INNATE PHAGOCYTOSIS & NEURODEGENERATION
See full list of projectsLeukocyte surface and functional biomarkers for prognosis of age-related macular degeneration
Supervisors: Dr. Ben J. Gu, Prof. Robyn Guymer, Prof. Erica Fletcher, Prof. James S. Wiley
Project Site: Florey Institute, Kenneth-Myer Building
Contact: E: ben.gu@florey.edu.au Ph: 03 9035 6317
Project description: Age-related macular degeneration (AMD) is a multifactorial disease and is a leading cause of irreversible vision loss in Australia. AMD at its early stage is characterized by accumulation of debris (lipid rich drusen) in retina, which is believed due to reduced clearance capacity. While AMD can be easily diagnosed with high resolution retina imaging, early prognosis biomarkers are needed to identify people with high risk for preventive treatment. Our previous study has shown that genetic variants leading to defective phagocytosis are risk factors for AMD. In this study, we will measure the phagocytosis ability of monocytes and monocyte subsets from AMD patients as well as age-matched healthy controls, using a real-time tri-colour flow cytometry method developed by our group. Meanwhile, the monocyte surface expression of scavenger receptors, e.g. P2X7, TREM-2, SCARA1 and CD36, will be examined. Cell surface biomarkers will be examined on peripheral blood leukocytes from patients and healthy controls. The sensitivity and specificity of promising parameters will be analysed and validated in a follow-up study. This study will not only identify a useful pattern for early prognosis of AMD, but also provide insights on the pathogenesis and development of this disease.
Identifcation of serum glycoproteins inhibiting innate immunite - also offered as MBiomedSc
Supervisors: Dr Ben Gu, Professor James Wiley
Project Site: Ion channel & Human Disease, Florey Neuroscience Institutes, Level 1, Kenneth-Myer Building, Parkville
Contact: Ben Gu T: 03 9035 6317 E: ben.gu@florey.edu.au James Wiley E: james.wiley@florey.edu.au
Project description: Innate immunity is the first line defense of host against invading pathogens. Phagocytosis of non-opsonized particles (bacteria or viruses not coated by immunoglobulin, complement, etc) is an important part of innate immunity. Our recent findings show that innate phagocytosis is completely abolished by a group of serum glycoproteins, i.e. serum inhibits innate immunity. These proteins play an important role in regulation of innate immunity and the most potent protein remains unknown. Identifying this protein will lead to a new therapies to boost resistance against infectious diseases.
Techniques involved are chromatography, cell culture, flow cytometry, electrophoresis, western blotting and mass spectrometry.
How does the brain remove the excess number of neurons during development and ageing - also offered as MBiomedSc
Supervisors: Dr Ben Gu, Professor James Wiley
Project Site: Ion channel & Human Disease, Florey Neuroscience Institutes, Level 1, Kenneth-Myer Building, Parkville
Contact: Ben Gu T: 03 9035 6317 E: ben.gu@florey.edu.au James Wiley E: james.wiley@florey.edu.au
Project description: Many more neurons are produced during development than are present in the adult brain. Also many neurons are lost during aging, however the process of innate phagocytosis, which removes unwanted and superfluous neurons is poorly defined. The unwanted neurones enter apoptosis but subsequent clearance of these dying cells is important for our body to avoid autoimmunity or inflammation in the brain. Apoptotic cells express unique markers which enable them to be recognized and engulfed by phagocytes. The knowledge of these unique markers is limited at present to certain cell membrane lipids, e.g. phosphatidylserine. Recent novel finding from our laboratory suggests that a unique protein epitope is expressed early in apoptosis and this is recognized by P2X7 receptors on phagocytes. This project will examine how apoptotic cells are recognized and cleared by phagocytes both in health and in disease. This result will have relevance to many neurological diseases as well as early neurodevelopment.
Techniques involved are cell culture, immunoprecipitation, western blotting, flow cytometry, peptide screen, molecular biology and mass spectrometry.
Identify the transcriptional regulatory factors of the P2X7 receptor - also offered as MBiomedSc
Supervisors: Dr Ben Gu, Professor James Wiley
Project Site: Ion channel & Human Disease, Florey Neuroscience Institutes, Level 1, Kenneth-Myer Building, Parkville
Contact: Ben Gu T: 03 9035 6317 E: ben.gu@florey.edu.au, James Wiley E: james.wiley@florey.edu.au
Project description: P2X7 is an ATP-gated purinergic receptor and plays a broad role in infection, inflammation, autoimmunity, neurodegeneration and oncogenesis. Several isoforms of P2X7 have been identified to be associated with cancer or other diseases. High expression of non-functional P2X7 has also been found in a broad range of tumour tissues. However, the transcriptional regulatory factors leading to these isoforms and non-functional P2X7 are unclear. This project will identify the transcriptional factors in the P2X7 promoter region, and how these transcriptional factors regulate production of P2X7 isoforms and non-functional P2X7. The results will provide insights on how cancer cells avoid removal by innate immunity.
Techniques involved include molecular biology, including primer extension, transfection, fluorescent super electrophoresis mobility shift assay and chromotin-immunoprecipitation, as well as cell culture, flow cytometry.
Do circulating microvesicles from patients with multiple sclerosis (MS) disrupt the blood brain barrier (BBB)? - also offered as MBiomedSc
Supervisors: Dr. Ben J. Gu, Prof. James S. Wiley
Project Site: Florey Institute, Kenneth-Myer Building
Contact: Dr Brn Gu E: ben.gu@florey.edu.au Ph: 03 9035 6317
Project description: Breakdown of the blood brain barrier (BBB) precedes clinical symptoms of new lesions of MS and it is possible that high numbers of microvesicles in multiple sclerosis (MS) plasma are related to episodes of disruption of the BBB. The integrity of BBB will be studied using an in vitro model examining lymphocyte transmigration across confluent monolayers of cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs) are grown to confluent monolayers in tissue culture plates and peripheral blood lymphocytes added to each well and incubated for 2-4 h. The HUVEC layer is washed 5 times with saline media, then fixed and examined by phase-contrast microscopy. Cells beneath the monolayer appear phase dark while adherent cells above appear phase light. The number of adherent and migrated cells are counted to give an index of efficiency of migration. To assess if microvesicles impair the integrity of the endothelial monolayer, the migration assay will be performed both in the absence and presence of plasma containing known concentrations of platelet derived microvesicles. Meanwhile, the lysosomal β-hesosaminidase activity will be measured in platelet poor plasma from 20 MS patients and 20 controls using a standard colourimetric assay. The microvesicle counts, β-hesosaminidase activity and the impact on lymphocytes transendothelial migration will be analysed in correlation. Results could provide evidence for a mechanism by which peripheral blood leukocytes infiltrate to brain in MS.
Techniques involved include cell culture, ultra-centrifugation, flow cytometry, fluorescent microscopy and biochemistry.