| 1. |
- Rydberg, Lennart, 1944, et al.
(författare)
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An ELISA technique for quantitation of human xenoantibodies binding to pig cells: application in patients with pig kidneys extracorporeally connected to the circulation.
- 1998
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Ingår i: Xenotransplantation. - 0908-665X. ; 5:2, s. 105-10
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Tidskriftsartikel (refereegranskat)abstract
- A quantitative ELISA technique for determination of human anti-pig xenoantibody number in serum samples has been established using pig lymphocytes and pig/rabbit erythrocytes as target cells and a pool of serum from human blood group AB donors. The number of low affinity antibodies binding to the cells was determined by quantitation following the use of aqueous washing of the cells and separation of bound and unbound antibodies with the phthalate oil method. The efficiency of different soluble Gal(alpha)1-3Gal-terminating di- and tri-saccharides to inhibit antibody binding was tested and found to vary between 70-90% at a saccharide concentration of 10 mg/ml. The assay was used to evaluate the antibody changes in two patients who, after plasmapheresis treatments, had pig kidneys extracorporeally connected to their blood circulation. The number of anti-pig IgM/IgG antibodies bound to each pig lymphocyte were reduced from 5,600/13,200 to 1,300/3,100 in patient 1 and from 1,200/6,500 to 500/2,100 in patient 2 by three consecutive daily plasmapheresis treatments. Although the lymphocytotoxic titers were reduced to very low levels, the antibody numbers still present in the blood of patient 1 caused a hyperacute rejection of the pig kidney. However, the antibody levels in patient 2 did not cause rejection of this kidney during 15 min perfusion time. A strong anti-pig antibody response 3 weeks after the perfusion experiment was found in patient 1 as shown by 27,600/245,300 IgM/IgG molecules bound to pig lymphocytes corresponding to an increase of lymphocytotoxic titer from 8 to 512. The second patient showed a much weaker immune response with 1,400/19,800 IgM/IgG antibodies corresponding to a lymphocytotoxic titer increase from 8 to 32. The use of this quantitation technique enables more accurate investigation of antibody binding to xenogenic target cells than conventional titration techniques.
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| 2. |
- Magnusson, Stefan, et al.
(författare)
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Biotinylated platelets have an impaired response to agonists as evidenced by in vitro platelet aggregation tests.
- 1998
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Ingår i: Thrombosis research. - 0049-3848. ; 89:2, s. 53-8
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Tidskriftsartikel (refereegranskat)abstract
- Biotinylation of platelets, using a water soluble biotin analogue which reacts with primary amines, has been proposed to be a reliable technique for study of in vivo survival of platelets and their subpopulations. The information about the influence of this technique on platelet function has been limited. In the present work we studied the effect of in vitro biotinylation on platelet function and activation. Washed human platelets, at a concentration of 1 x 10(9)/L, were biotinylated with five different concentrations of sulfo-NHS-biotin or NHS-LC-biotin, ranging from 0 to 5 mM. The degree of platelet activation during and after biotinylation was monitored by measuring the externalization of P-selectin, and the platelet function was evaluated by aggregometry. It was observed that biotinylated platelets, in a dose dependent manner, displayed an impaired aggregation response. A slight increase in platelet membrane P-selectin occurred during the labelling procedure.
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| 3. |
- Magnusson, Stefan, et al.
(författare)
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Soluble saccharides block the inhibition of agonist-induced human platelet aggregation observed after in vitro incubation of human platelet-rich plasma with porcine aortic endothelial cells.
