| 1. |
- Kardeby, Caroline, 1989-, et al.
(författare)
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Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα
- 2019
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Ingår i: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 3:3, s. 275-287
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Tidskriftsartikel (refereegranskat)abstract
- Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.
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| 2. |
- Påhlsson, Peter, 1962-, et al.
(författare)
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Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line
- 2001
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 396:2, s. 187-198
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Tidskriftsartikel (refereegranskat)abstract
- Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(▀-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(▀-galactosyl(1-4)a-galactosyl)-sn- glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Gala1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.
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| 3. |
- Aldén, Anna, et al.
(författare)
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HPLC analysis of carbohydrate deficient transferrin isoforms isolated by the Axis-Shield %CDT method
- 2005
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Ingår i: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981 .- 1873-3492. ; 356:1-2, s. 143-146
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Tidskriftsartikel (refereegranskat)abstract
- Background: Carbohydrate-deficient transferrin (CDT) is elevated during prolonged overconsumption of alcohol and CDT is considered to be the most specific biochemical marker for alcohol overconsumption. However, an accurate method for analysing CDT is necessary because the test is frequently used for example in legal matters. Methods: Patient serum samples were analysed with the Axis-Shield %CDT and eluates were pooled together. Transferrin was purified from the pool by affinity chromatography and further analysed with HPLC to determine the ratios of different transferrin isoforms. Results: In the eluates using the Axis-Shield %CDT method, a substantial amount of trisialo transferrin was found, which is generally not considered a CDT isoform. Conclusions: The fact that trisialo transferrin is present may generate falsely elevated CDT results and it could at least partly explain the discrepancy between results of the Axis-Shield %CDT assay and HPLC in routine analysis. © 2005 Elsevier B.V. All rights reserved.
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| 4. |
- Bengtson, Per, 1971-, et al.
(författare)
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Characterization of EBV-transformed B-cells established from an individual homozygously mutated (G329A) in the FUT7 alpha1,3-fucosyltransferase gene
- 2005
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Ingår i: Scand J Immunol. - : Wiley. ; 62:3, s. 251-8
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Tidskriftsartikel (refereegranskat)abstract
- The alpha1,3-fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis x (SLe(x)) on human leukocytes. Recently, individuals were characterized carrying a missense mutation (G329A; Arg110-Gln) in the FUT7 gene encoding this enzyme. The mutated FUT7 construct produced a Fuc-TVII enzyme with impaired activity compared with the wildtype enzyme. Polymorphonuclear granulocytes from an individual carrying this mutation homozygously also showed a reduced expression of SLe(x). In the present study, we have established Epstein-Barr virus-transformed B-cell lines from this individual (SIGN) and from an individual not carrying the mutation (IWO). The cell lines were confirmed to be of B-cell origin by flow cytometry analysis. IWO cells interacted with E-selectin in an in vitro flow chamber analysis whereas SIGN cell did not. However, when SIGN cell was transiently transfected with wildtype FUT7 cDNA, interaction with E-selectin could be restored. Cell surface expression of the SLe(x)-related epitopes recognized by antibodies CSLEX-1, KM-93 and HECA-452 was elevated on IWO cells compared with that on SIGN cells, consistent with a role of these antigens in E-selectin recognition. These cell lines will be useful in further characterization of E-selectin ligands and encourage further studies on the consequences of the FUT7-G329A mutation in vivo.
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| 5. |
- Bengtson, Per, et al.
