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Relation between human decay‐accelerating factor (hDAF) expression in pig cells and inhibition of human serum anti‐pig cytotoxicity: value of highly expressed hDAF for xenotransplantation

Relation between human decay‐accelerating factor (hDAF) expression in pig cells and inhibition of... Abstract: Background: Although the successful production of α1,3‐galactosyltransferase‐knockout (GT‐KO) pigs has increased expectations of clinical xenotransplantation, additional modifications of genetically engineered pigs are still being explored, because even GT‐KO pigs are incapable of inhibiting the host's immunological response completely. One of the potential candidates is a complement‐regulatory protein, such as human decay‐accelerating factor (hDAF). However, there are few reports on how high the expression level of hDAF in pig cells would be required for suppression of complement activation. The purpose of this study was to examine the relationship between the level of hDAF expression and its inhibitory effect on human serum cytotoxicity. Methods: An expression (pCAGGS) vector containing the hDAF gene was transfected into pig fibroblasts using an electroporation system (Gene Pulser II). Forty‐eight to fifty‐two hours after transfection, the cells were stained with FITC‐labeled anti‐hDAF antibody and then applied to the cell sorter. hDAF‐transfected cells with various expression levels were collected by gating on fluorescence intensity. The level of hDAF expression was determined relative to that in human control endothelial cells. Collected cells expressing ×1, ×5, ×10, ×15 and ×30 hDAF were incubated into 96‐well plates for 16 h, and the cells were subjected to 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay. Results: hDAF expression levels in transfected cells at the time of MTT assay (16 h after sorting) were comparable to those immediately after sorting. hDAF expression in pig cells five times higher than in human endothelial cells was effective in inhibiting complement‐dependent cytotoxicity of most human sera. However, 15‐ to 30‐fold expression of hDAF was required for effective inhibition of human sera with the highest cytotoxic capacity. Conclusions: A much higher level of hDAF expression in pig cells than previously considered necessary might be required to provide additional benefit in inhibiting antibody‐mediated rejection. Genetically engineered pigs that express very high levels of hDAF would be beneficial for xenotransplantation. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Xenotransplantation Wiley

Relation between human decay‐accelerating factor (hDAF) expression in pig cells and inhibition of human serum anti‐pig cytotoxicity: value of highly expressed hDAF for xenotransplantation

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References (34)

Publisher
Wiley
Copyright
Copyright © 2007 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0908-665X
eISSN
1399-3089
DOI
10.1111/j.1399-3089.2006.00365.x
pmid
17214706
Publisher site
See Article on Publisher Site

Abstract

Abstract: Background: Although the successful production of α1,3‐galactosyltransferase‐knockout (GT‐KO) pigs has increased expectations of clinical xenotransplantation, additional modifications of genetically engineered pigs are still being explored, because even GT‐KO pigs are incapable of inhibiting the host's immunological response completely. One of the potential candidates is a complement‐regulatory protein, such as human decay‐accelerating factor (hDAF). However, there are few reports on how high the expression level of hDAF in pig cells would be required for suppression of complement activation. The purpose of this study was to examine the relationship between the level of hDAF expression and its inhibitory effect on human serum cytotoxicity. Methods: An expression (pCAGGS) vector containing the hDAF gene was transfected into pig fibroblasts using an electroporation system (Gene Pulser II). Forty‐eight to fifty‐two hours after transfection, the cells were stained with FITC‐labeled anti‐hDAF antibody and then applied to the cell sorter. hDAF‐transfected cells with various expression levels were collected by gating on fluorescence intensity. The level of hDAF expression was determined relative to that in human control endothelial cells. Collected cells expressing ×1, ×5, ×10, ×15 and ×30 hDAF were incubated into 96‐well plates for 16 h, and the cells were subjected to 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay. Results: hDAF expression levels in transfected cells at the time of MTT assay (16 h after sorting) were comparable to those immediately after sorting. hDAF expression in pig cells five times higher than in human endothelial cells was effective in inhibiting complement‐dependent cytotoxicity of most human sera. However, 15‐ to 30‐fold expression of hDAF was required for effective inhibition of human sera with the highest cytotoxic capacity. Conclusions: A much higher level of hDAF expression in pig cells than previously considered necessary might be required to provide additional benefit in inhibiting antibody‐mediated rejection. Genetically engineered pigs that express very high levels of hDAF would be beneficial for xenotransplantation.

Journal

Xenotransplantation – Wiley

Published: Jan 1, 2007

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