| 1. |
- Coddens, Annelies, et al.
(författare)
-
Recognition of blood group ABH type 1 determinants by the FedF adhesin of F18-fimbriated Escherichia coli.
- 2009
-
Ingår i: The Journal of biological chemistry. - 0021-9258. ; 284:15, s. 9713-26
-
Tidskriftsartikel (refereegranskat)abstract
- F18-fimbriated Escherichia coli are associated with porcine postweaning diarrhea and edema disease. Adhesion of F18-fimbriated bacteria to the small intestine of susceptible pigs is mediated by the minor fimbrial subunit FedF. However, the target cell receptor for FedF has remained unidentified. Here we report that F18-fimbriated E. coli selectively interact with glycosphingolipids having blood group ABH determinants on type 1 core, and blood group A type 4 heptaglycosylceramide. The minimal binding epitope was identified as the blood group H type 1 determinant (Fucalpha2Galbeta3GlcNAc), while an optimal binding epitope was created by addition of the terminal alpha3-linked galactose or N-acetylgalactosamine of the blood group B type 1 determinant (Galalpha3(Fucalpha2)Galbeta3GlcNAc) and the blood group A type 1 determinant (GalNAcalpha3(Fucalpha2)-Galbeta3GlcNAc). To assess the role of glycosphingolipid recognition by F18-fimbriated E. coli in target tissue adherence, F18-binding glycosphingolipids were isolated from the small intestinal epithelium of blood group O and A pigs and characterized by mass spectrometry and proton NMR. The only glycosphingolipid with F18-binding activity of the blood group O pig was an H type 1 pentaglycosylceramide (Fucalpha2Galbeta3GlcNAc-beta3Galbeta4Glcbeta1Cer). In contrast, the blood group A pig had a number of F18-binding glycosphingolipids, characterized as A type 1 hexaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer), A type 4 heptaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), A type 1 octaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer), and repetitive A type 1 nonaglycosylceramide (GalNAcalpha3(Fucalpha2)Galbeta3GalNAcalpha3-(Fucalpha2)Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer). No blood group antigen-carrying glycosphingolipids were recognized by a mutant E. coli strain with deletion of the FedF adhesin, demonstrating that FedF is the structural element mediating binding of F18-fimbriated bacteria to blood group ABH determinants.
|
|
| 2. |
- Diswall, Mette, 1979, et al.
(författare)
-
Antigen-binding specificity of anti-αGal reagents determined by solid-phase glycolipid-binding assays. A complete lack of αGal glycolipid reactivity in α1,3GalT-KO pig small intestine.
- 2011
-
Ingår i: Xenotransplantation. - : Wiley. - 1399-3089 .- 0908-665X. ; 18:1, s. 28-39
-
Tidskriftsartikel (refereegranskat)abstract
- Diswall M, Gustafsson A, Holgersson J, Sandrin MS, Breimer ME. Antigen-binding specificity of anti-αGal reagents determined by solid-phase glycolipid-binding assays. A complete lack of αGal glycolipid reactivity in α1,3GalT-KO pig small intestine. Xenotransplantation 2011; 18: 28-39. © 2011 John Wiley & Sons A/S. Abstract: Background: αGal-specific lectins, monoclonal and polyclonal antibodies (Abs) are widely used in xenotransplantation research. Immunological assays such as immunohistochemistry, flow cytometry, Western blot and thin layer chromatography are often the only applicable characterization procedures when limited amount of tissue is available and biochemical characterization is impossible. Hence, detailed knowledge of the Ab/lectin carbohydrate-binding specificity is essential. Methods: The binding specificity of human blood group AB serum, three different affinity-purified human polyclonal anti-Gal Ab batches, and two anti-Gal mAb clones (TH5 and 15.101) as well as Griffonia simplicifolia isolectin B4 and Marasmius oreades agglutinin were examined for reactivity with glycolipid fractions isolated from human and pig (wild-type and α1,3GalT-KO) tissues using thin layer chromatogram and microtiter well binding assays. Results: All anti-Gal-specific reagents reacted with the pentaglycosylceramide Galα1,3nLc4, and several 6-12 sugar compounds in wild-type pig kidneys. However, their staining intensity with different αGal antigens varied considerably. Some, but not all, anti-Gal reagents cross-reacted with a pure iGb3 glycolipid reference compound. No reactivity with glycolipids isolated from α1,3GalT-KO pig small intestine or human tissues was found, confirming the specificity of the anti-Gal reagents in those tissues for α1,3Gal-epitopes produced by the α1,3GalT (GGTA1). Conclusions: Different anti-Gal reagents vary in their carbohydrate epitope specificity. Mono-/polyclonal Abs and lectins have different carbohydrate epitope fine specificity toward pig glycolipids as well as purified Galα1,3nLc4, and iGb3. Despite the difference in αGal specificity, all reagents were completely non-reactive with glycolipids isolated from α1,3GalT-KO pig small intestine.
|
|
| 3. |
- Diswall, Mette, 1979
(författare)
-
Biochemical studies of carbohydrate blood group antigens - Carbohydrate phenotype in relation to cellular glycosyltranferases.