(författare)
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Polymorphonuclear Leukocytes from Individuals Carrying the G329A Mutation in the α1,3-Fucosyltransferase VII Gene (FUT7) Roll on E- and P-Selectins
- 2002
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Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 169:7, s. 3940-3946
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Tidskriftsartikel (refereegranskat)abstract
- We recently identified several individuals carrying a missense mutation (G329A; Arg110-Gln) in the FUT7 gene encoding fucosyltransferase VII. This enzyme is involved in the biosynthesis of the sialyl Lewis x (Lex) epitope on human leukocytes, which has been identified as an important component of leukocyte ligands for E- and P-selectin. No enzyme activity was measurable in expression studies in COS-7 cells using the mutated FUT7 construct. One of the identified individuals carried this mutation homozygously. Flow cytometry analysis of polymorphonuclear leukocytes (PMN) from this individual showed a nearly complete absence of staining with mAbs directed against sialyl Lex and a diminished staining with an E-selectin IgG chimera. However, staining with P-selectin IgG chimera and Abs directed against P-selectin glycoprotein ligand-1 was not affected by the mutation. PMN from the homozygously mutated individual was further analyzed in an in vitro flow chamber assay. The number of rolling PMN and the rolling velocities on both E- and P-selectin were in the range of PMN from nonmutated individuals. FUT4 and FUT7 mRNA was quantified in PMN isolated from individuals carrying the FUT7 mutation. It was found that PMN from both FUT7 homozygously and heterozygously mutated individuals exhibited an elevated expression of FUT4 mRNA compared with PMN from FUT7 nonmutated individuals. The elevated expression of fucosyltransferase IV was reflected as an increased expression of the Lex and CD65s Ags on PMN from these individuals. The significance of the mutation was supported by transfection of BJAB cells.
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| 6. |
- Bergström, Maria, et al.
(författare)
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Lectin affinity capillary electrophoresis in glycoform analysis applying the partial filling technique
- 2004
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Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 809:2, s. 323-329
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Tidskriftsartikel (refereegranskat)abstract
- The study of protein glycosylation and its significance in biological interactions is a field of growing interest. This work demonstrates a lectin-based separation of protein glycoforms of α1-acid glycoprotein (AGP or orosomucoid) with capillary electrophoresis. Glycoform analysis was performed with a "partial filling technique" with the lectin Concanavalin A (Con A) as affinity ligand. Con A separated human AGP into two peaks, the first peak included AGP glycoforms without biantennary glycans, and the second peak represented the fraction that had one or more biantennary glycans. The applicability of the method was demonstrated with the analysis of AGP from clinical samples and AGP treated with N-glycosidase F. The AGP separation was also used as a reporter system to estimate the dissociation constant (KD) between Con A and a competing sugar. © 2004 Elsevier B.V. All rights reserved.
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| 7. |
- Landberg, Eva, 1966-, et al.
(författare)
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Changes in Glycosylation of Human Bile-Salt-Stimulated Lipase during Lactation
- 2000
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 377:2, s. 246-254
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Tidskriftsartikel (refereegranskat)abstract
- Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption–ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.
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| 8. |
- Liljeblad, Mathias, et al.
(författare)
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A Lectin Immunosensor Technique for Determination of α1-Acid Glycoprotein Fucosylation
- 2001
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 288:2, s. 216-224
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Tidskriftsartikel (refereegranskat)abstract
- The fucosylation of α1-acid glycoprotein (AGP), an acute-phase protein, is known to change in association with inflammatory diseases. Thus, fucosylation of AGP could be a potential diagnostic or prognostic marker. The change in fucosylation has previously been investigated using crossed affinoimmunoelectrophoresis, high-pH anion-exchange chromatography, and lectin ELISA. This study describes a surface plasmon resonance-based affinity biosensor assay for quantification of the fucosylation of AGP. Diluted EDTA plasma or serum was injected directly in a BIACORE 2000 biosensor. AGP was captured on the sensor surface using immobilized antibodies and a fucose-binding lectin from Aleuria aurentia was then used for the detection of fucosylation. The feature of the biosensor makes it possible to determine both the amount of bound AGP and the amount of bound lectin. Using a calibration curve it was possible to obtain a fucosylation ratio that was independent of AGP concentration. The assay was validated against a lectin ELISA and used to follow inflammation in patients with severe burns.
