ABSTRACT
Objective To investigate the optimal condition for the detection of anti-non-galactose (Gal) xenoantigen and antibody in human serum.Mehtods Peripheral blood mononuclear cell (PBMC) obtained from Wuzhishan miniature pig models with α-1,3-galactosyltransferase gene knockout (GTKO) were used as target cells,mixed and incubated with healthy human serum of different concentrations (4.8％,16.7％ and 100％) for 0.5,1.0,2.0,3.0 and 6.0 h,respectively.The abilities of PBMC to bind with IgM and IgG were detected by flow cytometry.Results At the serum concentration of 16.7％,the ability ofnon-Gal IgM to bind with PBMC was significantly enhanced from 0.5 h to 3.0 h incubation (P＜0.01),whereas no statistical significance was noted in terms of IgG (P＞0.05).Increasing serum concentration could also enhance the ability of non-Gal IgM to bind with PBMC.At the serum concentration of 100％ and incubation for 3 h,the ability of IgM to bind with PBMC was the highest among all groups (P＜0.01).At the serum concentration of 100％ and incubation for 6 h,the ability of IgG to bind with PBMC was significantly enhanced (P＜0.05).Prolonging incubation time and increasing serum concentration did not affect the activity of PBMC.Conclusions The optimal condition for detection of anti-non-Gal xenoantigen and antibody is determined.A quantity of 1×105 PBMC from pig should be incubated with 100％ human serum for 3 h for detection of IgM level,or incubated with 100％ human serum for 6 h for measurement of IgG level.This optimized condition contributes to screening the donor pigs which lowly express non-Gal antigen.
ABSTRACT
To establish a platform to monitor the immune rejection after abdominal aortic patch suture in a xenotransplantation model.Methods The carotid was excised from wild-type Bama pigs,cut into 2.5 cmx 1.0 cm pieces in shuttle shape and subsequently sutured to the abdominal aorta of cynomolgus monkeys.No immunosuppressive agent was administered.General conditions of the recipient monkeys were observed.The morphological changes of the graft artery were assessed by pathological examination at postoperative 1 year.Before and 7,14,28 and 49 d after surgery,the blood samples were collected from the recipient monkeys.The serum levels of IgM and IgG antibodies were quantitatively measured by the red blood cell and peripheral blood mononuclear cell (PBMC) from Bama pigs.The quantity of lymphocytes in the recipient monkeys was detected by routine blood test and flow cytometry.Results All 3 monkeys undergoing transplantation survived well.At postoperative 1 year,the lateral tissues of the vascular wall at the artery graft were seen in dark red color.Hematoxylin-eosin (HE) staining revealed a large quantity of red blood cell and platelet deposition,accompanied with lymphocyte infiltration.Using porcine red blood cell and PBMC as target cells,the serum levels of anti-pig IgM and IgG antibodies peaked at postoperative 28 d,and slightly declined at postoperative 49 d.The quantity of lymphocytes and T cell subset also peaked at postoperative 28 d and began to decrease at postoperative 49 d.Conclusions Artery patch suture is a simple and reliable xenotransplantation model.The recipients can maintain normal physiological state without the use of immunosuppressive agents.The grafts can effectively activate the immune system of the recipients,induce the production of anti-pig antibodies and provoke cellular immune rejection.Therefore,this model can be utilized to monitor the immune rejection throughout the xenotransplantation process.