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The possibility to alter the cell surface carbohydrate expression by insertion or deletion of glycosyltransferase genes is a powerful technique to study the biological function of selected carbohydrate antigens. However, shifting the equilibrium between competing glycosyltransferases might lead to unexpected phenotypic effects, such as accumulation of other carbohydrate antigens and exposure of antigen structures that normally are cryptic or present in minute amounts on the cell surface. The present work explores the relationship of glycosyltransferase repertoire (genotype) and the resulting cell membrane glycolandscape (phenotype) from a glycosphingolipid perspective using two model systems where glycosyltransferase genes have been deleted or introduced. In addition to contributing to basal glycobiology, the experimental models have the potential to serve clinical purposes in the xenotransplantation and microbial adhesion fields. Glycosylation is not template driven, explaining why the cell membrane carbohydrate expression cannot be predicted from genotyping, but rather requires phenotyping. For phenotyping in this sense, antibody/lectin recognition of cell membrane antigens is insufficient necessitating structural determination. Neutral and acidic glycolipids from tissues and cells were isolated by means of organic solvent extraction and repeated chromatography steps. Individual glycolipid components were purified by high performance liquid chromatography. The antigenic properties of the glycolipids were examined for reactivity with mono- and polyclonal antibodies, lectins and sera on thin layer chromatography plates. Structural elucidation was conducted by the combined use of mass spectrometry and proton nuclear magnetic resonance spectroscopy. Our studies report an indisputable correlation between glycosyltransferase gene setup and the resulting glycolandscape phenotype. The study is also indicative of the renowned complexity of glycosylation, e.g. the glycosyltransferase dependence on the underlying protein/lipid backbone and the species-, individual- and organ-specific glycosyltransferase activity. In addition, we have identified several novel glycosphingolipids in pig tissues for which function and importance remain to be elucidated.
|
|
| 4. |
- Diswall, Mette, 1979, et al.
(författare)
-
Glycolipid studies in small intestine and pancreas of alpha1,3-galactosyltransferase knockout miniature swine: alpha1,3GALT-KO animals lack alphaGAL antigens and contain novel blood group H compounds.
- 2008
-
Ingår i: Transplantation proceedings. - : Elsevier BV. - 0041-1345. ; 40:2, s. 543-6
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knock-out (GalT-KO) pigs have been produced. However, Galalpha1,3Gal (Gal) determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens that might interfere with the human immune response. METHODS: Glycolipids isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig were tested for immune reactivity with antibodies on thin-layer chromatograms after separation by high-performance liquid chromatography, and selected fractions were analysed by proton NMR spectroscopy. RESULTS: Immunostaining using purified human anti-Gal Abs revealed that tissues from WT animals express large amounts of Gal-antigens whereas GalT-KO tissues lacked these antigens. Proton NMR spectroscopy on small intestine fractions revealed both linear and branched nona- and decaglycosylceramides, respectively, with terminal Gal-epitopes. In corresponding GalT-KO fractions, Gal-epitopes seemed to be replaced by terminal alpha1,2fucoses. Two novel branched blood group H compounds was found in the GalT-KO intestine. CONCLUSIONS: The structural complexity of alphaGal-terminating antigens in the WT organs is very high. Knockout of alpha1,3GalT by gene-targeting results in elimination of Gal-determinants. In addition structurally novel alpha1,2fucose-terminated blood group H compounds were identified in the GalT-KO tissue. These compounds are not expected to be recognized by the human immune system.
|
|
| 5. |
- Diswall, Mette, 1979, et al.
(författare)
-
Structural characterization of alpha1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies.
- 2010
-
Ingår i: Xenotransplantation. - : Wiley. - 1399-3089 .- 0908-665X. ; 17:1, s. 48-60
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: alpha1,3-galactosyltranferase knockout (GalT-KO) pigs have been established to avoid hyperacute rejection in GalT-KO pig-to-human xenotransplantation. GalT-KO pig heart and kidney glycolipids were studied focusing on elimination of Gal-antigens and whether novel antigens would appear. Non-human primates are used as pre-clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT-KO hearts were compared with human serum regarding reactivity with pig glycolipids. METHODS: Neutral and acidic glycolipids were isolated from GalT-KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity-purified anti-Gal Ig, anti-blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT-KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy. RESULTS: GalT-KO heart and kidney lacked alpha3Gal-terminated glycolipids completely. Levels of uncapped N-acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x(2) antigen were increased. Human serum antibodies reacted with Gal-antigens and N-glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT-KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT-KO organs which were less abundant or lacking in the corresponding WT tissues. CONCLUSIONS: GalT-KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non-human primates have limitations as a human pre-clinical model for immune rejection studies.
|
|
| 6. |
- Diswall, Mette, 1979, et al.
