| Purpose: To detect the expression of Gal,Neu5 Gc and Sda antigens respectively on red blood cells and peripheral blood mononuclear cells and the levels of anti-Gal,Neu5 Gc and Sda antibodies in adult humans,infant humans and infant baboons by flow cytometry.To investigate the effect of cardiac surgery on development of anti-pig antibodies in infant humans.To evaluate the feasibility of infant baboons as an experimental model for TKO pig-infant humans xenotransplantation,providing the experimental data in vivo.Materials and Methods: Materials:40 serum donated by healthy adults;84 preoperative children with heart disease(naive group)and 25 postoperative children with heart disease(exposed group)serum were provided by department of Pediatric Cardiovascular Surgery at UAB;whole blood of different pigs and 40 infant baboon serum were provided by Revivicor,USA.Methods:(1)By FCM,the detection of Gal,Neu5 Gc,Sda antigen expression on human,baboon and pigs RBCs and PBMCs;(2)Binding of adult human,infant human(na?ve group),infant baboon serum Ig M and Ig G to WT and GTKO/CMAHKO/?4Gal NT2KO(TKO)pig RBCs were measured by flow cytometry.(3)Binding of adult human,infant human(naive group),infant baboon serum Ig M and Ig G to TKO pig CD3+T cells(gated from PBMCs)were measured by flow cytometry.(4)Binding of infant human(na?ve and exposed group)serum Ig M and Ig G to WT and TKO pig RBCs and CD3+T cells was measured by flow cytometry.(5)Serum of complement-dependent cytotoxicity was measured by WT and TKO PBMCs with Rabbit complement using sera of adult human,infant human.Results:(1)The detection of Gal,Neu5 Gc,Sda antigen expression in human,baboon and pigs RBCs and PBMCs: a.human do not express Gal,Neu5 Gc and Sda on RBCs and PBMCs;b.in contrast,Neu5 Gc was expressed in baboon on RBCs and PBMCs,but no Gal and Sda antigen expression;c.WT pigs expressed these three antigen.TKO pigs do not express these three antigens after knocking out the gene of the antigen.(2)Binding of adult human,infant human(na?ve group),infant baboon serum Ig M and Ig G to WT and TKO pig RBCs was measured: a.naive group infant serum(<12 months)binding with WT RBCs,positive Ig M binding(53%)and positive Ig G binding(78%)was detected in serum.When binding to TKO RBCs,no serum showed positive Ig M,and only one serum(1%)showed weakly positive Ig G binding;b.100% adult human serum showed positive Ig M/Ig G bound to WT RBCs,however,only(5%)adult human serum showed positive Ig M binding to TKO RBCs,no positive Ig G binding with TKO RBCs;c.100% infant baboon serum Ig M and Ig G were positively bound to WT RBCs;when binding with TKO RBCs,95% infant baboon serum was positive for Ig M binding,Ig G antibody binding was positive in 45% serum;(3)Binding of adult human and infant human(na?ve group)serum Ig M and Ig G to TKO pig CD3+T cells was measured: There was no significant difference in the binding of Ig M and Ig G between adult human(Ig M:1.031±0.06;Ig G:0.89±0.12)and infant human(Ig M: 1.096±0.05;Ig G:1.096±0.05)groups(p> 0.05),and these were no significant difference Ig M and Ig G(p>0.05)with negative control group(Ig M:1.094±0.07;Ig G:0.89±0.03)(excepted that the adult human serum Ig M was slightly lower than the negative control(p<0.05)).(4)Binding of infant human(na?ve and exposed group)serum Ig M and Ig G to WT and TKO pig RBCs and CD3+T cells was measured: a.in binding with WT RBCs,naive group infant serum: 50% Ig M binding positive,76% Ig G binding positive.Exposure group serum: 100% Ig M binding positive,96% Ig G binding positive.The binding level of Ig M and Ig G in exposed group was significantly higher than that in the naive group(p<0.05);b.TKO RBCs: Naive group serum: 0% Ig M binding positive,2% Ig G binding positive.Exposure group serum: 4% Ig M binding positive,4% Ig G binding positive.Though binding of Ig M in the exposed group was significantly higher than that in naive group(p<0.05),however,only one has weakly positive Ig M;Binding of Ig G was not significantly different between two groups(p>0.05);c.binding with TKO CD3+ T cells,There was no significant Ig M and Ig G difference between exposed group(11% Ig M binding positive,8% Ig G binding positive)and naive group(22% Ig M binding positive,58% Ig G binding positive)(p>0.05).(5)WT and TKO PBMCs were incubated with adult and infant human serum,and rabbit complement was added,to measure adult and infant human serum CDC: a.incubated with WT pig PBMC: The cytotoxic response of adult human serum(100% positive)was significantly higher than that of infant human(na?ve group)(50% positive)(p<0.05)and negative control group(p<0.05).However,in TKO pig PBMC,adult human serum(15% positive)cytotoxicity was significantly lower than that of the negative control group(p<0.05);the cytotoxic response of infant serum(40% positive,weak positive)was not significantly different with the adult and negative control groups(p>0.05).b.incubated with WT pig PBMC,the cytotoxic response of the exposed group(100% positive)was significantly higher than the naive group(67% positive)(p<0.05),while in TKO PBMC,there was no significant difference in the cytotoxic response between the exposed group(64% positive)and the naive group(40% positive)(p>0.05).Conclusion:(1)Infant humans do not express Gal,Neu5 Gc and Sda,they develop antibodies to all antigens,whose levels are low.Infant baboons do not express Gal and Sda,however,express Neu5 Gc,so produce anti-Gal and anti-Sda antibodies,whose levels are a little high.WT pigs express Gal,Neu5 Gc and Sda,however,TKO pigs which have been knocked out the genes of three antigens do not express Gal,Neu5 Gc,and Sda.(2)The cardiac surgery would not increase the anti-TKO pig antibody in infant humans.Organ/tissue/cell from GTKO/CMAHKO/?4Gal NT2 KO pig might be transplanted in infant in the absence of preformed antinon Gal/non Neu5Gc/non Sda antibodies.(3)The levels of antinon Gal/non Neu5Gc/non Sda(anti-TKO)antibodies in infant baboons is significant higher than that in infant humans,so infant baboons are not suitable as recipients model of TKO pig xenograft for infant humans. |
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