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.

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.

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.

Biochemical study of a recombinant soluble interleukin-2 receptor. Evidence for a homodimeric structure.

A truncated soluble form of the human interleukin-2 receptor p55 chain (T-S-IL-2R) was expressed to high levels in RODENT (mammalian) cells and affinity-purified. Its biochemical behavior was analyzed by polyacrylamide gel electrophoresis (PAGE), gel filtration, and sucrose gradient centrifugation. It migrated as a single 40-kDa band on sodium dodecyl sulfate-PAGE (reducing or nonreducing conditions), whereas it ran as a 80-kDa component on native PAGE or as a 86-kDa component on gel filtration. The combination of gel filtration and density gradient sedimentation gave a Stokes radius of 4.0 nm and a sedimentation coefficient of 3.72 S. The deduced molecular mass was 67 kDa, and the fractional ratio was 1.516. These data therefore indicated that the T-S-IL-2R was secreted as an homodimer of two noncovalently associated 40-kDa subunits. Cross-linking experiments using bifunctional reagents enabled the materialization of the dimeric structure on sodium dodecyl sulfate-PAGE. Stoichiometric binding studies using two monoclonal antibodies (mAbs 33B3.1 and 11H2) reacting with separate epitopes on the p55 chain also strongly supported the dimeric structure. Indeed, there was one binding site for the 33B3.1 mAb (and Fab fragment) per T-S-IL-2R 40-kDa subunit, whereas the 11H2 mAb (or Fab fragment) could bind only half a site per subunit, a result which could only be explained when assuming more than one subunit for the native T-S-IL-2R. Soluble interleukin-2 receptor species were also purified from culture supernatants of either L cells transfected with the full-length p55 cDNA or a normal alloreactive T cell clone. Similar biochemical behavior and reactivities with the two mAbs were found. Finally, cell-surface p55 chains expressed either by pgL21 or 4AS cells bound the 33B3.1 and 11H2 mAbs in a 2:1 ratio, suggesting that the p55 chains are also associated as homodimers when imbedded in the membrane.

Constitutive production of human interleukin for DA cells/leukemia inhibitory factor by human tumor cell lines derived from various tissues.

The cytokine HILDA (human IL for DA cells)/LIF (leukemia inhibitory factor), previously reported to trigger the proliferation of the DA1.a cell line was purified to homogeneity from the culture supernatant of the 5637 bladder carcinoma cell line by using a four step procedure, including cationic exchange chromatography at pH 6, Con A affinity chromatography, reverse phase HPLC, and gel filtration HPLC. The purified radiolabeled protein was analyzed in SDS-PAGE and a single band with a molecular mass of 43 kDa was revealed. The iodinated material was then tested in a radioreceptor assay using the M1 cell line as a second target cell line. The binding of the natural 125I hormone was abrogated in a dose dependent and specific fashion by either natural and rHILDA. The limit of signal detection of displacement for the radiolabeled ligand by unlabeled sample was found to be 0.05 ng/ml. The proliferative DA1.a assay and the M1 radioreceptor assay were used to analyze the HILDA content of 17 human tumor cell line culture supernatants, and 12 among them were spontaneously secreting a detectable level of LIF. The secreted amount of HILDA was largely enhanced by treating the cell line with 30 to 80 nM PMA; in these conditions a 10 to 20x increase in the detected amount was observed. The specificity of the detected signal was reinforced by analyzing the mRNA expression of HILDA in the tumor cell lines. The secreting cell lines were found to express various forms of LIF transcripts with a major specie at 3.8 kb.

Characterization and NH2-terminal amino acid sequence of natural human interleukin for DA cells: leukemia inhibitory factor. Differentiation inhibitory activity secreted by a T lymphoma cell line.

