Effect of plasma fractions from patients with focal and segmental glomerulosclerosis on rat proteinuria.

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.

A novel pentaglycosylceramide in ostrich liver, IV4-beta-Gal-nLc4Cer, with terminal Gal(beta1-4)Gal, a xenoepitope recognized by human natural antibodies.

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.

A novel synthesis of alpha-D-Galp-(1-->3)-beta-D-Galp-1-O-(CH2)3-NH2, its linkage to activated matrices and absorption of anti-alphaGal xenoantibodies by affinity columns.

Pig organs transplanted into primates are rapidly rejected because of the interaction between Gal alpha(1-->3)Gal epitopes carried by the graft and natural antibodies (anti-alphaGal antibodies) present in the blood of the recipient. This report describes a simplified synthesis of the xenogeneic disaccharide and its linkage to activated gel matrices. The digalactosides alpha-D-Galp-(1-->3)-alpha,beta-D-Galp-OAll were synthesized by the condensation of the trichloroacetimidoyl 2,3,4,6-tetra-O-benzyl-beta-D-galactopyranoside donor with the 3,4-unprotected allyl 2,6-di-O-benzyl-alpha- or beta-D-galactopyranoside acceptor precursor. Deallylation and hydrogenolysis led to the free digalactoside, whereas hydrogenolysis alone resulted in the 1-O-propyl digalactoside. Both products were tested by inhibition ELISA of natural anti-Gal alpha(1-->3)Gal antibodies. The alpha-D-Galp-(1-->3)-beta-D-Galp-OPr was found to be the best inhibitor. Thus, the allyl group of the partially benzylated alpha-D-Galp-(1-->3)-beta-D-Galp-OAll was engineered, via the hydroxy-, the tosyloxy- and the azidopropyl intermediates, into an aminopropyl group amenable to binding to N-hydroxysuccinimide-activated agarose gel matrices in order to obtain specific immunoabsorption columns. Columns made of gel substituted with 5 micromol of disaccharide per milliliter were found efficient for the immunoabsorption of anti-alphaGal antibodies from human plasma.

Interaction of anti-HLA antibodies with pig xenoantigens.

BACKGROUND: Many patients with renal failure are condemned to long-term dialysis with little prospect of transplantation because they are highly sensitized with immunoglobulin G (IgG) directed against class I human leukocyte antigens (HLA) of virtually all donors. Xenotransplantation could represent an attractive solution providing their alloantibodies (alloAb) do not recognize porcine motifs. Hitherto there has been no in vivo demonstration of any cross-reactivity and the objective of this work was to investigate this problem using a technique of extracorporeal pig kidney perfusion as a model of clinical xenografting. METHODS: Pig kidneys were perfused ex vivo with plasma from both a group of highly sensitized patients and healthy individuals. Sequential plasma samples were analyzed for the titer of anti-Galalpha1-3Gal antibody (Ab) (major natural xenoreactive Ab) by enzyme-linked immunosorbent assay and anti-HLA class I Ab against a cell panel. At the end of perfusion, kidneys were perfused with a citric acid buffer to elute bound Ab. RESULTS: Galalpha1-3Gal Ab were shown to decrease rapidly in the plasma (in less than 10 min) and then reached a plateau. A fractional decrease in anti-HLA Ab was also found in some of the perfused plasma samples. Anti-Gal Ab were readily detected in all citric acid perfusates and anti-HLA Ab in 8 of 10. The HLA specificities of eluted Ab were mainly concordant with the originally designated specificities for each patient. CONCLUSION: Anti-HLA class I Ab presumably cross-react with pig class I homologues. However, some plasma samples did not cross-react, suggesting that negatively cross-matched pig kidneys could be identified in the pig population for xenotransplantation in these patients. Further studies are required to precisely describe these cross-reactivities and to understand their functional significance in xenotransplantation.

Forssman penta- and tetraglycosylceramide are xenoantigens of ostrich kidney and liver.

The heterophile antigens Galalpha1-->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.

Characterization of two monoclonal antibodies against porcine VCAM-1.

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.

Characterization of a murine monoclonal antibody specific for swine beta1 integrin.

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.

Antihuman immunoglobulin affinity immunoadsorption strongly decreases proteinuria in patients with relapsing nephrotic syndrome.

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.

Characterization of a polyclonal anti-Galalpha1-3Gal antibody from chicken.

A polyclonal antibody was raised against the Galalpha1-3Gal carbohydrate epitope, which is expressed by all mammals (except man and the closest primate species) by immunizing hens with rabbit erythrocyte membranes. IgY was isolated from egg yolks, and affinity-purified on a Galalpha1-3Gal-Synsorb column. Two percent of the initial IgY fraction was recovered. The specificity of the affinity-purified antibody was characterized by: absorption with human, rabbit and pig erythrocytes; by using Synsorb columns; by inhibition with different saccharides; and by immunostaining of glycolipids separated on thin layer chromatograms. A weak reactivity was found toward blood group B or blood group Pk determinant, depending on the assay system used. Such reactivities were abolished after absorption by the appropriate sorbents, yielding a polyclonal anti-Galalpha1-3Gal antibody with narrow specificity.

