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  • 1. Aldén, Anna
    et al.
    Ohlson, Sten
    Påhlsson, Peter
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Rydén, Ingvar
    HPLC analysis of carbohydrate deficient transferrin isoforms isolated by the Axis-Shield %CDT method2005Ingår i: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 356, nr 1-2, s. 143-146Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 2.
    Bengtson, Per
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Larson, Cecilia
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Larson, Göran
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Identification of a Missense Mutation (G329A; Arg110→ Gln) in the Human FUT7 Gene2001Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 34, s. 31575-31582Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The human FUT7 gene codes for the α1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLex) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLex and SLex-related epitopes. One individual showed lower levels of SLex expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg110 → Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. TheFUT7 Arg110 is conserved in all previously cloned vertebrate α1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of ≤1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLex and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome but had no history of recurrent bacterial infections or leukocytosis.

  • 3.
    Bengtson, Per
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Larson, Göran
    Department of Clinical Chemistry and Transfusion Medicine, Institute of Laboratory Medicine, Sahlgrenska University Hospital, Göteborg, Sweden .
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Polymorphonuclear Leukocytes from Individuals Carrying the G329A Mutation in the α1,3-Fucosyltransferase VII Gene (FUT7) Roll on E- and P-Selectins2002Ingår i: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 169, nr 7, s. 3940-3946Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 4.
    Bengtson, Per
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Zetterberg, H.
    Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Mellberg, T.
    Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Larson, G.
    Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Characterization of EBV-transformed B-cells established from an individual homozygously mutated (G329A) in the FUT7α1,3-fucosyltransferase gene2005Ingår i: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 62, nr 3, s. 251-258Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The α1,3-fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis x (SLex) 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 SLex. 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 SLex-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.

  • 5.
    Bengtson, Per
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi.
    Zetterberg, H
    Påhlsson, Peter
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi.
    Larson, G
    EBV transformed B cells from an individual homozygously mutated (G329A) in the FUT7 gene do not roll on E- or P-selectins2003Ingår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 17, nr 5, s. A989-A989Konferensbidrag (Övrigt vetenskapligt)
  • 6.
    Bengtson, Per
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Zetterberg, Henrik
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfursion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Mellberg, Tomas
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfursion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Larson, Göran
    Institute of Laboratory Medicine, Department of Clinical Chemistry and Transfursion Medicine, Sahlgrenska University Hospital, Göteborg, Sweden.
    EBV-transformed B-cells from an individual homozygously mutated (G329A) in FUT7 do not roll on E-selectinManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The α1,3 fucosyltransferase VII (Fuc-TVII) is involved in the biosynthesis of E- and P-selectin ligands such as sialyl Lewis 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 was not able to produce an active Fuc-TVII enzyme in transfection studies and polymorphonuclear granulocytes from an individual carrying the mutation homozygously showed severely reduced expression of sialyl Lewis x. In the present study we have established Epstein-Barr virus transformed B-celllines 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 were transfected with wild type FUT7 cDNA interaction with E-selectin could be restored. Cell surface expression of the sLex related epitopes recognized by antibodies CSLEX-1, KM-93 and HECA-452 was elevated on IWO cells compared to SIGN cells, consistent with a role of these antigens in E-selectin recognition. Despite high expression of PSGL-1 neither of the cell lines interacted with P-selectin under flow. These cell lines may be useful in the further characterization ofEselectin ligands and the obtained results encourage further studies on the consequences of the FUT7-G329A mutation in vivo.

  • 7. Bergstrom, M
    et al.
    Ryden, I
    Påhlsson, Peter
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi.
    Ohlson, S
    Separation of protein glycoforms with affinity capillary electrophoresis2003Ingår i: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 13, nr 11, s. 76-Konferensbidrag (Övrigt vetenskapligt)
  • 8.
    Bergstrom, Maria
    et al.
    Linnaeus University.
    Åström, Eva
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Ohlson, Sten
    Linnaeus University.
    Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography2012Ingår i: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 885, s. 66-72Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coil as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked alpha 1-3 to GIcNAc interacted with higher affinity compared to fucose linked alpha 1-6 or alpha 1-4 and the obtained dissociation constants (K-d) were in the range of 10 mu M for all AAL forms. Tetra- and pentasaccharides with fucose in alpha 1-2, alpha 1-3 or alpha 1-4 had K-d values ranging from 0.1 to 7 mM while a large alpha 1-6 fucosylated oligosaccharide had a K-d of about 20 mu M. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.

