomyces griseus and is active against multi-drug resistant MRSA strains.

We aim to develop forms of indolmycin that are more potent against MRSA by feeding Streptomyces griseus with variant amino acids. In addition, we will perform structure-based studies to elucidate the molecular mechanism of anti-MRSA activity in indolmycin. This will allow for rational design of more effective forms of indolmycin.

Ultimately, this research could give rise to novel antibiotics to treat infections such as MRSA that are developing resistance to our current toolkit.

Altered glutamate dynamics in a mouse model of Alzheimer’s disease: Novel early biomarkers with therapeutic potential

Michael Smith Foundation for Health Research/The Pacific Alzheimer Research Foundation Post-Doctoral Fellowship Award

 

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. With no cure available, it imposes a major burden on society. In AD, a protein called amyloid beta accumulates into plaques in the brain. This event is an early and predictive marker for AD and can be detected up to 20 years before clinical symptoms arise.

 

We are exploring the dysfunction and hyperactivity of various cell types in the presence of amyloid plaques. We will investigate the possible role of altered glutamate dynamics in mouse models of AD. Fluorescent labelling will shed light on changes in glutamate signalling in awake and behaving AD mice. We will also test whether Ceftriaxone can restore normal glutamate dynamics in these mice.

 

Ultimately, our work with AD mouse models and novel glutamate imaging could shed light on possible drug targets and enable early intervention for people with Alzheimer’s disease.

End of Award Update

Source: CLEAR Foundation

 

What did we learn?

During our study it became clear that amyloid deposits have a drastic impact on glutamate signaling. This early cellular signaling deficit was only visible using in vivo 2-Photon imaging and a region-specific analysis of real time glutamate transients in direct proximity to amyloid plaques. We found areas of chronically high glutamate levels directly surrounding amyloid plaques and adjacent areas in which glutamate signaling was impaired. In larger scale wide-field real-time imaging experiments this effect was lost, indicating the importance of region-specific effects on cellular function in the early stages of the disease. Moreover, we used luminescent conjucated oligothiophens (provided by Dr. Peter Nilson, Linköping University) to visualize prefibrillary forms of amyloid. We found a direct overlap of these prefibrillary forms of amyloid surrounding the dense core plaque and a decrease in astrocytic GLT-1 transporter. We hypothesized that this decrease in GLT-1 is responsible for the observed glutamate dysfunction that was found in our experiments using stimulation experiments in awake and anesthetized animals. We thus used Ceftriaxone to restore GLT-1 expression in our mouse model of AD. We provided longitudinal imaging data of glutamate transients before and after Ceftriaxone treatment and could show that this partially restores glutamate signaling.

 

Why is this knowledge important?

As prefibrillary species are present in the brain before dense core plaques are formed it is important to further our understanding of their impact on cellular function. Moreover, it is essential to develop strategies that aim at the earlies stage that amyloids have on cellular function as these can be treated. Once neurons and other cells undergo cell death, strategies of recovery are much harder to implement. Our results provided a new therapeutical target that not only treats the disease on a symptomatic level but in an amyloid centric way that prevents/delays cellular dysfunction at an early stage in the disease.

 

What are the next steps?

We are currently expanding our research to investigate the impact of prefibrillary amyloid species on vessel function to search for more potential therapeutic targets.

 

Publications

The role and regulation of pannexin ion channels during cytotoxic edema

A primary and often fatal consequence of stroke, traumatic brain injury, and other brain insults is edema: an increase in brain tissue water content. Cytotoxic edema is a component of this process and occurs when excess ions and water enter across the neuronal plasma membrane -the semi-permeable barrier separating the intra- and extracellular space. This increase in cell volume causes membrane swelling and ultimately results in cell death.    

Presently, the cascade of events by which neuronal swelling triggers cell death remains obscure. Preliminary evidence from Dr. Brian MacVicar's lab (the host) indicates that swelling triggers cell death by activating pannexins- a class of large transmembrane ion channels. Following activation, pannexins form large pores in the membrane and allow ions and small molecules to diffuse between the intra- and extracellular compartments. Consequently, pannexins can initiate cell death by collapsing the transmembrane electrochemical gradient and/or promoting the loss of essential cellular components. The precise mechanism by which swelling triggers the opening of pannexins is unknown. Interestingly, these ion channels can be mechanically activated by membrane stretch. Moreover, membrane stretch also leads to the production of reactive oxygen species (ROS)-a group of harmful chemical agents that can directly activate pannexins.    

