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Background: The use of stem cells in regenerative medicine and transplantation may require grafting of cells that will challenge the recipient's immune system. Our knowledge of tissue antigen expression in human embryonic stem cells (hESC) and during their differentiation is limited, especially regarding histo-blood group AB(O)H antigens., Methods: Nine different hESC lines, and hESC-derived hepatocyte- and cardiomyocyte-like cells, were blood group ABO genotyped and A/B antigen expression was studied by immunohistochemistry., Results: This study reveals, for the first time, that A and B antigens in hESC were expressed according to the ABO genotype and that the antigens had a different cellular/sub-cellular distribution. In addition, several genotype A hESC lines stained positive with one anti-B antibody. Furthermore, studies of hepatocyte- and cardiomyocyte-like cells of different maturation state, originating from a blood group B hESC line, showed that hepatocyte-like cells expressed B antigens whereas cardiomyocyte-like cells were negative., Conclusion: 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.

Background: The use of thin easily revascularized cutaneous nerve autografts, which has been the gold standard, or the alternative use of nerve allografts or artificial grafts for nerve reconstructing have all their pros and cons. Nerve xenotransplantation may offer a potential alternative. In a potential pig to human nerve xenograft transplantation set-up several porcine antigen barriers have to be considered such as carbohydrate antigens system like the blood group A/O, the Galalpha1-3Gal (alphaGal) and the Hanganutziu-Deicher (HD) antigens. The swine leukocyte protein antigens system may also have to bee considered. The knowledge of the antigen expression on pig peripheral nerves is today limited. The present study describes the distribution of glycolipid based carbohydrate xenoantigens in ischiadicus nerve from blood group A and O pigs., Methods: Glycolipid fractions were separated on thin layer chromatography plates and immunostained with human AB sera, biotinylated Griffonia simplicifolia isolectin B4, monoclonal antibodies reacting with the HD antigen and with blood group A antigens based on different core saccharide structures. In addition, the subcellular distribution of alphaGal and HD antigens were studied by light- and electron-microscopical immunohistochemistry. The total amount of neutral glycolipids was 15 mg/g tissue for both blood group A and O nerves with mono-glycosylceramides as the dominating component., Results and Conclusions: The total amount of acidic glycolipids (gangliosides and sulpholipids) was 9 mg/g tissue for both the blood group O and A nerves with sulphatides as the dominating components. Analyses of the glycolipid fractions showed strong expression of both the alphaGal and the HD antigens in nerves from both blood group A and O pigs. In addition, small amounts of blood group A antigens were expressed in nerves from blood group A pigs. Staining of neutral glycolipids from blood group A pigs using monoclonal antibodies reacting with A antigen having different core structures suggested that the A epitope expressed on pig ischiadicus nerves is based on the type 1 core chain structure. Light and electron microscopical studies on the alphaGal and HD-antigen distribution revealed that the neural cells were alphaGal antigen negative. Endothelial cells of blood vessels, and lymphatic and perineural cells expressed alphaGal antigen. Both endothelial cells and myelinized axons revealed positively labelled for the HD antigen.

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.

Objective: There is a relative shortage of donor organs for clinical transplantation, and the use of animal organs is being considered. A clinical trial was performed connecting pig kidneys to the circulation of a dialysis patient., Material and Methods: A pig kidney was, after plasmapheresis, extracorporeally connected to the circulation of a volunteer dialysis patient. The patient was of blood group B and the pig of blood group A., Results: The experiment gave rise to a strong humoral immune response where the xenoantibodies were shown to be of immunoglobulin G (IgG), IgM and IgA immunoglobulin classes, recognizing mainly the Gal alpha1-3Gal epitope and the anti-A antibodies was exclusively of IgM type, recognizing the blood group A trisaccharide. Immunohistological examinations of blood group A pig kidneys revealed that blood group A antigens are located in the distal tubules, thin and thick tubules of Henle and the epithelium of the collecting ducts but absent in proximal tubules, glomeruli, large vessels and capillaries. In the perfused kidney, a patchy destruction of tubular cells was found and these segments stained positive for blood group A antigens and had a codeposition of human IgM antibodies and complement components. Tubular segments which were apparently normal were all negative for blood group A antigens but strongly expressed the Gal alpha1-xenoantigen., Conclusion: In this patient, challenged simultaneously with carbohydrate antigen epitopes representing both the ABO and the xenobarrier, the humoral immune response differed concerning the immunoglobulin classes induced. The low remaining anti-A titre after plasmapheresis was probably sufficient to cause destruction of A antigen-positive tubular cells, while the corresponding Gal alpha1-xenoantigen-positive cells were structurally intact. This case confirms that in future xenotransplantation, matching for the ABO system has to be undertaken in the same way as in human allotransplantation.

