Longitudinal analysis of the expression of human decay accelerating factor (HDAF) on lymphocytes, in the plasma, and in the skin biopsies of transgenic pigs

Abstract

Abstract: Pigs transgenic for the regulators of human complement activation, such as decay accelerating factor (DAF), membrane cofactor protein (MCP), and CD59, have recently been produced as a means to overcome the organ shortage for clinical transplantation. Histological investigations in DAF transgenic pigs have demonstrated that large amounts of the transgenic protein are expressed on the endothelium of the organs, where hyperacute rejection is known to initiate. Several papers have recently appeared in the literature that show considerable variability in the expression of a transgene over the lifespan of a transgenic animal. In order to evaluate the consistency in the expression of the transgene in our human DAF (HDAF) transgenic pigs, we have analyzed 22 newborn transgenic pigs and 5 control littermates (group A) over a 6‐month period and 11 long‐term surviving transgenic animals (group B). In all the animals from group A, HDAF expression was investigated on peripheral blood mononuclear cells (PBMC) and in the plasma as a free circulating molecule at weekly intervals in the first month of life and monthly thereafter. In the same animals, HDAF expression was also analyzed on endothelial cells using sequential ear biopsies. Moreover, HDAF expression was evaluated on the endothelial cells of our longest surviving transgenic animals (group B) and compared with that observed in the biopsy taken at birth. Over the observation period, considerable variability was found in the blood of most animals from group A, both in the percentage of cells expressing the transgene and the mean number of HDAF molecules per cell. In the same animals, free circulating HDAF levels were shown to be very high at birth, progressively declining to a nadir reached between the second and the fifth week of life. No statistically significant correlation could be found between these three hematological parameters. Over the duration of the study, consistent expression of HDAF in the tissues was, however, found at the different time points in all the newborn animals from group A. Moreover, very high levels of HDAF, at least comparable to those observed at birth, were still detected in the skin biopsies of all the animals from group B after more than 18 months of life (mean: 25.18 ± 3.46; range: 21–31 months). Thus, our data show that over an observation period of up to 6 months, there is consistency in the expression of HDAF on the endothelium of serially collected skin biopsies from HDAF transgenic pigs and that levels of HDAF similar to those observed at birth are expressed by these transgenic animals after more than 2 years of life. Hematological parameters such as expression of HDAF on PBMC or measurements of free circulating HDAF in the plasma do not seem to correlate with the expression of HDAF on the endothelial cells.