(författare)
-
Studies on glycolipid antigens in small intestine and pancreas from alpha1,3-galactosyltransferase knockout miniature swine.
- 2007
-
Ingår i: Transplantation. - : Ovid Technologies (Wolters Kluwer Health). - 0041-1337. ; 84:10, s. 1348-56
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knockout (GalT-KO) pigs have been produced. Galalpha1,3Gal determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens. This is the first biochemical study of carbohydrate antigens in GalT-KO pig organs. METHODS: Neutral and acidic glycolipids were isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig. Glycolipid immune reactivity was tested on thin-layer chromatograms. Small intestine neutral glycolipids were separated by high-performance liquid chromatography and selected fractions were analyzed by proton nuclear magnetic resonance spectroscopy. Total gangliosides were quantified on thin-layer chromatograms and in microtiter wells. RESULTS: Using Galalpha1,3nLc4 glycolipid reference, total Galalpha1,3Gal glycolipid antigens in the WT animal was estimated at about 30 microg (small intestine) and 3 microg (pancreas) per gram of dry tissue. Galalpha1,3Gal determinants were not detected in GalT-KO tissues at a detection limit of less than 0.25% (small intestine) and 0.5% (pancreas) of the WT tissues. Isoglobotriaosylceramide (iGb3) was absent but trace amounts of Fuc-iGb3 was found in both GalT-KO and WT pig small intestine. Blood group H type 2 core saccharide compounds were increased in GalT-KO pancreas. Total amount of gangliosides was decreased in GalT-KO tissues. The alpha1,3-galactosyltransferase acceptor, N-acetyllactosamine determinant, was not increased in GalT-KO tissues. Human serum antibodies reacted with WT organ Galalpha1,3Gal antigens and gangliosides, of which the ganglioside reactivity remained in GalT-KO tissues. CONCLUSIONS: Knockout of porcine alpha1,3-galactosyltransferase gene results in elimination of Galalpha1,3Gal-terminated glycolipid compounds. GalT-KO genetic modification did not produce new compensatory glycolipid compounds reactive with human serum antibodies.
|
|
| 7. |
- Diswall, Mette, 1979, et al.
(författare)
-
The alpha1,3GalT knockout/alpha1,2FucT transgenic pig does not appear to have an advantage over the alpha1,3GalT knockout pig with respect to glycolipid reactivity with human serum antibodies.
- 2014
-
Ingår i: Xenotransplantation. - : Wiley. - 1399-3089 .- 0908-665X. ; 21:1, s. 57-71
-
Tidskriftsartikel (refereegranskat)abstract
- The human H-transferase (α2FucT) was introduced in Gal-negative pigs to produce pig organs not only free from Gal-antigens, but also in which the uncapped N-acetyllactosamine precursor had been transformed into non-xenogenic blood group H type 2 compounds. This work is the first descriptive analysis of glycolipids from the GalT-KO/FucT-TG pig. The aim was to investigate the cell membrane antigens in GalT-KO/FucT-TG tissues to explore its efficacy as an organ donor. Also, detailed knowledge on the correlation between the cellular glycosyltransferase configuration and the resulting carbohydrate phenotype expression is valuable from a basic glycobiological perspective.
|
|
| 8. |
- Elsaid, H. O. A., et al.
(författare)
-
Reduced α-galactosidase A activity in zebrafish (Danio rerio) mirrors distinct features of Fabry nephropathy phenotype
- 2022
-
Ingår i: Molecular Genetics and Metabolism Reports. - : Elsevier BV. - 2214-4269. ; 31
-
Tidskriftsartikel (refereegranskat)abstract
- Fabry disease (FD) is a rare genetic lysosomal storage disorder, resulting from partial or complete lack of alpha-galactosidase A (α-GAL) enzyme, leading to systemic accumulation of substrate glycosphingolipids with a broad range of tissue damage. Current in vivo models are laborious, expensive, and fail to adequately mirror the complex FD physiopathology. To address these issues, we developed an innovative FD model in zebrafish. Zebrafish GLA gene encoding α-GAL enzyme presents a high (>70%) homology with its human counterpart, and the corresponding protein has a similar tissue distribution, as evaluated by immunohistochemistry. Moreover, a similar enzymatic activity in different life stages could be demonstrated. By using CRISPR/Cas9 technology, we generated a mutant zebrafish with decreased GLA gene expression, and decreased expression of the specific gene product in the kidney. Mutant animals showed higher plasma creatinine levels and proteinuria. Transmission electron microscopy (TEM) studies documented an increased podocyte foot process width (FPW) in mutant, as compared to wild type zebrafish. This zebrafish model reliably mirrors distinct features of human FD and could be advantageously used for the identification of novel biomarkers and for an effective screening of innovative therapeutic approaches. © 2022 The Authors
|
|
| 9. |
- Le Bas-Bernardet, S, et al.