- 1998
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Ingår i: Transplant international : official journal of the European Society for Organ Transplantation. - 0934-0874. ; 11:5, s. 345-52
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Tidskriftsartikel (refereegranskat)abstract
- Platelet aggregation is a prominent feature in the hyperacute process of vascularized allografts and xenografts. In a study of extracorporeal connection of pig kidneys to the blood circulation of human volunteers, we observed in one case considerable destruction of human platelets in the pig kidney without signs of hyperacute rejection or microthrombi formation. In the present study, we have investigated the agonist-induced aggregation of human platelets in mixtures with porcine aortic endothelial cells (PAEC). In vitro incubation of human platelet-rich plasma (PRP) with PAEC inhibited platelet aggregation induced by ADP, collagen and arachidonic acid in a time-dependent manner and partially inhibited adrenalin-induced aggregation. Aggregation of the human platelets could not be induced by high concentrations of ADP (20 microM) to overcome the inhibition capacity of the PAEC. The PAEC inhibiting effect could be transferred by the supernatants of PAEC/PRP and PAEC/PPP incubation mixtures. Preincubation of the PAEC with aspirin, but not with NG-methyl-L-Arg, reduced the aggregation inhibitory effect. Control experiments mixing human umbilical vein endothelial cells (HUVEC) and human PRP or mixing porcine PRP and PAEC did not elicit any inhibition of ADP-induced platelet aggregation. The aggregation inhibition effect could partially be blocked by preincubation of PRP with soluble Gal alpha 1-3Gal, Gal alpha 1-3 beta 1-4GlcNAc, lactose, galactose, and glucose, but not by lactosamine, galactosamine, or glucosamine. The Gal alpha 1-3Gal disaccharide was most effective in blocking aggregation inhibition, and to a similar extent as its ability to block the human anti-pig lymphocytotoxicity reaction. In conclusion, the data indicate that PAEC, upon stimulation by human anti-pig xenoantibodies in a nondynamic system, inhibits agonist-induced human platelet aggregation, and that this effect is probably at least partially caused by prostacyclin released from the PAEC.
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| 4. |
- Bengtsson, Anders, 1954, et al.
(författare)
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Extracorporeal ("ex vivo") connection of pig kidneys to humans. III. Studies of plasma complement activation and complement deposition in the kidney tissue.
- 1998
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Ingår i: Xenotransplantation. - 0908-665X. ; 5:3, s. 176-83
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Tidskriftsartikel (refereegranskat)abstract
- The complement system is one of the important factors involved in the hyperacute rejection of xenografts. This report deals with the activation of the complement system in a clinical trial where pig kidneys were extracorporeally connected to two volunteer dialysis patients who were pretreated with plasmapheresis in order to substantially reduce anti-pig xenoantibodies. The clinical data of the perfusion experiments and the patients humoral immune response to pig xenoantigens have been reported in detail (Xenotransplantation 1996; 3:328-339, 340-353). Three consecutive daily plasmapheresis treatments of the patients reduced the plasma complement protein (C3, C4, and C5) concentrations to 8-27% of the baseline values. The perfusion of the pig kidney connected to patient 1 was terminated at 65 min due to graft rejection and this patient was not hemodynamically affected by the experiment. The second experiment was terminated at 15 min due to an anaphylactic like reaction of the patient. In patient 1 a slight reduction of plasma C3, C4, and C5 and an increase of C5a and SC5b-9 occurred, while C3a decreased during the perfusion. Patient 2 had an increase of all complement parameters, most prominent for C4d and SC5b-9, which occurred concomitant with the appearance of the anaphylactic like side effects. In general, plasma levels of PMN elastase, IL6 and IL8 increased in both patients during the perfusion. Immunohistochemical investigation of the kidney tissues revealed deposition of human complement factors C1q, C4c, and C3c in a congruent pattern with the vasculature of the kidney in patient 1. In kidney 2 only trace amounts of C1q and C3c were found. Both kidneys were negative for properdin. Therefore, in this experimental set up with extracorporeal connection of pig kidneys to the human circulation the human complement cascade is activated mainly through the classical pathway.
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| 5. |
- Hallberg, Eva C., 1966, et al.
(författare)
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Chemical and lectin-gold electron microscopical studies of the expression of the Galalpha1-determinant in the pig aorta.
- 1998
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Ingår i: Xenotransplantation. - 0908-665X. ; 5:4, s. 246-56
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Kudryashov, V, et al.
(författare)
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Characterization of a mouse monoclonal IgG3 antibody to the tumor-associated globo H structure produced by immunization with a synthetic glycoconjugate.