For the present proposal, we will test the hypothesis that pannexin activation is a crucial step underlying cell death following cytotoxic edema. Furthermore, we hypothesize that pannexins are activated by neuronal swelling through direct mechanical stimulation and/or the production of ROS.   

These hypotheses will be tested in acutely prepared rat brain slices using advanced microscopy/imaging and electrophysiology techniques. As there are few effective treatments for edema, this research could reveal new avenues for therapeutic intervention following a variety of brain insults. Considering the implications of this project for basic biomedical and clinical research, it will be essential to diffuse and disseminate our knowledge to a variety of communities. This will be done largely through symposiums/presentations at the Society for Neuroscience as well as publication in peer-reviewed scientific journals.

A primary and often fatal consequence of brain insults such as stroke and traumatic injury is edema: an increase in brain tissue water content. Cytotoxic edema is a component of this process, which occurs at the level of individual brain cells, or neurons. The cells swell up as excess ions and water enter, causing them to die. This project will build on earlier work carried out under the project supervisor, which suggests that cytotoxic edema is caused by the action of pannexins.

Pannexins are activated through unknown mechanisms when the cell membrane is caused to stretch, either chemically through the production of reactive oxygen species or mechanically. Following activation, pannexins form large pores in the cell membrane that allow ions and small molecules to pass through. They are believed to cause cell death by collapsing the transmembrane electrochemical gradient and/or by promoting the loss of essential cellular components.

We will study tissue samples using advanced imaging and electrophysiology techniques to test the hypotheses that:

  1. Pannexin activation is a crucial step underlying neuronal cell death in the brain following cytotoxic edema
  2. Pannexins are activated by neuronal swelling through direct mechanical stimulation and/or the production of reactive oxygen species

New lines of research for therapies for damage to the brain are greatly needed and some could arise from this work.

The mechanism and significance of the synaptogenic activity of amyloid precursor protein

Michael Smith Foundation for Health Research/The Pacific Alzheimer Research Foundation Post-Doctoral Fellowship Award

Amyloid Precursor Protein (APP) is a cell surface protein that has been mostly studied in the context of Alzheimer’s disease. Much about its normal function remains unknown. APP can cause connections to form between brain cells by an unknown mechanism. We believe this happens through an interaction with synaptic organizing proteins (organizers).

This project will investigate the possibility that APP forms synapses by interacting with major organizers in the brain, namely neurexins and receptor protein tyrosine phosphatases (RPTPs).

To test this, we will use a combination of cell cultures and mouse models. We will test whether APP binds to neurexins and RPTPs and whether binding to these organizers is required for the connection-forming activity of APP. We will also compare brain cell connections in normal mice to those in mice that express an altered form of APP that cannot bind to organizers.

This study may shed light on the function of APP through detailing how it can help form connections between brain cells. If defects in connections between brain cells contribute to neurodegeneration in Alzheimer’s disease, these results could shed light on the mechanisms behind that as well.

Upstream regulators of the Hippo signaling pathway in liver development and cancer

Liver cancer is the fifth most common cancer and the leading cause of cancer deaths worldwide, primarily because of late diagnosis and scanty therapeutic options. Animal studies have demonstrated that the Hippo intracellular signalling pathway is critical in regulating liver size and liver cell fate and is a potential tumour suppressor in the liver.

Loss of cell-cell adhesion is associated with the progression and poor prognosis of liver cancer. In this project, we will explore whether loss of cell-cell adhesion regulates Hippo signalling in liver cells. We are particularly interested in how loss of cadherin molecules can regulate the Hippo signalling pathway and subsequently contribute to liver development and cancer. The findings generated from this proposal will increase our understanding of the underlying molecular mechanisms involved in liver development and tumourigenesis.