(författare)
-
Bortezomib, C1-Inhibitor and Plasma Exchange Do Not Prolong the Survival of Multi-Transgenic GalT-KO Pig Kidney Xenografts in Baboons.
- 2015
-
Ingår i: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. - : Elsevier BV. - 1600-6143. ; 15:2, s. 358-70
-
Tidskriftsartikel (refereegranskat)abstract
- Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n=2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.
|
|
| 10. |
- Löfling, Jonas, et al.
(författare)
-
Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants.
- 2008
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 18:7, s. 494-501
-
Tidskriftsartikel (refereegranskat)abstract
- Many microbes bind and adhere via adhesins to host cell carbohydrates as an initial step for infection. Therefore, cell lines expressing Lewis b (Le(b)) determinants were generated as a potential model system for Helicobacter pylori colonization and infection, and their expression of blood group Lewis determinants was characterized. CHO-K1 cells were stably transfected with selected glycosyltransferase cDNAs, and two Le(b) positive clones, 1C5 and 2C2, were identified. Expression of Lewis (Le(a), Le(b), Le(x), and Le(y)) determinants was analyzed by flow cytometry of intact cells, SDS-PAGE/Western blot of solubilized glycoproteins, and thin layer chromatography immunostaining of isolated glycolipids (GL). Binding of H. pylori to cells was examined by microscopy and quantified. Flow cytometry showed that 1C5 and 2C2 were Le(a) and Le(b) positive. 1C5 expressed Le(b) on O-linked, but not N-linked, glycans and only weakly on GLs. In contrast, 2C2 expressed Le(b) on N-, O-glycans, and GLs. Furthermore, both clones expressed Le(a) on N- and O-glycans but not on GLs. 2C2, but not 1C5, stained positively for Le(y) on N-linked glycans and GLs. Both clones, as well as the parental CHO-K1 cells, expressed Le(x) on GLs. A Le(b)-binding H. pylori strain bound to the 1C5 and 2C2 cells. In summary, two glycosyltransferase transfected CHO-K1 cell clones differed regarding Lewis antigen expression on N- and O-linked glycans as well as on GLs. Both clones examined supported adhesion of a Le(b)-binding H. pylori strain and may thus be a useful in vitro model system for H. pylori colonization/infection studies.
|
|
| 11. |
- Mölne, Johan, 1958, et al.
(författare)
-
Blood group ABO antigen expression in human embryonic stem cells and in differentiated hepatocyte- and cardiomyocyte-like cells.
- 2008
-
Ingår i: Transplantation. - 1534-6080. ; 86:10, s. 1407-13
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The use of stem cells in regenerative medicine and transplantation may require grafting of cells that will challenge the recipient's immune system. Our knowledge of tissue antigen expression in human embryonic stem cells (hESC) and during their differentiation is limited, especially regarding histo-blood group AB(O)H antigens. METHODS: Nine different hESC lines, and hESC-derived hepatocyte- and cardiomyocyte-like cells, were blood group ABO genotyped and A/B antigen expression was studied by immunohistochemistry. RESULTS: This study reveals, for the first time, that A and B antigens in hESC were expressed according to the ABO genotype and that the antigens had a different cellular/sub-cellular distribution. In addition, several genotype A hESC lines stained positive with one anti-B antibody. Furthermore, studies of hepatocyte- and cardiomyocyte-like cells of different maturation state, originating from a blood group B hESC line, showed that hepatocyte-like cells expressed B antigens whereas cardiomyocyte-like cells were negative. CONCLUSION: Since clinical stem-cell therapy is likely to be performed with immature progenitor cells, blood group ABO compatibility of donor cells/recipients should be favorable to avoid unnecessary rejection problems caused by ABO incompatibility. The in vitro loss of B antigens in a genotype B hESC line indicates that loss of ABH antigens occurs early during human embryogenesis since these antigens are lacking in adult cardiomyocytes.
|
|
