I will always add my new comments at the top of this current page, so you do not have to scroll down.
--Mark Hill 19:10, 26 May 2009 (EST) You know that all your information is on the "Talk" page? Is this because it is still draft? Best to put it on the front.
--Mark Hill 11:02, 16 May 2009 (EST) Project background information.
Aims of the project
Hypothesis: That delirium is caused by or associated with apoptosis of neurons, and is the major mechanism of neuronal loss in dementia. This project aims to investigate whether delirium is associated with inflammation and apoptosis, how the severity of apoptosis influences health, cognitive and physical functional outcomes and how delirium interacts with dementia at a neuronal and patient level.
Since the late 19th century delirium has been viewed as a transient disorder that resolves completely with appropriate treatment of underlying risk factors. Delirium is still generally understood to be due to temporary cerebral metabolic dysfunction associated with abnormal neurotransmitter levels. Although the definition of delirium has been a work in progress, the central features have not changed. However, recent research has revealed that the sequelae of delirium are frightening, with higher mortality and subsequent cognitive and ADL disability in survivors. The outcomes of delirium are worse than those of other neurological diagnostic groups such as stroke suggesting that ascribing the pathophysiology to temporary metabolic abnormalities is an inadequate explanation. Rather, these permanent but diffuse deficits suggest that neuronal loss is occurring across the cerebrum. No mechanism has yet been identified for this.
Project Plan Student tasks over 32 weeks. Please note that students will be enrolled in extra Faculty courses at the same time as the ILP. For more information on structure of the ILP over the 32 week timeframe.
This ILP combines clinical and laboratory work to understand and investigate the phenomenology and pathophyisology of delirium. The ILP is based in the Geriatric Medicine Unit at POWH and the Cell Biology Laboratory at UNSW. In Phase 1 students will learn about the variety of presentations and manifestations of delirium on the ward, complete a literature review and undertake courses to ensure safe laboratory work. Students are encouraged to read widely around the topic and devlop an appreciation of the clinical variety fo delirium.
In Phase two students will be involved in investigation of patients with delirium, including neuropsychological assessment, venepuncture for blood and lumbar puncture to collect cerebrospinal fluid. Students will manage the specimens safely into -80 storage. They will learn ELISA assay techniques theory and practice and conduct their own laboratory work on these and previously collected specimens. They will also be involved with a variety of other trials we are conducting to assess the various aspects of delirium, including prevention, treatment and interaction with dementia.
Phase three should see completion of experimental work and follow-up of participants previously enrolled. Students will write a report of their work with a view to publication.
This ILP project will contribute to a world-first research effort to understand one of the scourges affecting older patients in hospitals around the world. Students will take a leading role in our search through the five apoptotic pathways as well as new leads that have been identified in chemokines to identify how these pathways affect different types of delirium.
Students will gain an understanding of the clinical and pathophysiological features of delirium and of mechanism of inflammation and cell death. Students will gain a hands-on working knowledge of laboratory techniques and an appreciation of how to take ideas from the bedside to the benchtop.
Inouye SK. Delirium in older persons. N Engl J Med 2006; 354: 1157-65. McCusker J, Cole M, Dendukuri N, Belzile E, Primeau F. Delirium in older medical inpatients and subsequent cognitive and functional status: a prospective study. CMAJ 2001; 165: 575-583. Takuma K, Yan SS, Stern DM, Yamada K. Mitochondrial dysfunction, endoplasmic reticulum stress, and apoptosis in Alzheimer’s disease. J Pharmacol Sci 2005; 97: 312-316.
I was just wondering in terms of elisa, if the kit says it is able to detect "protein in cell lysates, supernatants, whole blood, and serum" does CSF fall into any of those categories?
