Thin layer chromatograms of ostrich liver neutral glycosphingolipids were immunostained with human sera. In addition to the expected staining of the Forssman pentaglycosylceramide by some sera, more polar and less abundant unknown glycolipids could be stained. Among them, the shortest carbohydrate chain glycolipid was purified and structurally characterized by mass spectrometry, proton NMR and methylation analysis. It was a novel pentaglycosylceramide of the neolactoseries terminated with the Gal(beta1-4)Gal determinant which is not expressed in mammalian species. Human antibodies affinity-purified on a synthetic Gal(beta1-4)Gal(beta1-4)Glc-Sepharose column recognized the newly characterized Gal(beta1-4)Gal-terminated pentaglycosylceramide, and, in addition, longer chain glycolipids. Occurrence of antibodies directed at the Gal(beta1-4)Gal epitope was studied by ELISA on 108 human sera. Anti-Gal(beta1-4)Gal antibodies were predominantly IgM, and their distribution was similar to that of anti-Gal(alpha1-3)Gal and anti-Forssman IgMs. It was concluded that anti-Gal(beta1-4)Gal are natural antibodies, not previously identified in man. They can be considered as xenoantibodies directed at species which express Gal(beta1-4)Gal-terminated carbohydrate chains.

BACKGROUND Patients suffering from focal and segmental glomerulosclerosis (FSGS) and in whom this disease recurs after transplantation are likely to have an active form of the disease and to have a factor(s) (such as, albuminuric factor) present in their blood that alters glomerular permeability for albumin. METHODS We used a sequential 50 and 70% ammonium sulfate (AS) precipitation of plasma from patients with relapsing FSGS and non-FSGS nephrotic syndrome (NS), in addition to plasma from healthy individuals, to obtain both an immunoglobulin (Ig)-rich fraction (50% AS precipitate) and a non-Ig fraction (70% AS supernatant). These fractions were injected intra-arterially or intravenously/intraperitoneally into Sprague-Dawley rats, and proteinuria (g protein/mmol creatinine) was measured for 24 hours. Ig fractions eluted from immunoadsorption onto protein A were also tested. A biochemical characterization was then carried out on the 70% AS supernatants by ultrafiltration on 30 and 50 kD cut-off membranes and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Differentially stained bands were sequenced. RESULTS The 70% AS supernatants from FSGS patients induced proteinuria when injected intra-arterially into normal rats. This effect was significantly different (P < 0.05) from that observed when similar fractions were prepared from the plasma of patients suffering from non-FSGS NS, but was not different from that observed with fractions from healthy individuals and even with an injection of saline solution. Injections of other plasma fractions did not induce a significant proteinuria in the FSGS group versus the non-FSGS NS group. SDS-PAGE of 70% AS supernatants revealed a protein of 23 kD that was more concentrated in AS supernatants from FSGS plasma than the other plasma samples and that was identified by microsequencing as apolipoprotein A1. After sequential ultrafiltration of 70% AS supernatants on 30 and 50 kD cut-off membranes, a second band of 43 kD was found at a much higher concentration in the FSGS samples than in non-FSGS NS and healthy individuals samples. This band is likely to correspond to a candidate albuminuric factor recently reported by another group [1], and was identified by microsequencing as alpha1 acid glycoprotein or orosomucoid. Consequently, purified orosomucoid from the plasma of FSGS, non-FSGS NS patients, or healthy individuals was injected intra-arterially into rats. No differences were found between the proteinuria induced in each group. CONCLUSIONS These data strongly suggest that in vivo injection of material into the rat is not a reliable model for testing plasma fraction activity and that the 43 kD orosomucoid is not likely to be the albuminuric factor.

