McMaster University

McMaster T-cell Epitope
Centre (MTEC)

Scope of Search

Facilities

  • Project 1
  • Project 2

Project 1

Larché laboratory

Comprises over 1600 square feet of recently renovated (2006) wet laboratory floor space including two tissue culture suites. Three Class II laminar flow cabinets and four CO2 incubators are available for tissue culture purposes. The laboratory also includes two -80oC freezers, three -20oC freezers & two refrigerator/freezers, two refrigerated Allegra Beckman Coulter bench top centrifuges and a fume cupboard. Specialist items of equipment include a Luminex 200 platform, a PerkinElmer LKB Microbeta Counter, a StemCell Technologies RoboSep magnetic automated cell separation robot, an ImmunoBioSys Bioreader 5000i ELIspot/plaque assay plate reader and a PerkinElmer Victor 3 multipurpose plate reader. The RoboSep robot can be used to isolate specific populations of cells for example components of peripheral blood mononuclear cells preparations with a high degree of purity and reproducibility. The Larché laboratory is a well established cellular immunology laboratory with extensive experience of culturing human blood cells and primary bronchial epithelial cells. The laboratory has an adjacent office (70 sq ft) and the laboratory/offce complex has 5 fully networked, secure desktop computers and a wireless network, 3 printer/scanners and a direct fax machine.


Larché Office

Comprises 120 sq ft at McMaster site and 200 sq ft at the FIRH site. Both offices are within easy walk to the laboratories and clinical facilities. Both offices house fully networked and secure desktop computers with printers and scanners. Contribution of the scientific environment to this project: McMaster University has sufficient infrastructure to support the proposed project including a dedicated Human Immune Testing Suite (HITS) and a further Flow Cytometry Core facility juxtaposed. The Human Immune Testing Suite (HITS) is a GLP compliant facility designed to be a 1-stop shop for clinical trials of human immunology. McMaster University has strength and depth in immunology, allergy and asthma research including the following faculty: Ali Ashkar – innate immunity, John Bienenstock- mucosal immunology/allergy, Dawn Bowdish – innate immunity, Jonathan Bramson – viral and cancer immunology, Steve Collins – mucosal immunity, Mike Cyr - Clinical Immunology & Allergy, Judah Denburg – allergy, eosinophils, Jack Gauldie – immunology, asthma, fibrosis, Gail Gauvreau – allergy, asthma, Derek Haaland - Clinical Immunology & Rheumatology, Mark Inman – murine models of asthma, Manel Jordana – allergy, murine models, Paul Keith - Clinical Immnology & Allergy, Martin Kolb, pulmonary fibrosis, Mark Larché – immunology, allergy, asthma, autoimmunity, transplantation, Maggie Larché - Inflammation & rheumatology, Mary Messieh - Clinical Immunology & Allergy, Karen Mossman – viral immunology, Paul O’Byrne – asthma, Carl Richards – arthritis & cancer immunology, Ken Rosenthal – viral immunity, Martin Stampfli – viral immunity, COPD, Susan Waserman - peanut allergy/general clinical allergy/immunology, Zhou Xing – tuberculosis immunity These individuals collaborate extensively. For example, the PI has ongoing collaborations with Ashkar (intrinsic adjuvanticity of allergens), Bienenstock (multiple sclerosis), Bowdish ((intrinsic adjuvanticity of allergens), Denburg (biology of TSLP in asthma and allergy), Gauvreau (the current application and immunological mechanisms of anti-OX40L), Haaland (rheumatoid arthritis, scleroderma, SLE), Inman (the current applcaition and several studies in murine models of asthma), Jordana (peanut allergy), Kolb (Scleroderma), (Maggie) Larché (Scleroderma, rheumatoid arthritis), O'Byrne (current application, anti- OX40L), Waserman (peanut allergy).