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| 9. |
- Liljeblad, Mathias, et al.
(författare)
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Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance
- 2000
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Ingår i: Glycoconjugate Journal. - 0282-0080 .- 1573-4986. ; 17:5, s. 323-329
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Tidskriftsartikel (refereegranskat)abstract
- It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE® 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.
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| 10. |
- Liljeblad, Mathias, et al.
(författare)
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Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique
- 2002
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Ingår i: Biosensors & bioelectronics. - 0956-5663 .- 1873-4235. ; 17:10, s. 883-891
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Tidskriftsartikel (refereegranskat)abstract
- A method based on a surface plasmon resonance technique for detection of changes in concentration and glycosylation of proteins in cell culture supernatant is described. The method was used to analyze α1-acid glycoprotein (AGP) produced by a human hepatoma cell line (HepG2). Cell culture supernatant was injected to a BIACORE 2000 instrument and AGP was captured on the sensor chip by immobilized antibodies. The captured glycoprotein was then analyzed for content of carbohydrate epitopes using three different lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA). The method was used to analyze changes in concentration and glycosylation of AGP produced by HepG2 cells grown with or without three different cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and transforming growth factor β-1 (TGFβ1). Using the described method it was shown that when HepG2 cells were grown in the presence of IL-6 both AGP concentration and fucosylation increased. When HepG2 cells instead were grown in the presence of TGFβ1 AGP fucosylation increased whereas AGP concentration decreased.
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| 11. |
- Pupkaite, Justina, 1988-
(författare)
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Collagen Hydrogels for Regenerative Medicine
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The need for regenerative therapies to repair damaged or deteriorated organs and tissues, such as heart, skin, and cornea, is rising due to donor shortage and aging of the world’s population. Many proposed regenerative therapeutic approaches include a combination of cells, bioactive compounds, and hydrogels. Although collagen hydrogels have shown a lot of promise in regenerative medicine research, there are still challenges in their design and application strategies. Therefore, this thesis describes the development of novel collagen hydrogel designs for improved use in tissue bonding, cell delivery, and myocardial infarction therapy applications.Firstly, a visible-light crosslinked collagen hydrogel for tissue photobonding was developed. Methacrylated collagen hydrogel was crosslinked using the photoinitiator rose Bengal and visible light. The properties of the resulting hydrogel were tunable by changing the hydrogel composition. Biomimetic and ex vivo skin models were used to demonstrate the ability of the hydrogel to bond tissues whose edges are separated. Additionally, using the hydrogel led to less scarring compared to traditional sutures in a mouse skin incision model.Secondly, collagen was modified with thiol groups to design a hydrogel crosslinked using the thiol-Michael addition click reaction for cell encapsulation and delivery. The hydrogels demonstrated excellent shear-thinning and self-healing properties, allowing for injection after the crosslinking was complete. Additionally, the hydrogels showed minimal swelling and maintained their shape in an aqueous buffer for a prolonged period. Cell encapsulation and delivery using the hydrogels was demonstrated in vitro with mesenchymal stromal cells and endothelial cells.Thirdly, recombinant human collagen III hydrogels were prepared by crosslinking the collagen with EDC and NHS. The hydrogels contained either 1% or 2% collagen. Therapeutic strategies for these hydrogels were investigated, including the timing and dosage of the treatment, in a mouse MI model. Comparing 1% hydrogel injection at a single early time point (3 h) and three time points (3 h, 7 and 14 days) post-MI revealed improved cardiac function, reduced scar size and inflammation, and increased vascularization in the single injection group. Additionally, increasing the collagen III dose to 2% in the hydrogel at a single early time point (3 h) injection did not confer any additional functional improvement compared to 1% and resulted in scar size and vascular density comparable to control (PBS injection). In summary, this work contributes to the development of collagen hydrogel therapies for regenerative medicine by presenting a visible-light crosslinked collagen hydrogel for tissue bonding, a novel click-crosslinked collagen hydrogel with excellent shear-thinning properties for cell delivery, and the use of a recombinant human collagen III hydrogel in post-MI therapy, highlighting the importance of optimizing the timing and dosage of biomaterial therapies.