The heterophile antigens Gal alpha 1-->3Gal and N-glycolylneuraminic acid are the major obstacle to grafting mammal organs, especially from pig, to man. Lack of expression of these common xenoantigens by birds has raised interest in ostrich as a potential organ donor for xenotransplantation. Glycosphingolipids of ostrich liver and kidney were investigated for their carbohydrate determinants. Both organs were found similar in their glycolipid composition with three major species, mono-, di-, and pentaglycosylceramide. The pentaglycosylceramide was characterized as the Forssman antigen. In both organs, the ceramide portion was highly hydroxylated with prevalence of alpha-hydroxylated fatty acids, C18 phytosphingosine in kidney and C18 sphingosine in liver Forssman glycolipid, These data indicate that hydroxylation of kidney glycosphingolipids, which is found in mammals, has been maintained since the divergence of birds from other vertebrates. Characterization of a minor glycolipid as a Forssman tetraglycosylceramide built on the galabiosylceramide core indicates that the Forssman tetraglycosylceramide also exists in vivo. Its precursors, galactosyl- and galabiosylceramide, were characterized in kidney and liver. The Forssman antigen is the third heterophile antigen against which man raises natural antibodies. Its localization in the vascular endothelium and connective tissue makes ostrich an unpromising organ or cell donor for xenotransplantation to man.

Immunization of mice with TNF alpha-activated porcine endothelial cells led to the characterization of two monoclonal antibodies (MAbs), 5F3 and 8A7, specific for porcine VCAM-1. Upon flow cytometry, both antibodies increasingly labeled endothelial cells according to their degree of activation. They bound a band of MW 80 kDa on Western blots of endothelial cells, which is the apparent molecular weight of porcine VCAM-1. It was determined by surface plasmon resonance that the antibodies are directed to different antigenic sites. It was also found that 5F3 competes for binding the antigen with a MAb previously characterized as binding domain 1 of porcine VCAM-1. Subsequently, 5F3, but not 8A7, was found to inhibit the adhesion of human B lymphocyte Ramos cells to porcine endothelial cells in vitro. These antibodies, which do not cross-react with human VCAM-1, might be useful for diagnostic or therapeutic purposes in xenotransplantation.

996 Idiopathic focal and segmental glomerulosclerosis (FSGS) is one of the most frequent causes of nephrotic syndrome (NS). Its immediate relapse after kidney transplantation suggests the presence of a circulating factor (albuminuric factor, AF), able to rapidly alter the glomerular permselectivity for albumin. We showed that removing patients' Ig by adsorption onto Prot-A or immunoaffinity (sheep anti-human Ig) columns stongly (but transiently) decreases the proteinuria after relapse, suggesting a link between AF and Ig. Injection of patients' plasma into rats did not induce significant proteinuria, however, fractions of Prot-A column eluate with a MW lower than that of an intact Ig, inconsistently induced albuminuria. One explanation for this inconsistency could be that AF is sensitive to low pH, another could be that Ig complexed AF is inefficient. We used a sequential 50-70% ammonium sulfate (AS) precipitation of patients' plasma to obtain a semi-purified supernatant with a very low level of Ig and albumin. We have injected fractions obtained from 6 FSGS relapsing patients, 3 non FSGS nephrotic patients after transplantation and 4 healthy individuals, intraaortically into uninephrectomized Sprague Dawley rats. FSGS relapsing fractions were found to induce a selective proteinuria (albumin = 76± 20%), measured as 0.11± 0.03 at day O vs 0.21± 0.06 g albumin/mmol creatinine at 24 h. This effect was significantly different (p < 0.01) from that observed when similar fractions prepared from plasma of patients suffering from non FSGS nephrotic syndrome and receiving the same immunosupressive treatment, were injected (0,14± 0,05 at day O vs 0,16± 0,04 at 24 h). SDS-PAGE electrophoresis of these semi-purified fractions revealed, among several bands shared by FSGS and non FSGS nephrotic syndrome, one protein of 23 Kd which was more abondant in FSGS supernatants than in those from healthy individuals and non-FSGS NS plasma. Our results show that 50-70% AS supernatants from relapsing FSGS lacking in Ig reproducibly induced albuminuria in rats. This test is now being used to predict FSGS relapse following transplantation and biochemical characterisation of the active moiety in the AS supernatant is underway.