The distribution of the Galalpha1-3Gal antigen (Galalpha) in cultured adult porcine islets (API) and fetal porcine pancreatic islet-like cell clusters (ICC) was studied using immunoelectron microscopy. API and ICC were cultured for 1 and 5 days, respectively, and immunogold labeled using human affinity isolated anti-Galalpha1-3Gal antibody, GS-IB4 lectin and antibodies against islet pancreatic hormones, vimentin, and von Willebrand factor. Differentiated endocrine cells were Gala-negative, but, in ICC, some immature endocrine cells were slightly Gala-positive. The Gala-expression in API was much weaker compared to ICC. In both API and ICC, the Gala antigen was expressed on duct epithelial cells, acinar cells, and endothelial cells. In ICC, strong Gala expression was observed on flattened cells covering their surfaces. These cells were identified as centroacinar cells originating from intra-islet ducts. In conclusion, although mature endocrine cells of cultured API and ICC lack the Gala-xenoantigen, several other cellular compounds are strongly Gala positive, which may contribute to xenorejection of these grafts.

Depletion of anti-pig xenoantibodies reduces cell cytotoxicity of human serum to pig endothelial cells and lymphocytes. The aim of this study was to test, in a pig kidney xenoperfusion model, the ability of soluble alpha Gal terminated disaccharides to prevent the hyperacute rejection process in an organ. Porcine kidneys were perfused with whole human blood lacking saccharide and blood supplemented with Gal alpha 1-3GAL, Gal alpha 1-2Gal and lactose. Parameters evaluated were, urine production, renal blood flow, vascular resistance, renal clearance, blood cell counts, xenoantibody titers, complement activation and histopathology. The blood flow was higher in the Gal alpha 1-3Gal (155 +/- 31 ml/min x 100 g-1 kidney tissue) group compared to Gal alpha 1-2Gal (138 +/- 16), lactose (92 +/- 78) and controls (69 +/- 16), When calculated as percent of the blood flow value at 1 min, the blood flow at 30 min was 157% for the Gal alpha 1-3Gal and for 187% the Gal alpha 1-2Gal. The corresponding values for the lactose and control groups were 102% and 74%, respectively. Urine production in the lactose/control groups was lower (0.7 ml/min x 100 g-1 kidney tissue) compared to Gal alpha 1-3Gal (3.0) and Gal alpha 1-2Gal (3.7). Urine sodium excretion was reduced in the lactose/control groups, compared to the Gal alpha alpha 1-groups during the perfusions. An increase in urine potassium excretion was found in the Gal alpha alpha 1-groups while a reduction occurred in the lactose/control experiments. An initial 40-50% reduction in platelet count was observed in all groups while the leukocyte count showed a continuous decrease. Immunohistochemistry revealed less deposition of IgM, IgG, C3 and C1 q in the Gal alpha alpha 1-saccharide groups compared to the lactose/control groups. Soluble Gal alpha a1-disaccharides improved both functional and histological parameters. However, significant pathological changes were still present indicating that this approach to inhibit HAR must be used in combination with additional therapeutic approaches such as solid phase xenoantibody immunoadsorption and blocking of complement activation.