Flow Cytometry Facilities at McMaster University/St. Joseph's Healthcare

  1. The McMaster Flow Cytometry facility which serves as a core resource to the entire McMaster research community. The facility offers 2 analytical instruments: a 9-color, 2-laser FACS Canto and a 15-color, 4-laser LSRII. The facility has also recently purchased a 4-laser 16-color BD Aria III.
  2. The Human Immune Testing Suite (HITs) at McMaster University is a self-contained, 600 ft2, GLPcompliant research laboratory restricted to the immunological analysis of human clinical samples. At the core of this facility is a dedicated 4-laser LSRII flow cytometer equipped with a high-throughput sample loader and a Biomek NX robotic liquid handler, which performs all of the staining operations. Through the implementation of standardized methodology and robotic technology, this facility ensures maximum reproducibility by minimizing potential user error. The implementation of the robotic device also permits medium-level throughput and, importantly, hands-free operation. HITs is also equipped with a CTL ELISPOT reader, a BD Medimachine for isolation of lymphocytes from whole tissues and all standard laboratory instrumentation, including: level 2 biosafety cabinets, incubators, microscopes, centrifuges, freezers, and cell separation equipment. HITs currently has a liquid nitrogen tank with capacity for 22 000 vials and is aquiring two additional tanks to increase our total capacity to >120 000 vials. All tanks have external reservoirs that maintain the nitrogen levels automatically. The temperatures on the tanks are monitored by visual inspection twice daily and 24 hour monitoring is provided by McMaster security. HITs is a member of the Cancer Immunotherapy Consortium harmonization group and participates regularly in their harmonization panels to maintain best practices. In addition to flow cytometry-based applications, HITs employs phage display technology for identification of antibody targets/epitopes. HITs has participated in 3 major clinical studies that involved reception, cataloging and storage of >1500 clinical specimens. The first trial took place from 2003 – 2007 and involved the processing of peripheral blood specimens from 127 patients infected with West Nile virus. Specimens were taken on a monthly basis over a 12-month period following the onset of symptoms, amounting to a total of 1524 specimens. On average, the PBMC obtained from each specimen yielded 5 vials of PBMC for cryopreservation amounting to a total of approximately 7600 cryovials banked in our facility. We continue to work with those cryopreserved specimens and find excellent viability and functionality in our assays. The second epidemiological trial started in 2009 and will continue until 2011. This latter study involves sampling of nursing home patients’ blood prior to influenza vaccination to identify markers of vaccine failure. In the 2009 season, 138 blood specimens were processed over a period of 6 weeks and stored as PBMC and plasma. On average, each specimen yielded 4 vials of PBMC for cryopreservation and 3 vials of plasma for a total of 552 PBMC cryovials and 414 plasma vials. Sample vials have been thawed and demonstrated excellent viability and functionality. An additional 400 patients will be recruited to this study in 2010 and 2011. The final study involved an investigation of changes in immunity against HER-2 over the course of Herceptin treatment. In this study, 50 breast cancer patients were recruited prior to initiation of Herceptin treatment. Peripheral blood samples were taken at baseline, 6 months and 12 months after the onset of treatment. PBMCs and plasma were prepared from all of the blood specimens (typically 5 PBMC vials and 3 plasma vials) yielding a total of approximately 750 PBMC cryovials and 450 plasma vials.

3. At St. Joseph's Healthcare, the PI is also a part owner of a 2-laser FACS-Canto with Apple Desktop computer running WinMDI and FloJo (Treestar).

Project 2:

The clinical study will be performed in two hospitals with dedicated clinical research areas that have been performing similar clinical research for many years. The clinical space has waiting areas for subjects, and the wet lab space has all of the equipment needed for specimen processing, storage and preparation for shipping to the collaborating site in Seattle.