Approximately 20 to 30% of patients with idiopathic nephrotic syndrome and focal glomerulosclerosis experience a relapse of their nephrotic syndrome after transplantation. Previously, it has been shown that ex vivo immunoadsorption on protein A strongly (although transiently) reduces proteinuria in relapsing patients. To investigate whether the factor(s) that give rise to albuminuria are bound directly to protein A in the immunoadsorption procedure or are part of a complex with Ig, four patients with relapse of focal glomerulosclerosis presenting as nephrotic syndrome after transplantation were treated, sequentially, using a (non-protein A) anti-Ig affinity column and a protein A column. This study reports that the effect on proteinuria of immunoadsorption using an anti-Ig immunoaffinity column is comparable in its magnitude and kinetics to that of immunoadsorption on protein A. The two procedures were also equally effective in depleting the relapsing patients' plasma of a factor capable of altering the albumin permselectivity of isolated glomeruli in vitro. This study demonstrates for the first time that immunoglobulins have a role in the nephrotic syndrome. In addition, the fact that the two different immunoadsorption procedures both resulted in the removal of the same putative albuminuric factor in these patients and that no autoreactivity of eluted immunoglobulins was observed on human tissues strongly suggests that the factor or factors that may be responsible for immediate nephrotic syndrome after transplantation are bound to an immunoglobulin. However, no firm evidence can be yet provided against a direct role of immunoglobulins.

Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160-1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM160-1A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human beta(1) integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine alpha(2) integrin. Therefore, the NaM160-1A3 antibody was directed against porcine beta(1) integrin and allowed the purification of the complex alpha(2) beta(1), also termed Very Late Antigen 2 (VLA-2). It did not recognize human beta(1) integrin.

81 We have investigated the behavior of transgeneic kidneys for the human DAF molecule perfused with plasmas of hyperimmunized patients against HLA molecules, in an ex vivo perfusion system. Indeed these latter patients could be potential candidates for renal xenograft since their constant high level of anti-HLA antibodies (Ab) renders then unlikely to be considered for an allograft. The use of transgeneic pig organs could be an attractive alternative solution, since they have been shown to be protected against hyperacute rejection, providing these alloantibodies do not cross-react with xenoantigens. We have selected 6 haemodialyzed patients of our waiting list and having anti-HLA Ab reaction on at least 80% of a panel of representative alloreactive cells and stable in time. All patients have accepted to undergo two plasmaphereses allowing us to obtain at least 4 liters of plasma per patient. DAF transgeneic kidneys were perfused for 2.5 hours, after a cold ischemia time of 6 hours, in a perfusion system with a separate cartridge oxygenator circuit and a constant monitoring of the perfusion pressure. Biopsies were performed before and at the end of the perfusion. Samples of plasma were sequentially harvested from the perfusion circuit during the procedure to measure the level of anti-Gal α 1-3 Gal and anti-HLA Ab. Finally, the organs were frozen in order to eluate and study the trapped Ab. Control organs were obtained from non transgeneic animals of the same genetic background and control plasmas were obtained from non immunized individuals. The histology at the end of perfusion, showed essentially a diffuse tubulopathy without any glomcrular or vascular abnormalities for transgenic as well as control organs. Electronic microscopy showed no abnormalities. Anti-Gal Ab decreased quickly to reach a plateau after 30 minutes of perfusion. On the contrary, the level of anti-HLA Ab remained stable, suggesting that these Ab did not cross-react with xeno epitopes and especially SLA molecules. Moreover, kidneys, once perfused, were processed to eluate the bound Ab (acid elution) which were identified as anti-Gal Ab; none of these were anti-HLA Ab. These results indicate that hyperimmunized patients would not be disadvantaged toward xenotransplantation as they are toward allotransplantation.