Two novel isoneolacto-undecaglycosylceramides carrying Galalpha1-->3Lewis(x) on the 6-linked antenna and N-acetylneuraminic acidalpha2-->3 or Galactose alpha1-->3 on the 3-linked antenna, expressed in porcine kidney.

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Galalpha1-->3Gal, anti-type 2 lactosamine and anti-Lewis(x) antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI'3(alphaGal)2-iso-nLc8Cer, and two were novel structures differing by the number of Galalpha3Lewis(x) determinants: VI3VI'3(alphaGal)2V'3alphaFuc-iso-nLc8, and VI3VI'3(alphaGal)2 V3V'3(alphaFuc)2-iso-nLc8. The single Galalpha3Lewis(x) determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI'3(NeuAc)2-iso-nLc8, and VI3NeuAcVI'3alphaGal-iso-nLc8. A novel structure was discovered presenting a Galalpha3Lewis(x) determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI'3alphaGalV'3alphaFuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney alpha3fucosyltransferase synthesizes the Gala3Lewis(x) determinant, acting on the 6-linked before the 3-linked Galalpha3neolactosamine, and appears unable to synthesize the sialosylated Lewis(x) determinant on neolactoseries glycolipids.

A novel glycosphingolipid expressed in pig kidney: Gal alpha 1-3Lewis(x) hexaglycosylceramide.

Immunodetection of thin layer chromatograms of neutral glycosphingolipids of pig kidney cortex with a polyclonal antibody directed against the Gal alpha 1-3Gal determinant revealed several glycosphingolipids reacting with different intensities. A minor glycosphingolipid was isolated by preparative high performance thin layer chromatography. It was characterized as a type 2 hexaglycosylceramide with the following structure Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer by fast atom bombardment- and desorption-chemical ionization-mass spectrometry, methylation analysis and hydrolysis with alpha-galactosidase followed by immunostaining with an anti-Lewis(x) monoclonal antibody. The proton NMR spectrum was found compatible with the proposed structure. Two other glycosphingolipids carrying the new determinant were partially characterized as an octa- and a branched-dodecaglycosylceramide. The expression of the Gal alpha 1-3 Lewis(x) determinant appeared to be developmentally regulated as it increased with age. The characterization of Gal alpha 1-3Le(x) in pig kidney indicates a new epitope capable of recognition by human natural antibodies in the context of xenotransplantation of pig organs to man. It also adds new members to the family of Le(x)-based glycolipids.

Simultaneous expression by porcine aorta endothelial cells of glycosphingolipids bearing the major epitope for human xenoreactive antibodies (Gal alpha 1-3Gal), blood group H determinant and N-glycolylneuraminic acid.

Glycosphingolipids were isolated from primary cultures of porcine endothelial cells labelled with 14C-galactose or 14C-glucosamine. They were characterized by their mobility on thin layer chromatogram, their sensitivity to exoglycosidases, and their labelling with antibodies. In addition to the major glycosphingolipids, globotetra- and globotriaosylceramide, minor ones were identified as penta- and heptaglycosylceramide of the neolactoseries terminated by either Gal alpha 1-3Gal- (xenoreactive epitope) or Fuc alpha 1-2Gal- (H determinant). Two gangliosides were found, GM3 and GD3, and N-glycolylneuraminic acid was their major sialic acid. Therefore, porcine endothelial cells differ from human endothelial cells by expression of glycosphingolipids that are absent in man: two Gal alpha 1-3Gal-terminated glycolipids recognized by human natural antibodies, and two N-glycolylneuraminic acid-terminated gangliosides which are potent immunogens.

Free ceramides of Echinococcus multilocularis metacestodes.

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.

Structure and genetic polymorphism of blood group A-active glycosphingolipids of the rat large intestine.

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.

Biochemical characterization of blood group-active glycosphingolipids.

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.

Gangliosides of Echinococcus multilocularis metacestodes.

Gangliosides, glycosphingolipids with sialic acid, were found in metacestodes of Echinococcus multilocularis in low quantities. All gangliosides were resolved after preparative high-performance thin layer chromatography into four fractions. Cholera toxin was specifically bound to the major ganglioside, allowing the identification of it as a GM1. Precise structure of the four fractions was determined by sequential degradation by exoglycosidases, gas chromatography, electron impact mass spectrometry and liquid secondary ion-mass spectrometry. Beside GM1, the other fractions were GM3, GD1a and, at a lesser percentage, GM2, all belonging to the same a-ganglio-series. The ceramide part of these parasite gangliosides contained sphingosine associated to unsaturated n24, saturated n24 and n16 fatty acids.