Gauvreau laboratory

Located at McMaster University, it comprises over 1500 square feet of clincal laboratory floor space in addition to 1000 square feet of wet laboratory floor space. The clinical laboratory complex has a dedicated suite for subject examination and invasive procedures including blood draw and bone marrow aspiration. This suite is equipped with emergency medications and resuscitation equipment, and is complete with a research washroom and shower room. Clinical supplies are stocked in a room next to the procedure suite. Dedicated staff will maintain the procedure suite and keep it stocked with clinical supplies and medications. There are 2 separate ventilated rooms for inhalation challenges (allergen and methacholine) and sputum induction, and an adjacent full pulmonary function laboratory with spirometers and a plethysmograph, all meeting the requirements of the American Thoracic Society. The laboratory is staffed by technicians with over 30 years of experience with pulmonary function testing. The clinical area has one -80°C freezer, 2 refrigerator/freezer units, all with continuous temperature logs and central temperature failure alarms. Critical equipment is connected to an emergency power generator in the event of electricity failure. An exercise laboratory with treadmill, cycle ergometer and metabolic cart is also contained within the clinical laboratory complex. The wet laboratory complex across the hall from the clinical laboratory complex has a dedicated tissue culture room with a Class II laminar flow cabinet, CO2 incubator and Olympus inverted microscope with camera, an adjacent room for processing of biological specimens. An adjacent room is customized for molecular biolology experiments, and another houses bench top centrifuges and ultracold freezers. Microscopy and imaging is conducted in a separate adjacent room. The wet laboratory has two -80°C freezers, 3 refrigerator/freezers, two refrigerated Herus bench top centrifuges, two refrigeratedeppendorf centrifuges, a Shandon III cytocentrifuge, 3 light microscopes with cell counting software, a Miltenyi MACs cell sorting magnet, Perkin Elmer ELISA plate reader, iCycler Thermal Cycler and software,water purification system, autoclaves, FluoroChem 5500 gel documentation system, a fume cupboard and 2 fumehoods. The MACs sytstem can be used to isolate specific populations of cells for example components of peripheral blood mononuclear cells preparations with a high degree of purity and reproducibility. All equipment is maintained and calibrated as per manufacturer instructions. A technician is assigned for overseeing regular maintenance of all equipment in the clinical and wet laboratory space, temperature logs, cleaning and repair of this equipment, as is required for industry-sponsored clinical research. The Gauvreau laboratory is a well established cellular immunology laboratory with extensive experience of culturing humanblood cells and bone marrow cells and quantifying cell populations by flow cytometry and microscopy from human airway samples of induced sputum, BAL and airway mucosal biopsies. The laboratory has an adjacent office (200 sq ft) for students and technicians with 6 fully networked, secure desktop computers and a wireless network and 3 printer/scanners. This clinical/wet laboratory unit regularly conducts investigatorinitiated and industry-sponsored clinical studies using the allergen challenge model, and has performed over 150 allergen challenges and over 300 methacholine challenges in the last year. Bone marrow aspirates are also conducted within clinical trials, with approximately 40 samples collected and handled over the past year.


Gauvreau office

Located at McMaster University beside the clinical laboratory and the wet laboratory, it occupies 140 square feet of floor space and houses a fully networked and secure computer with printer and scanner. Administrative space immediately adjacent to the office contains secure document storage, desk space for clinical study monitors/auditors, a meeting area, and secure desktop computers connected to a wireless network and printer/scanner/fax machine. With the exception of bronchoscopy, the clinical study will be carried out in dedicated research units at McMaster University which are suitable for the administration of all study specific procedures required by this protocol. The bronchoscopy will be carried out in the dedicated endoscopy suite at St Joseph’s Healthcare Hamilton, where Dr Neighbour can conveniently monitor subjects before and after bronchoscopy procedures. This unit provides clinical endoscopy services and performed 15,000 endoscopy procedures in the last year, 960 of which were bronchoscopy procedures. Both of the units have separate fully equipped office areas with desk space for each of the key personnel listed in the clinical core proposal. Immediately adjacent to each clinical research area is a wet lab area which contains all of the equipment for initial sample processing and storage. There is a dedicated biohazard transportation service to allow safe transfer of samples between the two sites, and to allow delivery of the samples to the flow cytometry facility based at McMaster University. The sputum samples for Project 2 will be processed at the dedicated sputum laboratory at St Joseph's Healthcare Hamilton. This facility acts as a central lab providing slide staining and reading for national and international sites with numbers of sites ranging from 5-45. This lab also processes all of the sputum samples from our internal studies, and is subject to accreditation by the Department of Laboratory Medicine. The laboratory is staffed by Certified Medical Laboratory Technologists who have been employed in this laboratory for over 15 years. This laboratory also processes 1500 clinical sputum samples per year. Both of the research units have separate fully equipped office areas with desk space and computer access for each of the key personnel listed in the clinical core proposal. All of the laboratory areas that will be utilized in this proposal contained approved biohazard safety cabinets. McMaster University has a dedicated biohazard safety committee ensures that all staff and student have up-todate training in the legally mandated training. In addition the committee reviews all proposals to ensure compliance with biohazard safety standards.


Flow Cytometry Facilities at McMaster University

1. The McMaster Flow Cytometry facility which serves as a core resource to the entire McMaster research community. The facility offers 2 analytical instruments: a 9-color, 2-laser FACS Canto and a 15-color, 4-laser LSRII. The facility has also recently purchased a 4-laser 16-color BD Aria III.