Since the turn of the century, multiple attempts to transplant animal organs to man have failed, due to hyperacute rejection which irremediably damages the graft within minutes to hours. Interest for xenotransplantation was revived during the lace eighties as a mean to alleviate the shortage of human organs resulting from the success of allotransplantation and the decrease of available human organs. Pig is considered as the more suitable organ donor because of ethical, physiological and economical considerations. However, transplantation between members of discordant (or distantly related) species, such as pig to human, results in hyperacute rejection in unmodified recipients. Hyperacute rejection is associated with codeposition of recipient xenoreactive natural antibodies and complement on the endothelium of the donor organ, and endothelial cell activation. During recent years, a partial deciphering, at the molecular and cellular level, of the complexe mechanisms of xenograft rejection has led to the development of strategies aimed at making pig organs compatible to man. Such strategies include treatment of the host and engineering of transgenic pigs. The control of hyperacute rejection, which may be considered a near reality, opens the way for new research, and makes possible new attempts of xenografts to man with realistic chances of temporary success.

La transplantation d'organes animaux a l'homme a ete tentee a de multiples reprises depuis le debut du siecle mais s'est toujours soldee par des echecs. La xenogreffe avec des organes de porc suscite cependant un tres grand interet. En raison de l'eloignement phylogenique des deux especes, un greffon porcin vascularise ne peut resister a l'intensite du rejet plus de quelques dizaines de minutes. On a recemment identifie les acteurs de la reaction immediate de rejet suraigu : antigenes de l'endothelium du donneur, anticorps naturels et complement de l'hote. Les reactions du rejet secondaire commencent aussi a etre analysees. Parallelement, l'isolement et l'utilisation d'ilots de Langerhans porcins comme alternative a l'insulinotherapie chez les patients diabetiques sont l'objet de recherches intensives. La xenotransplantation necessite non seulement la mise au point de traitements specifiques du rejet, mais aussi la production d'animaux, modifies par transgenese, dont les organes seraient, a terme, toleres par l'homme.

653 About 20-30% of patients with idiopathic nephrotic syndrome (INS) with focal glomerulosclerosis experience a relapse of their INS after transplantation (Tx). The pathogenesis of INS is unknown but several observations suggest an alteration of glomerular permselectivity by plasmatic factor(s). We have previously shown that ex vivo immunoadsorption(IA) on Protein A (Excorim, Sweden) strongly reduces proteinuria (mean 83± 11%), though transiently (28±20days), in 14 relapsing patients(N engl J Med 1994). In order to investigate whether the factor that gives rise to albuminuria are bound directly to Protein A in the IA procedure or as part of a complex with Ig, 4 patients with relapse of focal glomerulosclerosis presenting as INS after Tx were treated using an other anti-Ig support: Ig-Therasorb, Germany. Two of them were also treated by Protein A IA. The unique difference between the 2 procedures was that the treated plasma volume was less important with anti-Ig than Protein A IA (30% less). Despite this difference, the effect on proteinuria of IA using an anti-Ig was superimposable in its magnitude (reduction of 90 ± 7%) and kinetics(return to a threshold of 3g within 10 to 30 days) to that of IA on protein A. The two procedures were also equally effective in depleting the relapsing patients' plasma of a factor capable of altering the albumin permselectivity of isolated glomeruli in vitro (Kidney Int 1997), but there was no significant activity in the eluates. It seems highly improbable that two different and almost specific Ig ligands might both have such an action without an Ig being involved. In addition, the molecular weight of the active fraction after injection into rats and on in vitro test on isolated glomeruli was not compatible with an intact Ig (ie less than 100kd). As there is no specific Ig deposit in the FSGS glomeruli, it is conceivable that traces of autoantibodies, at a too low concentration to be detected, are involved. Alternativelly, Ig may interact with a non renal antigen resulting in a production of active factor. In any case, this study demonstrates that Ig have a direct role in the INS or is a carrier for this factor(s).

