P. L. Dabkowski

Bio: P. L. Dabkowski is an academic researcher from Austin Hospital. The author has contributed to research in topics: Antibody & Transplantation. The author has an hindex of 7, co-authored 9 publications receiving 819 citations.

Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 1-3)Gal epitopes.

Abstract: A major problem with pig-to-human-tissue xenograft studies is that humans have natural antibodies to pig cells; these antibodies would cause hyperacute rejection if pig tissues were xenografted to humans. Here we show that most of human IgM antibodies present in the serum of healthy donors and reactive with pig cells react with galactose in an (alpha 1-3) linkage with galactose--i.e., Gal(alpha 1-3)Gal. Absorption studies demonstrated that the antibodies detected the same or similar epitopes on the surface of pig erythrocytes, blood and splenic lymphocytes, and aortic endothelial cells (EC). The antibodies were sensitive to 2-mercaptoethanol (2ME) treatment, did not bind to protein A or G, and were present in the high molecular weight fraction of serum; they are clearly IgM antibodies. Further, the antibodies did not react with human ABO blood group substances and are not related to human blood group A or B, which carry a terminal galactose. The reaction of human serum with pig erythrocytes was specifically inhibited by mono- and disaccharides: D-galactose, melibiose, stachyose, methyl-alpha-D-galactopyranoside, and D-galactosamine but not by D-glucose or methyl-beta-D-galactopyranoside; demonstrating that the reaction is with galactose in an alpha and not a beta linkage. A cDNA clone encoding the murine alpha-1,3-galactosyltransferase (which transfers a terminal galactose residue with an (alpha 1-3) linkage to a subterminal galactose) was isolated by polymerase chain reaction (PCR), cloned, and transfected into COS cells, which are of Old World monkey origin and, like humans, do not express Gal(alpha 1-3)Gal. After transfection, COS cells became strongly reactive with human serum and with IB4 lectin [which reacts only with Gal(alpha 1-3)Gal]; this reactivity could be removed by absorption with pig erythrocytes. As most of the antibody reacting with pig cells can be removed by absorption with either melibiose or Gal(alpha 1-3)Gal+ COS cells, most of these react with Gal(alpha 1-3)Gal. These findings provide the basis for genetic manipulation of the pig alpha-1,3-galactosyltransferase for future transplantation studies.

Characterization of cDNA clones for porcine α(l,3)galactosyl transferase: The enzyme generating the Galα(l,3)Gal epitope

Abstract: Galoα(l,3)Gal is a terminal carbohydrate found on many glycosylated cell surface molecules of species other than humans and Old World monkeys, and is produced by the α(l,3)galactosyl transferase enzyme's adding galactose to a substrate. We have previously shown, by the transfection of COS cells with the cloned mouse α(l,3)galactosyl transferase, that most human anti-pig antibodies react with Galα(l,3)Gal. Using cross-species hybridization with the mouse α(l,3)galactosyl transferase cDNA, bacteriophage λ, gt11 and λgt10 pig cDNA libraries were screened and overlapping clones isolated which encode the pig α(l,3)galactosyl transferase. Sequencing of the clones demonstrated a single open reading frame coding for a protein with high homology to murine (75% identity) and bovine (82% identity) α(l,3)galactosyl transferases. Southern blot analysis shows the porcine α(l,3)galactosyl transferase gene to be a single copy gene, and northern analysis demonstrated an mRNA of 3.9 kb. After splicing the clones to produce a single full length clone, transfection of Galα(l,3)Gal- COS cells led to strong reactivity with human serum and with the IB4 lectin (which reacts only with Galα(l,3)Gal), indicating that the expression of the transferase led to the expression of Galα(l,3)Gal. The cloning of the cDNA gene for the pig α(l,3)galactosyl transferase is the first step in the production of a transgenic pig lacking the transferase and therefore the Galα(l,3)Gal epitope; such animals could serve as donors for human transplantation.

Properties of the amniotic membrane for potential use in tissue engineering.

Abstract: An important component of tissue engineering (TE) is the supporting matrix upon which cells and tissues grow, also known as the scaffold. Scaffolds must easily integrate with host tissue and provide an excellent environment for cell growth and differentiation. Most scaffold materials are naturally derived from mammalian tissues. The amniotic membrane (AM) is considered an important potential source for scaffolding material. The AM represents the innermost layer of the placenta and is composed of a single epithelial layer, a thick basement membrane and an avascular stroma. The special structure and biological viability of the AM allows it to be an ideal candidate for creating scaffolds used in TE. Epithelial cells derived from the AM have the advantages of stem cells, yet are a more suitable source of cells for TE than stem cells. The extracellular matrix components of the basement membrane of the AM create an almost native scaffold for cell seeding in TE. In addition, the AM has other biological properties important for TE, including anti-inflammatory, anti-microbial, anti-fibrosis, anti-scarring, as well as reasonable mechanical property and low immunogenicity. In this review, the various properties of the AM are discussed in light of their potential use for TE.