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Biological Procedures Online

, Volume 5, Issue 1, pp 182–188

Received: 05 June 2003Revised: 06 August 2003Accepted: 26 August 2003DOI: 10.1251-bpo60

Cite this article as: Mahajan, S.D., Aalinkeel, R., Schwartz, S.A. et al. Biol. Proced. Online 2003 5: 182. doi:10.1251-bpo60

Abstract

CD8+ cytotoxic T lymphocyte CTL activity is currently believed to be one of the key immunologic mechanisms responsible for the prevention or attenuation of HIV-1 infection. The induction of CD8+ T cell activation may also result in the production of soluble or non-classical lytic factors that are associated with protection from infection or slower disease progression. Traditionally, CD8+ CTL responses have been measured by the classic chromium release assay, monitoring the ability of T cells Effector cells to lyse radiolabelled HLA — matched -target cells- that express the appropriate antigen-MHC complex. This method is not only labor intensive, semi quantitative assay at best, but also needs fresh, non-cryopreserved cells. Recently, cytokine specific ELISPOT assays or tetrameric MHC-I- peptide complexes have utilized to directly quantitate circulating CD8+ effector cells, and these assays are more sensitive, quantitative and reproducible than the traditional CTL lysis assay and can also be performed on cryopreserved cells. Although these are reproducible assays for the assessment of soluble antiviral activity secreted by activated T cell populations they can be extremely expensive to perform. We have used FACS Analysis to measure Granzyme B release as a function of cell mediated cytotoxicity. This method helps quantitate the CTL activity and also identifies the phenotype of the cells elucidating this immune response. The method described not only monitors immunological response but also is also simple to perform, precise and extremely time efficient and is ideal for screening a large number of samples.

Indexing termsCD8-Positive T-Lymphocytes HIV Killer Cells, Natural AbbreviationsCTLcytotoxic T lymphocyte

HIVHuman Immunodeficiency Virus

NKnatural killer

PIpropidium iodide

7AAD7-aminoactinomycin D

PBMCsperipheral blood mononuclear cells

E:TEffector: Target

R1 - R2Region 1-Region 2

FL-1fluorescence axis 1

FL-2fluorescence axis 2

SSCside scatter

PEphycoerythrin

Published: September 5, 2003

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Autor: Supriya D. Mahajan - Ravikumar Aalinkeel - Stanley A. Schwartz - Ram P. Chawda - Madhavan P. N. Nair

Fuente: https://link.springer.com/







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