Pig kidneys were extracorporeally "ex vivo" connected to the circulation of two volunteer male dialysis patients (Breimer et al., this issue). The patients were pretreated by daily plasmapheresis for 3 consecutive days, which reduced the anti-pig lymphocytotoxic titer from 8 to 2 in the first patient and from 8 to 1 in the second patient. The anti-pig hemagglutinating titers were reduced from 32 to 4 in the first patient and from 2 to 1 in the second patient. No drugs, except heparin, were given. The perfusion lasted for 65 min in patient 1 and the experiment was terminated due to increased vascular resistance in the pig kidney. Ultrastructural investigation showed a picture similar to a hyperacute vascular rejection. Immunohistochemical studies showed a weak staining of IgM antibodies, but no IgG in the small arteries and glomeruli. The pig kidney of patient 2 was perfused for 15 min and the experiment terminated due to serious side effects of the patient. Light and electron microscopical investigation showed virtually no structural changes of the kidney tissue and immunostaining for human antibodies was negative. In both patients, serum samples collected 2-5 weeks postperfusion showed a strong anti-pig antibody titer rise (up to 512) which thereafter declined but stabilized on a higher level than before the experiment. The antibody response in the two patients was different. In patient 1, the major anti-pig antibodies directed to carbohydrate antigens were of IgG (IgG1 and IgG2 subclasses) type, while the IgM response was less prominent and virtually no IgA antibodies were produced. Despite the short duration of the perfusion in patient 2, a humoral immune response was seen that was mainly confined to the IgA immunoglobulin class (IgA1 subclass). Blood group glycospingolipid fractions, prepared from the contralateral kidney of the donor pigs, were used for immunostaining with patient serum samples. In both patients, the antibodies produced after the perfusion, mainly recognized the Galα1-3Gal epitope both as part of the "linear B" pentasaccharide but also on more complex carbohydrate structures. Patient 1 was HLA-immunized before the experiment due to a kidney allograft and had a panel reactivity of 85% before the perfusion. No change in the panel reactivity of HLA-antibodies was found after the perfusion experiments. Patient 2 had no HLA antibodies before and remained negative after the perfusion. Patient serum samples collected before and after the perfusion were tested for reactivity against human endothelial cell lines. No antibodies were generated.

The Galalpha1-3Gal (alphaGal) antigen is considered the main xenoantigen in the pig to human species combination but other porcine antigens have to be considered such as the swine lymphocyte antigen (SLA), the blood group A/O and the Hanganutziu-Deicher (H-D) antigens. The H-D antigens are N-glycolyl-neuraminic acid (NeuGc) terminated gangliosides that are widely distributed in mammalian species but absent in humans. Upon exposure to a vascularized pig organ, the human recipient can be immunized by direct interaction with the pig tissue or/and by transfer of tissue/cells from the organ into the recipient. In the present work, we describe the release of cells from porcine kidneys upon perfusion and the expression of glycolipid based alphaGal, blood group A/O and H-D antigens in pig lymphocytes. Pig kidneys were flushed with 20 ml of NaCl or Lidocain containing 5000 U heparin, and thereafter perfused with 3000-ml perfusion solution and the cells released were counted and examined microscopically. Neutral glycolipid and ganglioside fractions were extracted from purified pig lymphocytes. The extracted components were characterized by thin layer chromatography, degradation and mass spectrometry. The expression of alphaGal and H-D epitopes on cells released from pig kidneys and purified pig lymphocytes were studied by immune electron microscopy. A total amount of about 300 x 106 leukocytes, mainly lymphocytes were released in the perfusate from the kidneys, of which about 100 x 106 cells were eluated in the 600 to 2400 ml perfusate fraction. Immunelectron microscopical analysis with Griffonia simplicifolia isolectin B4 showed staining of pig leukocytes and other cells, morphologically similar to endothelial cells, released in the perfusate. The purified porcine lymphocytes contained 930 microg neutral glycolipid (4.2 microg/mg cell protein) of which 95% was glycolipids with one to four sugar residues. Immunostaining of the neutral glycolipid fractions revealed alphaGal terminated compounds migrating in the five and 10 to 12 sugar regions and blood group A compounds in the six and eight sugar regions. Two major gangliosides NeuGc-GM3 and NeuGc-GD3 were found in the pig lymphocytes. In a patient extracorporeally xenoperfused with a pig kidney, an increased staining of both alphaGal terminated structures as well as the H-D reactive gangliosides were found in the post-perfusion serum samples. In summary, leukocytes, mainly lymphocytes are released from pig kidneys during perfusion which may contribute to immunization of human xenograft recipients.