Background: The Gal alpha1-3Gal antigen (Gal alpha) is the primary target for human natural anti-pig xenoantibodies. The presence of Gal alpha has been shown in porcine endothelial cells (ECs) using light microscopy, whereas the expression of Gal alpha in other cell structures in the porcine kidney is only partially characterized., Methods: Immunogold electron microscopy of pig kidney cryosections was performed using Griffonia simplicifolia isolectin B4 and affinity isolated human anti-Gal alpha1-3Gal antibodies., Results: The most intense expression of Gal alpha was found on the apical and basolateral portions of the plasma membrane of the proximal convoluted tubule segments 1 and 2 cells, whereas segment 3 and 4 cells were negative. A strong staining was found in peritubular capillary ECs and in the inner medullary and papillary collecting duct cells. Moderate labeling of ECs and subendothelium was observed in large blood vessels, whereas glomerular ECs reacted weakly. Additionally, glomerular parietal epithelial cells, connecting tubule cells, and some cortical collecting duct cells were labeled. Among interstitial cells, a part of type-1 cells and all type-2 cells were labeled, whereas others were negative., Conclusions: By immune electron microscopy, a detailed information of the Gal alpha antigen distribution in porcine nephrons and blood vessels has been revealed, which clarifies conflicting data obtained by light microscopy. In addition, expression of the Gal alpha antigen in the renal interstitial cells was documented for the first time. These data are of importance for the understanding of xenoantibody-mediated hyperacute rejection, for interpretation of pig kidney xenograft biopsies, and for generating transgenic pigs lacking the Gal alpha epitope.

In the xenotransplantation research field, pig aortic endothelial cells are frequently used in different model systems, e.g., for the study of the xenoantibody-antigen reaction. The Gal(alpha1),3Gal determinant is the major target for human xenoreactive antibodies in pig tissue. Characterisation of the Gal(alpha1)- distribution in pig aortic endothelial cells is thus important for understanding the reaction occurring at the endothelial cells during the xenorejection. We have determined the complete structure of the major Gal(alpha1),3Gal terminated glycolipid, Gal(alpha1),3nLc4Cer. Structural studies were performed on isolated glycosphingolipids by mass spectrometry and NMR spectroscopy. The results show a predominance of the pentasaccharide among the Gal(alpha1)-terminated glycolipids but also the presence of several Gal(alpha1)-terminated glycolipids with extended carbohydrate core chains. Ultrastructural localisation of the Galalpha1-antigen in pig aorta was done by lectin-gold electron microscopic studies of aortic wall sections using the Griffonia simplicifolia isolectin B4. Gal(alpha1)-determinants are predominantly localised on the luminal surface of pig aortic endothelial cells and endothelial cells of vasa vasorum and, to a lesser extent, vascular subendothelium.

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.

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., (© 1996 Munksgaard.)

Under analysis is an experience with the removal of renal transplants from 102 corpses after heart arrest. For the removal two methods were used: nephrectomy by block with the following perfusion (45 donors) and nephrectomy after a preliminary intracorporal intra-aortal perfusion of the kidneys (57 donors). In the first group of observations 47 organs (52%) were good for transplantation (well washed kidneys with good turgor), in the second one--91 organs (80%). The perfusion "in situ" gave both valuable kidneys much more often than the routine technique of taking. The removal of kidneys following the preliminary cold perfusion should be considered the method of choice when working with the non-heart-beating donors.

270 fine needle aspiration and 41 puncture biopsies were fulfilled after 92 cadaveric renal transplantations to diagnose the status of the grafts. With the clinical and ultrasonographic data, the first method proved to be effective in diagnosing acute graft rejection in 79.9% of cases and in determining the status of graft reversibility or irreversibility, but the fine needle aspiration biopsy failed to differentiate the cellular or vascular types of the rejection. The method was also effective in diagnosing the tubular necrosis, acute cyclosporin nephrotoxicity, infectious transplant complications. The paracentetic biopsy essentially supplements the fine needle one and allows one to diagnose chronic cyclosporin nephrotoxicity and chronic transplant rejection. It appears to be the only method revealing the magnitude of graft rejection during oligoanuria caused by ischemic graft lesion. It was concluded that the results of fine needle aspiration biopsy were the most informative within the first postoperative month because later on it becomes difficult to obtain biopsy specimens adequate to interpret the results correctly.

The authors review possibilities of thin needle aspiration biopsy of renal allotransplants in the diagnosis of cyclosporin nephrotoxicity. 151 thin needle aspiration biopsy specimens were explored in 55 patients following kidney transplantation. According to the authors, this method when applied in the early postoperative period may turn an informative one. However, if the time elapsed after operation exceeds 3 months, they recommend performing puncture biopsy of renal transplants.