- 1998
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Ingår i: Glycoconjugate journal. - 0282-0080. ; 15:3, s. 243-9
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Tidskriftsartikel (refereegranskat)abstract
- Globo H (Fuc alpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 3Gal alpha1 --> 4Galbeta1 --> 4Glc) is a carbohydrate structure that shows enhanced expression in many human carcinomas. From mice immunized with a globo H-KLH (keyhole limpet hemocyanin) synthetic conjugate an IgG3 monoclonal antibody (mAb VK-9) was derived that recognizes the globo H structure. Serological analysis showed that the minimal structure recognized by this mAb was the tetrasaccharide sequence Fuc alpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 3Gal. An isomeric structure with an internal alphaGalNAc linkage was also recognized but less efficiently. mAb VK-9 did not react with many related structures, such as galactosylgloboside, globoside, H type 1, H type 2 blood group structures or fucosyl-gangliotetraosyl ceramide, but did react weakly with globo A ceramide. Not only did mAb VK-9 react with carbohydrate-protein conjugates but it could also recognize globo H-ceramide and human tumor cells expressing globo H. These results suggest that globo H-KLH could be explored as a vaccine in the treatment of carcinoma patients.
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| 7. |
- Olling, A, et al.
(författare)
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Electrospray ionization and collision-induced dissociation time-of-flight mass spectrometry of neutral glycosphingolipids.
- 1998
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Ingår i: Rapid communications in mass spectrometry : RCM. - 0951-4198. ; 12:10, s. 637-45
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Tidskriftsartikel (refereegranskat)abstract
- A series of native naturally occurring neutral glycosphingolipids has been analysed by electrospray ionization tandem mass spectrometry using a hybrid magnetic sector-TOF instrument. The collision-induced dissociation products of precursor ions were detected by an orthogonal acceleration time-of-flight mass spectrometer as the second analyser. Glycosphingolipids, with mono- to hexa-saccharide chain lengths with different ceramide constituents, were studied. The result of electrospray ionization in the positive ion mode generally showed singly charged molecular ions with Na+ as adduct, [M + Na]+. The sensitivity of the electrospray ionization was greatly enhanced by addition of NaCl, LiCl (forming [M + Li]+) or KCl (yielding [M + K]+) to the sample. A comparison between the collision-induced dissociation of precursor molecular ions of monoglycosylceramides, using Na+, Li+ and K+ as adducting species, showed that the intensity of the fragment ions and the extent of the daughter ion fragmentation of the molecular ions, are dependent on the type of adduct used. The daughter ion spectra of Li+ adduct ions showed intense sequence fragment ions, both of the saccharide chain and the ceramide moiety, and were superior to those obtained using Na+ or K+. The collision-induced dissociation spectra of the [M + Li]+ ions, of glycosphingolipids containing di- to hexasaccharides, are also presented. Proposed possible fragments, resulting from the CID of the molecular ions [M + Li]+ of monoglycosylceramides, are shown.
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| 8. |
- Patience, C, et al.
(författare)
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No evidence of pig DNA or retroviral infection in patients with short-term extracorporeal connection to pig kidneys.
- 1998
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Ingår i: Lancet. - 0140-6736. ; 352:9129, s. 699-701
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Tidskriftsartikel (refereegranskat)abstract
- The xenotransplantation of organs and tissues, in particular those from pigs, is viewed as a means to alleviate the shortage of human donor organs and cells available for transplantation and also as a therapy for other diseases. The potential microbiological hazards of xenotransplantation have recently attracted much attention. One concern is over pig endogenous retroviruses (PERV). Until the possible consequences of infection by PERV are better understood it is unlikely that a significant number of porcine xenotransplants will proceed. However, a small number of patients have already been treated with or exposed to living porcine cells or tissue, and investigation of these patients may provide valuable information.
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| 9. |
- Strokan, V, et al.
(författare)
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Characterisation of human natural anti-sheep xenoantibodies.
- 1998
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Ingår i: Xenotransplantation. - 0908-665X. ; 5:2, s. 111-21
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
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