Free ceramides were isolated and purified from the metacestodes of Echinococcus multilocularis. Two different fractions were obtained by preparative thin-layer chromatography. Their structure was determined by gas chromatography and electron impact mass spectrometry of trimethylsilylated derivatives. The ceramide with the higher thin-layer chromatographic migration rate contained exclusively erythro-sphinganine associated with saturated C16, C18 and very-long-chain fatty acids (up to C30) and unsaturated C24 fatty acid. The second ceramide contained 90.3% sphingosine and 9.7% sphinganine associated with saturated C16 and C24 and unsaturated C18 and C24 fatty acids. These findings were discussed with regard to the structure and metabolic pathway of neutral and acid glycosphingolipids found in the metacestodes.

Glycosphingolipids are quantitatively minor components of cell lipids. However, their segregation in the outer leaflet of the plasma membrane confers to these membranes specific structural and immunological properties. Current methods of extraction, purification and analysis of blood cell glycolipids are presented. Valuable structural data may be obtained by a combination of chemical and enzymatic degradations with thin-layer chromatography and immunological detection by monoclonal antibodies of known specificity. Examples of physical characterization by Mass Spectrometry and Proton Magnetic Resonance Spectroscopy are also presented.

Study of blood group A- and B-active glycosphingolipid content of the epithelium of the large intestine of 16 strains of inbred rats led to the discovery of two related strains, SHR and WKY, devoid of A-active glycolipids, whereas all strains expressed B-active glycolipids. This finding evidenced a new A/non-A genetic polymorphism in the rat. Blood group A-active glycolipids were isolated from the large intestine of F344 rats and purified by affinity chromatography on immobilized Helix pomatia lectin. Three glycolipid fractions were separated by preparative thin-layer chromatography and characterized by electron-impact mass spectrometry of their permethylated and permethylated-LiAlH4-reduced derivatives. They were identified as a tetraglycosylceramide (A-4), a hexaglycosylceramide (A-6), and a difucosylated heptaglycosylceramide (A-7) with small amounts of monofucosylated octaglycosylceramide (A-8). Methylation analysis and fragmentation indicated that A6 and A-8 had a lacto- and A-7 a neolactotetraosylceramide core, respectively, identical to the core structures of B-6 and B-7 previously characterized in the large intestine of WF rats (Angström et al. (1987) Biochim. Biophys. Acta 926, 79-86). Upon methylation analysis, B-6 and B-7 purified from SHR (A-deficient) and F344 (A-expressing) were found identical to those of WF rats. This result indicated that precursor substrates for the synthesis of A-active glycolipids were available in SHR rats and thus the genetic deficiency of A-active glycolipid expression probably originated in a defect of the termination of the blood group A determinant by the alpha-3-N-acetylgalactosaminyltransferase.

Neutral and acid glycosphingolipids of Echinococcus multilocularis metacestodes that were obtained after intraperitoneal infection of Meriones unguiculatus have been analyzed by thin layer chromatography. Neutral and acid glycosphingolipids accounted for 95% and 5% of total glycosphingolipids, respectively. 12 different fractions were observed in the neutral glycosphingolipids extracts of the parasite. The most important was a monohexosylceramide fraction accounting for 56.4% of neutral glycosphingolipids. 9 different fractions were detected in gangliosides (acid glycosphingolipids). The fact that these glycosphingolipids were specific to the parasite was established by the analysis of different cell populations of the host. Glycosphingolipids were purified from control and parasite-infected gerbil blood cells as well as from peritoneal exudate cells of healthy gerbils after a non-specific immunostimulation. The chromatograms obtained with these extracts were totally different from the parasite. In addition, parasitosis was found to have no effect on the host blood cell glycosphingolipids.