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Halina Miller-Podraza

Publications and source records attributed to Halina Miller-Podraza.

6 recordsLinked to original sources
PubMed2006

N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible.

Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.

Antibodies↗
PubMed2005

Novel binding epitope for Helicobacter pylori found in neolacto carbohydrate chains: structure and cross-binding properties.

Helicobacter pylori is a bacterium that colonizes the stomach of a majority of the global human population causing common gastric diseases like ulcers and cancer. It has an unusually complex pattern of binding to various host glycoconjugates including interaction with sialylated, sulfated, and fucosylated sequences. The present study describes an additional binding epitope comprising the neolacto internal sequence of GlcNAcbeta3-Galbeta4GlcNAcbeta. The binding was detected on TLC plates as an interaction with a seven-sugar ganglioside of rabbit thymus. The glycolipid was purified and characterized as Neu5Gcalpha3Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3-Galbeta4Glcbeta1Cer with less than 10% of the fraction carrying a repeated lacto (type-1) core chain, Galbeta3Glc-NAcbeta3Galbeta3GlcNAcbeta. After stepwise chemical and enzymatic degradation and structural analysis of products the strongest binder was found to be the pentaglycosylceramide GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer, whereas the hexa- and tetraglycosylceramides were less active, and the trihexosylceramide was inactive. Further studies revealed that the terminal GlcNAcbeta of the pentaglycosylceramide may be exchanged for either GalNAcbeta3, GalNAcalpha3, or Galalpha3 without loss of the activity. Calculated minimum energy conformers of these four isoreceptors show a substantial topographical similarity suggesting that this binding is a result of a molecular mimicry. Although the glycoconjugate composition of human gastric epithelial cells is not known in detail it is proposed that repeating N-acetyllactosamine units of glycoconjugates may serve as bacterial attachment sites in the stomach.

Animals↗
PubMed2005

Interaction of Helicobacter pylori with sialylated carbohydrates: the dependence on different parts of the binding trisaccharide Neu5Ac{alpha}3Gal{beta}4GlcNAc.

We have recently shown that binding of Helicobacter pylori to sialylated carbohydrates is dependent on the presence of the carboxyl group and the glycerol chain of Neu5Ac. In this work we investigated the importance of GlcNAc in the binding trisaccharide Neu5Acalpha3Galbeta4GlcNAc and the role of the N-acetamido groups of both Neu5Ac and GlcNAc. An important part of the project was epitope dissection, that is chemical derivatizations of the active carbohydrate followed by binding studies. In addition we used a panel of various unmodified carbohydrate structures in the form of free oligosaccharides or glycolipids. These were tested for binding by hemagglutination inhibition assay, TLC overlay tests, and a new quantitative approach using radiolabeled neoglycoproteins. The studies showed that the N-acetamido group of Neu5Ac is important for binding by H. pylori, whereas the same group of GlcNAc is not. In addition, Fuc attached to GlcNAc, as tested with sialyl-Lewis x, did not affect the binding. Free Neu5Ac was inactive as inhibitor, and Neu5Acalpha3Gal turned out to be active. The binding preference for neolacto structures was confirmed, although one strain also was inhibited by lacto chains. The combined results revealed that an intact Neu5Ac is crucial for the interactions with H. pylori. Parts of Gal also seem to be necessary, whereas the role of the GlcNAc is secondary. GlcNAc does influence binding, however, primarily serving as a guiding carrier for the binding epitope rather than being a part of the binding structure.

Carbohydrate Metabolism↗
PubMed2004

Isolation and partial characterization of Gal alpha-containing polyglycosylceramides from porcine tissues.

Mammalian cell surface carbohydrate antigens are present both as glycoproteins and glycolipids. Of the glycolipids, polyglycosylceramides (PGC) have very long carbohydrate chains extending out from the cell surface. Hereto, Gal alpha-terminating xenoantigens in pig tissues have been identified in glycoproteins and short chain glycolipids but no studies of the complex PGC have been performed. In this communication, we describe the isolation and partial characterization of PGC from pig erythrocytes, small intestinal mucosa, kidney and liver. The mucosa, kidney and liver PGC fractions contained a complex pattern of Gal alpha antigens as shown by immunostaining using the Griffonia Simplicifolia isolectin B(4) while no reactivity was found with the erythrocyte PGC fractions. The mucosa PGC fractions stained strongly for blood group A antigens while the erythrocyte PGC fractions were negative. The presence of Gal alpha-terminating PGC compounds in porcine tissue adds further complexity to the distribution of this xenoantigen. Due to the long carbohydrate chains, PGC will be important targets for the Gal alpha xenoantibodies in pig to human xenotransplantation.

Animals↗
PubMed2003

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures.

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.

Binding Sites↗