Winnie Pao

IgY antiporcine endothelial cell antibodies effectively block human antiporcine xenoantibody binding.

Avian IgY antibodies are structurally different from mammalian IgGs and do not fix mammalian complement components or bind human Fc receptors. As these antibody‐mediated interactions are believed to play significant roles in both hyperacute rejection (HAR) and acute vascular xenograft rejection (AVXR), IgY antibodies to xenoantigen target epitopes may inhibit these rejection processes. In this report, we show that chicken IgY antibodies to α‐Gal antigen epitopes and to other porcine aortic endothelial cell (PAEC) antigens block human xenoreactive natural antibody binding to both porcine and rat cardiac tissues and porcine kidney tissues. Chicken IgY antibodies blocked complement‐mediated lysis of PAECs by human serum, and inhibited antibody‐dependent cell‐mediated lysis of PAECs by heat‐inactivated human serum plus peripheral blood leukocytes. Binding of IgY to porcine endothelial cells did not affect cell morphology nor expression of E‐selectin. These results suggest that avian IgYs could be of potential use in inhibiting pig‐to‐human xenograft rejection.

Synthetic peptides which inhibit the interaction between C1q and immunoglobulin and prolong xenograft survival

Background. Acutevascular xenograft rejection (AVXR), also termed delayed xenograft rejection (DXR), occurs when hyperacute rejection (HAR) is prevented by strategies directed at xenoreactive natural antibodies and/or complement activation. We have hypothesized that AVXR/DXR is initiated in part by early components of the complement cascade, notably C1q. We have developed synthetic peptides (termed CBP2 and WY) that interfere with the interaction between C1q and antibody. Methods. CBP2 and the WY-conjugates were used as inhibitors of immunoglobulin aggregate binding to solid phase C1q. Inhibition of complement activation by the peptides of the classical system was determined using lysis assays with sensitized sheep red blood cells or porcine aortic endothelial cells as targets and of the alternate complement pathway using guinea pig red blood cells as targets. Two transplant models were used to study the effects of administering peptides to recipients: rat heart transplant to presensitized mouse, and guinea heart transplant to PVG C6-deficient rats. Results. CBP2 and WY-conjugates inhibited immunoglobulin aggregate binding to C1q. The peptides also inhibited human complement-mediated lysis of sensitized sheep red blood cells and porcine aortic endothelial cells in a dose-dependent manner and the WY-conjugates prevented activation of the alternate complement pathway as shown by inhibition of guinea pig red blood cells lysis with human serum. In addition, the use of the peptides and conjugates resulted in significant prolongation of xenograft survival. Conclusions. The CBP2 and WY peptides exhibit the functional activity of inhibition of complement activation. These peptides also prolong xenograft survival and thus provide reagents for the study of the importance of C1q and other complement components in transplant rejection mechanisms.

Simple purification methods for an αgalactose-specific antibody from chicken eggs

For many applications avian antibody from egg yolk (IgY) offers advantages over the well-known mammalian antibodies. Different experimental techniques for the purification of IgY from chickens immunized with an alphagalactose-containing antigen (alphaGal-trisaccharide) were compared. These included ammonium sulfate precipitation, filtration with diatomaceous earth, treatment with deoxycholate, and thiophilic and affinity chromatography. Samples were tested for overall purity, protein and lipid content, and specific activity. Evaluated on the basis of these results and the simplicity of the process, the favored purification method is ammonium sulfate precipitation of diluted egg yolk directly followed by affinity chromatography. The high lipid content of IgY preparations is greatly reduced by either thiophilic or affinity chromatography. Affinity purification of ammonium sulfate precipitated material resulted in anti-alphaGal-trisaccharide IgY preparations with approximately 1% of the original protein content but approximately 100-fold higher specific activity for the alphaGal-trisaccharide epitope.

Distribution of, and immune response to, chicken anti‐αGal immunoglobulin Y antibodies in wild‐type and αGal knockout mice

Chicken antibodies (immunoglobulin Y; IgY) to the αGal epitope (galactose α‐1,3‐galactose) bind to αGal antigens of mouse and porcine tissues and endothelial cells in vitro and block human anti‐αGal antibody binding, complement activation and antibody‐dependent cell‐mediated lysis mechanisms. The activities and toxicity of anti‐αGal IgY have not been tested in vivo. In this study, we tested the effects of multiple injections of affinity‐purified anti‐αGal IgY (AP‐IgY) in both wild‐type (WT) and α‐1,3‐galactosyltransferase knockout (Gal KO) mice. WT and Gal KO mice were injected once, twice, three, or four times intravenously (i.v.) with AP‐IgY and killed at 1 hr or 24 hr. Mice displayed no toxicity to four injections of AP‐IgY. Heart, lung, liver, kidney, spleen and pancreatic tissue were evaluated using immunohistochemical techniques for the presence of the αGal epitope using the GSI‐B4 lectin, and for bound IgY, as well as mouse IgM and IgG. The binding of AP‐IgY antibodies to the endothelium of WT mouse tissues was essentially identical to the pattern of binding of the GSI‐B4 lectin after injection of WT mice and death at 1 hr. WT mice killed 24 hr after i.v. injection of AP‐IgY showed little remaining bound IgY in their endothelia, indicating that IgY is cleared over that time period. We also evaluated the blood drawn at the time of death for the presence of anti‐αGal IgY, anti‐IgY IgM and anti‐IgY IgG by enzyme‐linked immunosorbent assay. Anti‐αGal IgY was almost undetectable in WT mouse sera at all injection and killing times. In contrast, Gal KO mouse sera showed increasing anti‐αGal IgY levels until 24 hr after the fourth injection, when anti‐αGal IgY levels were almost undetectable. Anti‐IgY IgM and IgG levels in WT and Gal KO mouse sera showed a typical increase in anti‐IgY IgM 24 hr after the second injection (3 days after the first injection) and an increase in anti‐IgY IgG 24 hr after the third injection (5 days after the first injection). These results show that IgY binds to αGal epitopes in the WT mice and is cleared sometime over a 24‐hr time period and that IgY is an expected immunogen in mice eliciting a rather typical anti‐IgY IgM and IgG response.