Type Markers Suppliers Sensitivity Cost Overseas /domestic Usage 1) Sandwich ELISA Kit #7190 Cleaved Caspase-3 (Asp175) PathScan® - TBA O/S 1 2) Human Active Caspase-3 Quantikine ELISA Kit KM300 Caspase 3 R&D Systems 0.1 ng/mL TBA O/S 1 3) Human Caspase-3 BMS2012INST Caspase 3 Bender MedSystems 0.12 ng/ml Assay Range: 10 - 0.16 ng/ml TBA O/S 1 4) ELISA Kit Catalog # KHO1091 Caspase 3 Invitrogen 0.033 ng/mL $828 Domestic Supplier 1 5) Apoptosis Multi-Target Sandwich ELISA Kit #7105 p53 protein, phospho-p53 protein (Ser15), Bad, phospho-Bad (Ser112), Cleaved Caspase-3 (Asp175) and Cleaved PARP (Asp214)* PathScan - TBA O/S 1 6) RD 192072200R Human GFAP ELISA Gfap BioVendor Analytical Limit of Detection is calculated from the real GFAP values in wells and is 0.033ng/ml Assay Sensitivity takes the dilution of samples into consideration and is calculated according to the formula: Assay Sensitivity = Analytical Limit of Detection x sample dilution = 0.033ng/ml x 3 =0.1ng/ml TBA O/S 1 7) Sandwich enzyme immunoassay KA0024 Human GFAP (Glial Fibrillary Acidic Protein) Abnova Corporation - $985 O/S 1 8) Bcl-2 Sandwich ELISA Kit APT230 Bcl-2 Family Chemicon International 3.9 ng/mL Range of detection: 250 ng/mL to 3.9 ng/mL Detects human Bcl-2 protein in cell lysates, supernatants, whole blood, and serum. TBA Domestic supplier Australia +61 3 9839 2000 1 9) Human Cytochrome c EIA Kit 900-141 Cytochrome C Assay Designs/Stressgen Bioreagents 6.03 pg/ml Assay range: 28.13-900 pg/ml TBA Domestic Supplier 1 10) Human Cytochrome c EIA Kit 650.070.096 Cytochrome C Diaclone 0.05 ng/ml TBA 1 11) Human Cytochrome c EIA Kit RBMS263R Cytochrome C. BioVendor Laboratory Medicine, Inc. 0.05 ng/ml TBA 1 12) Immunoassay Kit Catalog #KHO1051 Cytochrome C Invitrogen <0.156 ng/mL $829 Domestic Supplier 1 13) Human CD178 / FasL ELISA Kit 850.750.096 Fas-L Diaclone/Tepnel 44pg/ml Range: 62.5-2000 $350 Domestic supplier 1 14) Human CD178 / FasL ELISA Kit 850.750.192 Fas-L Diaclone/Tepnel 44pg/ml Range: 62.5-2000 $525 2 1) http://www.cellsignal.com/products/7190.html 2) www.rndsystems.com/pdf/km300.pdf 3) http://www.biocompare.com/RequestInformation/8/892214/Human-Caspase-3-Instant-ELISA-Kit-from-Bender-MedSystems.html?q=1 4) http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&productID=KHO1091 5) http://www.cellsignal.com/products/7105.html 6) www.ibl-america.com/pdf/BioVender/RD192072200R_IBL.pdf 7) http://www.abnova.com/Products/Products_Detail.asp?strCountry=US&Catalog_ID=KA0024 8) http://www.millipore.com/searchsummary.do?tabValue=&q=bcl-2+elisa 9) http://www.assaydesigns.com/commerce/ccp1155-1155-cytochrome-c-28human29--eia-kit.htm 10) http://www.tepnel.co.uk/product-details.asp?ID=552&Cat=397 11) http://www.biovendor.com/country315/product/immunoassays/cytochrome-c-human-elisa 12) http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&entryPoint=adirect&productID=KHO1051&messageType=catProductDetail 13) http://www.diaclone-usa.com/diaclone/datasheet.asp?A=kits&S=850.750.192 14) http://www.diaclone-usa.com/diaclone/datasheet.asp?A=kits&S=850.750.192
Comparison of ELISA kits available on Apoptosis
Markers Suppliers Sensitivity Cost Overseas/domestic Usage
1) Sandwich ELISA Kit #7190
Caspase-3 PathScan® - TBA O/S 1
2) Human Active Caspase-3 Quantikine ELISA Kit KM300
Caspase 3 R&D Systems 0.1 ng/mL TBA O/S 1
3) Human Caspase-3 BMS2012INST
Caspase 3 Bender MedSystems 0.12 ng/ml TBA O/S 1 Assay Range: 10 - 0.16 ng/ml
4) Apoptosis Multi-Target Sandwich ELISA Kit #7105 p53 protein, PathScan - TBA O/S 1 phospho-p53 protein (Ser15), Bad, phospho-Bad (Ser112), Cleaved Caspase-3 (Asp175) and Cleaved PARP (Asp214)* 5) RD 192072200R Human GFAP ELISA
Gfap BioVendor Analytical Limit TBA O/S 1 of Detection is calculated from the real GFAP values in wells and is 0.033ng/ml Assay Sensitivity takes the dilution of samples into consideration and is calculated according to the formula: Assay Sensitivity = Analytical Limit of Detection x sample dilution = 0.033ng/ml x 3 =0.1ng/ml
6) Sandwich enzyme immunoassay KA0024 Human GFAP (Glial Fibrillary Acidic Protein)
Gfap Abnova Corporation - $985 O/S 1
1) http://www.cellsignal.com/products/7190.html 2) www.rndsystems.com/pdf/km300.pdf 3) http://www.biocompare.com/RequestInformation/8/892214/Human-Caspase-3-Instant-ELISA-Kit-from-Bender-MedSystems.html?q=1 4) http://www.cellsignal.com/products/7105.html 5) www.ibl-america.com/pdf/BioVender/RD192072200R_IBL.pdf 6) http://www.abnova.com/Products/Products_Detail.asp?strCountry=US&Catalog_ID=KA0024
- Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Initiator caspases (including 8, 9, 10 and 12) are closely coupled to proapoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6 and 7), which in turn cleave cytoskeletal and nuclear proteins like PARP, a-fodrin, DFF and lamin A, and induce apoptosis. Cytochrome c released from mitochondria is coupled to the activation of caspase-9, a key initiator caspase (1). Proapoptotic stimuli include the FasL, TNF-a, DNA damage and ER stress. Fas and TNFR activate caspases 8 and 10 (2), DNA damage leads to the activation of caspase-9 and ER stress leads to the calcium-mediated activation of caspase-12 (3). The inhibitor of apoptosis protein (IAP) family includes XIAP and survivin and functions by binding and inhibiting several caspases (4,5). Smac/Diablo, a mitochondrial protein, is released into the cytosol upon mitochondrial stress and competes with caspases for binding of IAPs. The interaction of Smac/Diablo with IAPs relieves the inhibitory effects of the IAPs on caspases.