2. The Human Immune Testing Suite (HITs) at McMaster University is a self-contained, 600 ft2, GLP compliant research laboratory restricted to the immunological analysis of human clinical samples. At the core of this facility is a dedicated 4-laser LSRII flow cytometer equipped with a high-throughput sample loader and a Biomek NX robotic liquid handler, which performs all of the staining operations. Through the implementation of standardized methodology and robotic technology, this facility ensures maximum reproducibility by minimizing potential user error. The implementation of the robotic device also permits medium-level throughput and, importantly, hands-free operation. HITs is also equipped with a CTL ELISPOT reader, a BD Medimachine for isolation of lymphocytes from whole tissues and all standard laboratory instrumentation, including: level 2 biosafety cabinets, incubators, microscopes, centrifuges, freezers, and cell separation equipment. HITs currently has a liquid nitrogen tank with capacity for 22 000 vials and is aquiring two additional tanks to increase our total capacity to >120 000 vials. All tanks have external reservoirs that maintain the nitrogen levels automatically. The temperatures on the tanks are monitored by visual inspection twice daily and 24 hour monitoring is provided by McMaster security. HITs is a member of the Cancer Immunotherapy Consortium harmonization group and participates regularly in their harmonization panels to maintain best practices. In addition to flow cytometry-based applications, HITs employs phage display technology for identification of antibody targets/epitopes. HITs has participated in 3 major clinical studies that involved reception, cataloging and storage of >1500 clinical specimens. The first trial took place from 2003 – 2007 and involved the processing of peripheral blood specimens from 127 patients infected with West Nile virus. Specimens were taken on a monthly basis over a 12-month period following the onset of symptoms, amounting to a total of 1524 specimens. On average, the PBMC obtained from each specimen yielded 5 vials of PBMC for cryopreservation amounting to a total of approximately 7600 cryovials banked in our facility. We continue to work with those cryopreserved specimens and find excellent viability and functionality in our assays. The second epidemiological trial started in 2009 and will continue until 2011. This latter study involves sampling of nursing home patients’ blood prior to influenza vaccination to identify markers of vaccine failure. In the 2009 season, 138 blood specimens were processed over a period of 6 weeks and stored as PBMC and plasma. On average, each specimen yielded 4 vials of PBMC for cryopreservation and 3 vials of plasma for a total of 552 PBMC cryovials and 414 plasma vials. Sample vials have been thawed and demonstrated excellent viability and functionality. An additional 400 patients will be recruited to this study in 2010 and 2011. The final study involved an investigation of changes in immunity against HER-2 over the course of Herceptin treatment. In this study, 50 breast cancer patients were recruited prior to initiation of Herceptin treatment. Peripheral blood samples were taken at baseline, 6 months and 12 months after the onset of treatment. PBMCs and plasma were prepared from all of the blood specimens (typically 5 PBMC vials and 3 plasma vials) yielding a total of approximately 750 PBMC cryovials and 450 plasma vials.


Contribution of the scientific environment to this project:

Both McMaster University and St Joseph’s Healthcare Hamilton are academic health centers with extensive collaboration between basic scientists and clinical researchers. Regular academic rounds and research data presentations are held and are attended by Faculty and trainees. These meeting allow for review of data and discussions around proposal development. The University provides support to researchers in the form of infrastructure and provision of adequate laboratory space to carry out the studies proposed in this application. McMaster University has sufficient infrastructure to support the proposed project including a dedicated Human Immune Testing Suite (HITS) and a further Flow Cytometry Core facility juxtaposed. The Human Immune Testing Suite (HITS) is a GLP compliant facility designed to be a 1-stop shop for clinical trials of human immunology. McMaster University has strength and depth in immunology, allergy and asthma research including the following faculty: Ali Ashkar – innate immunity, John Bienenstock- mucosal immunology/allergy, Dawn Bowdish – innate immunity, Jonathan Bramson – viral and cancer immunology, Steve Collins – mucosal immunity, Mike Cyr - Clinical Immunology & Allergy, Judah Denburg – eosinophils, Jack Gauldie – immunology, Immunology & Rheumatology, Mark Inman – murine models of asthma, Manel Jordana – allergy, in vivo models, Paul Keith - Clinical Immnology & Allergy, Martin Kolb, pulmonary fibrosis, Mark Larché – immunology, allergy, asthma, autoimmunity, transplantation, Maggie Larché - Inflammation & rheumatology, Mary Messieh - Clinical Immunology & Allergy, Karen Mossman – viral immunology, Paul O’Byrne – asthma, Carl Richards – arthritis & cancer immunology, Ken Rosenthal – viral immunity, Martin Stampfli – viral immunity, COPD, Susan Waserman - peanut allergy/general clinical allergy/immunology, Zhou Xing – tuberculosis immunity. These individuals collaborate extensively. For example, the PI has ongoing collaborations with Denburg (biology of TSLP in asthma and allergy), Larché (the current application and immunological mechanisms of anti-OX40L), Inman (the current application and pathophysiology of airway hyperresponsiveness in asthma), O'Byrne (current application, several other protocols testing investigational products on allergen responses).