Since clinical stem-cell therapy is likely to be performed with immature progenitor cells, blood group ABO compatibility of donor cells/recipients should be favorable to avoid unnecessary rejection problems caused by ABO incompatibility. The in vitro loss of B antigens in a genotype B hESC line indicates that loss of ABH antigens occurs early during human embryogenesis since these antigens are lacking in adult cardiomyocytes.

Removal of human preformed natural anti‐pig antibodies from the blood is a prerequisite before xenografting between pig and man can be performed. This work explores the effect of plasmapheresis and immunoadsorption (protein‐A sepharose) on the reduction and recurrence of anti‐pig antibodies in 14 patients. The anti‐pig antibody changes were evaluated by lymphocy to toxic, hemagglutinating, and endothelial cell ELISA techniques. The changes induced showed a similar pattern with all three techniques used. In addition, plasma from plasmapheresis treatments were perfused through pig kidneys and the reduction of anti‐pig antibodies was estimated by the mentioned in vitro techniques. The anti‐pig antibody titers could be reduced to low levels, but not completely eliminated, by 3–4 plasmapheresis sessions. The titers gradually returned to pretreatment levels or higher in a period of 1–2 weeks. A few patients showed signs of a more rapid resynthesis reaching pretransplant levels in 3–4 days. Protein A immunoadsorption satisfactory removed IgG but not IgM antibodies. In vitro perfusion of pig kidneys at 37°C showed a rapid reduction of anti‐pig antibody titers of 3–4 titer steps. The combination of 3–4 plasma exchanges followed by in vitro pig kidney perfusion completely removed all anti‐pig antibodies. Reduction of the anti‐pig lymphocyte and erythrocyte antibody titers by soluble oligosaccharides carrying terminal Galoc‐epitopes was only partly successful. A 40–60% inhibition was achieved by 5–10 mg saccharide/ml serum and no clear inhibition difference between di‐ and trisaccharides was found. Inhibition of plasma obtained after 3–4 plasmapheresis treatments with soluble Galα1‐di‐ and trisaccharides resulted in very low anti‐pig titers. Therefore one feasible pretreatment procedure, before pig to human xenotransplantation could be plasmapheresis for major reduction of anti‐pig antibody titer followed by neutralisation of the remaining antibodies by addition of soluble oligosaccharides or immunoadsorption with Galα‐1‐columns.

Currently, the pig species is regarded as the most likely organ donor for human xenotransplantation in the future. However, it cannot be granted that the pig will be the optimal species of choice. We have studied human anti-sheep antibodies in comparison with anti-pig antibodies. The anti-sheep lymphocytotoxic and hemagglutination titers were in the range 8 to 128 and 2 to 32, respectively, in single individuals, which were considerably lower than the anti-pig titers of these individuals. Perfusion of sheep kidneys with human blood reduced the anti-sheep xenoantibody titers to zero as measured by lymphocytotoxic, hemagglutination, and sheep aortic endothelial cell antibody binding assays. The perfused kidneys showed generalised depositions of human IgM and C3c in the vascular tree and focal depositions of C1q and fibrin. Obliteration of capillaries by human platelets and polymorphonuclear cells were observed. Total neutral glycolipid fractions were isolated from sheep intestinal, pancreatic, and kidney tissues. By using a chromatogram binding assay, a monoclonal anti-Forssman antibody identified a single compound with five sugar residues in all organs. Several glycolipid bands were stained in all organs by the Gal(alpha)1-specific lectin I-B4 from Griffonia (Bandeiraea) Simplicifolia. A human AB serum pool showed staining by both IgG and IgM antibodies of the Forssman and Gal(alpha)1-terminating components as well as some other, not structurally identified, components. The Forssman and Gal(alpha)1-reactivity in human sera could be eliminated by immunoadsorption using Forssman and Gal(alpha)1-3Gal-immunoadsorbent columns, respectively. Immunostaining of sheep kidney tissue sections showed the presence of Gal(alpha)1-terminating epitopes by immunoperoxidase and immunogold silver staining techniques. Proximal convoluted tubules showed a strong staining, while thin loops of Henle, collecting ducts, urothelium, and vessels showed a weaker staining. Distal convoluted tubules and thick loops of Henle were completely negative. In summary, human serum contains anti-sheep xenoantibodies reacting mainly with the Forssman and Gal(alpha)1-determinants in sheep tissues and the anti-sheep antibody titers are lower than the corresponding anti-pig titers.