Six human T lymphomas and an NK-like cell line were tested for their ability to produce HILDA, one of the two human growth promoting activities for the DA1.a cells. Among them, the HSB2 cell line turned out to be the only one secreting significant HILDA activity (200-400 units/ml) after activation with 50 nM phorbol myristate acetate. Subclones of the HSB2 cell line were obtained by limiting dilution experiments. One of them (2B3) was found to secrete 1,000-5,000 units/ml of HILDA after phorbol myristate acetate activation in the presence of 10% fetal calf serum and 200-500 units/ml in serum-free conditions. 2B3-HILDA was purified from serum-free conditioned medium by a four-step procedure including fast flow cationic exchange at pH 6, concanavalin A chromatography, reverse-phase high performance liquid chromatography and gel-filtration high performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified radiolabeled cytokine revealed a single band of Mr 43,000, which co-electrophoresed with the biological activity. The NH2-terminal amino acid sequence of the first residues of the protein were determined and found to be similar to the equivalent residues deduced from the molecularly cloned cytokine. Isoelectric point determination revealed some charge heterogeneity of HILDA, which focused to pH 8.5-9 after neuraminidase treatment. Carbohydrate content of the cytokine was studied by deglycosylation experiments which showed that the O-linked oligosaccharides represented 2,000-3,000 and that the N-linked sugars account for half of the apparent molecular weight of HILDA.

Biochemical characterization and purification of HILDA, a human lymphokine active on eosinophils and bone marrow cells.

We previously described a lymphokine termed HILDA (for human interleukin DA) produced by T-lymphocyte alloreactive clones after antigenic stimulation. This factor sustains the growth of a murine IL3-sensitive cell line (DA2). In addition, HILDA is a potent activator of eosinophils and displays a burst-promoting activity on human bone marrow. In the present study, HILDA was purified to homogeneity from T-cell clone supernatant using successively sequential concentration, concanavalin A (ConA) affinity chromatography with differential elution (alpha-D glucopyranoside and alpha-D mannopyranoside), high-performance liquid chromatography (HPLC) gel filtration and reverse-phase HPLC. The pure material appeared as a 38-kd glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions. Biologic activity could be recovered from SDS-PAGE gel slices corresponding to the 38-kd band. We conclude from the specificity of the DA-2 cell line and biochemical characteristics described that this lymphokine is different from other known factors produced by human T lymphocytes.

Preparative two-step purification of human IL-2 by HPLC and hydrophobic affinity chromatography.

Interleukin 2 (IL-2) has been purified by a protocol using gel filtration high performance liquid chromatography (HPLC) and hydrophobic affinity chromatography with blue-trisacryl M. Peripheral blood lymphocytes or tonsillar lymphocytes were stimulated with phytohemagglutinin (PHA). Serum free conditioned medium (CM) containing IL-2, other lymphokines and residual PHA molecules was analyzed after 3 variations of ammonium sulfate (AS) precipitation: (1) precipitation of CM with 50% AS yielded a precipitate containing most of the residual PHA but also a fraction of IL-2. (2) Precipitation with direct 80% AS of crude CM yielded both IL-2 and residual PHA. (3) A double step procedure (50% AS followed by 80% AS) yielded a precipitate containing IL-2 but free of residual lectin. HPLC purification of these various AS-precipitated materials or of lyophilized crude CM yielded 2 peaks with mitogenic activity as assayed with the CTLL2 murine clone or IL-2-dependent human Con A-stimulated lymphoblasts. IFN was easily separated from IL-2 and PHA, but BCGF still copurified with IL-2. Peak I (25 kDa) was enriched 400-fold for IL-2 while peak II (68 kDa) contained the residual PHA. The IL-2-containing fractions eluted from HPLC were further purified by blue-trisacryl M chromatography. The IL-2 eluted with 0.4 M NaCl. The entire protocol (HPLC followed by blue-trisacryl) led routinely to 8000-fold IL-2 enrichment. Preparative HPLC directly applied to lyophilized crude (CM) enriched IL-2 activity 400-fold with yield averaging 60% of the IL-2 input. The final material was free from interferon and IL-1, but BCGF still copurified with IL-2. The 2-step purified material (HPLC and blue-trisacryl) gave 2 bands in SDS-PAGE both of which contained IL-2.