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| 12. |
- Rydén, Ingvar, et al.
(författare)
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Diagnostic accuracy of a1-acid glycoprotein fucosylation for liver cirrhosis in patients undergoing hepatic biopsy
- 2002
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Ingår i: Clinical Chemistry. - 0009-9147 .- 1530-8561. ; 48:12, s. 2195-2201
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Tidskriftsartikel (refereegranskat)abstract
- Background: Increased fucosylation of serum glycoproteins has previously been reported in patients with liver disease. We analyzed a1-acid glycoprotein (AGP) fucosylation in serum samples from patients investigated for suspected liver disease to evaluate its value as a biochemical marker for liver cirrhosis. Methods: We used a novel lectin immunoassay adapted to the AutoDELFIA system to analyze AGP fucosylation in 261 consecutive patients admitted for liver biopsy at Malm÷ University Hospital in Southern Sweden. The results were compared with histopathologic findings. In addition, AGP fucosylation was compared with other biochemical markers described as useful in the diagnosis of liver cirrhosis. The biochemical markers were compared by ROC curve analysis. Results: AGP fucosylation was significantly (P <0.05) higher in patients with liver cirrhosis (n = 65) than in healthy controls (n = 72), patients with normal histology (n = 29), patients with steatosis only (n = 38), patients with viral or chronic hepatitis without cirrhosis (n = 71), and patients with other liver diseases without histologic signs of cirrhosis (n = 58). By calculating the AGP fucosylation index (AGP-FI = AGP fucosylation/AGP serum concentration), we obtained a high diagnostic accuracy. The areas under the ROC curves for AGP-FI were 0.83 and 0.74 for men and women, respectively, compared with 0.82 for hyaluronic acid and 0.77 for the aspartate aminotransferase/alanine aminotransferase ratio in both men and women. Conclusions: AGP fucosylation appears to be useful in identifying patients with liver cirrhosis among patients investigated for liver disease. The lectin immunoassay showed satisfactory reproducibility and is suitable for routine use in a clinical laboratory.
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| 13. |
- Rydén, Ingvar, et al.
(författare)
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Fucosylation of α1-acid glycoprotein (orosomucoid) compared with traditional biochemical markers of inflammation in recent onset rheumatoid arthritis
- 2002
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Ingår i: Clinica Chimica Acta. - 0009-8981 .- 1873-3492. ; 317:1-2, s. 221-229
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Tidskriftsartikel (refereegranskat)abstract
- Background: Fucosylation of α1-acid glycoprotein (AGP, orosomucoid) has previously been found to be increased in patients with rheumatoid arthritis. Furthermore, the degree of fucosylation has been suggested to reflect disease activity. Therefore, we investigated the fucosylation of AGP in 131 patients (96 women and 35 men) with recent onset rheumatoid arthritis (RA). We compared the results with traditional biochemical markers of inflammation, i.e. plasma concentrations of AGP (P-AGP), and C-reactive protein (P-CRP).Methods: AGP fucosylation measured with a novel lectin enzyme-linked immunosorbent assay (ELISA) was compared with a disease activity score (DAS28) and its components, and with P-AGP, and P-CRP at the time of diagnosis, and at a follow-up visit 1 year later.Results: Both men and women with RA had increased AGP fucosylation compared to healthy individuals. We found a weak correlation between AGP fucosylation and DAS28 only in men. In men with initially increased AGP fucosylation, the level of fucosylation correlated with the change in DAS28 during the first year following diagnosis. Conclusion: We conclude that AGP fucosylation is not superior to traditional markers of disease activity in RA. However, AGP fucosylation may give some additional information to traditional biochemical markers on the disease progression in men.
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| 14. |
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