  • 9. Bergström, Maria
    et al.
    Nilsson, Mikael
    Isaksson, Roland
    Rydén, Ingvar
    Påhlsson, Peter
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Ohlson, Sten
    Lectin affinity capillary electrophoresis in glycoform analysis applying the partial filling technique2004Ingår i: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 809, nr 2, s. 323-329Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 10.
    Gunnarsson, Peter
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Fornander, Louise
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Grenegård, Magnus
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Sialic acid residues play a pivotal role in alpha(1)-acid glycoprotein (AGP)-induced generation of reactive oxygen species in chemotactic peptide pre-activated neutrophil granulocytes2010Ingår i: INFLAMMATION RESEARCH, ISSN 1023-3830, Vol. 59, nr 2, s. 89-95Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have recently shown that terminal sialic acid residues are essential for alpha(1)-acid glycoprotein (AGP)-induced Ca2+ mobilization in neutrophils. The aim of the present study was to establish the importance of sialic acid residues on AGP in modulating human neutrophil functions, with emphasis on the generation of reactive oxygen species (ROS). ROS were measured by luminol-enhanced chemiluminescence in isolated human neutrophils. We found that AGP did not provoke ROS generation in resting or L-selectin presensitized neutrophils. Moreover, AGP did not affect the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS generation, but it slightly suppressed opsonized zymosan-induced responses. However, when the neutrophils were prestimulated with fMLP, the subsequent addition of AGP provoked a marked ROS response. Dose-response studies and time studies revealed that the ROS generating capacity of AGP was highest at a concentration of 0.05 mg/ml and when given 3-10 min after addition of fMLP. A desialylated form of AGP or pretreatment of neutrophils with 3- and 6-sialyllactose caused a substantially lower ROS response in neutrophils prestimulated with fMLP. Our data show that AGP can stimulate a second ROS response in fMLP preactivated neutrophils and that terminal sialic acid residues on AGP play a crucial role in this regard.

  • 11.
    Gunnarsson, Peter
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa. Linköpings universitet, Hälsouniversitetet.
    Levander, Louise
    Linköpings universitet, Institutionen för hälsa och miljö. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Grenegård, Magnus
    Linköpings universitet, Institutionen för medicin och hälsa. Linköpings universitet, Hälsouniversitetet.
    Sialic Acid Dependent and Independent Effects of alpha 1-Acid Glycoprotein (AGP) on Human Platelets2008Ingår i: 2008 Meeting of the Society for Glycobiology, 2008, Vol. 18, nr 11, s. 990-990Konferensbidrag (Övrigt vetenskapligt)
    Abstract [en]

    Objective: We have recently shown that terminal sialic acid residues are essential for α1-acidglycoprotein (AGP)-induced Ca2+ mobilization in neutrophils. The aim of the present studywas to establish the importance of sialic acid-residues on AGP in modulating humanneutrophil functions, with emphasis on the generation of reactive oxygen species (ROS).Material and methods: ROS were measured by luminol-enhanced chemiluminescence inisolated human neutrophils.

    Results: We found that AGP did not provoke ROS generation in resting or L-selectin presensitizedneutrophils. Moreover, AGP did not affect the N-formyl-methionyl-leucylphenylalanine(fMLP)-induced ROS generation but it slightly suppressed opsonized zymosaninducedresponses. However, when the neutrophils were pre-stimulated with fMLP, thefollowing addition of AGP provoked a marked ROS response. Dose-response studies and timestudies revealed that the ROS generating capacity of AGP was maximal at a concentration of0.05 mg/ml and when given 3-10 min after addition of fMLP. A desialylated form of AGP orpre-treatment of neutrophils with 3’- and 6’-sialyllactose caused a substantial lower ROSresponse in neutrophils pre-stimulated with fMLP.

    Conclusions: Our data show that AGP can stimulate a second ROS response in fMLP preactivatedneutrophils and that terminal sialic acid residues on AGP play a crucial role in thisregard.

  • 12.
    Gunnarsson, Peter
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Levander, Louise
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Grenegård, Magnus
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    The acute-phase protein alpha 1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (siglecs)2007Ingår i: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, ISSN 1530-6860, Vol. 21, nr 14, s. 4059-4069Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We studied whether the acute-phase protein alpha1-acid glycoprotein (AGP) induces rises in [Ca2+]i in neutrophils and sought to identify the corresponding AGP receptor (or receptors). We found that AGP elicited a minimal rise in [Ca2+]i in Fura-2-loaded neutrophils, and this response was markedly enhanced by pretreatment with anti-L-selectin antibodies. (The EC50 value of the AGP-induced Ca2+ response was 9 microg/ml.) Activation of phospholipase-C, Src tyrosine kinases, and PI3 kinases proved to be essential for the AGP-mediated increase in [Ca2+]i, whereas the p38 MAPK and SYK signaling pathways were not involved. Furthermore, antibodies against sialic acid binding, immunoglobulin-like lectin 5 (Siglec-5) and oligosaccharide 3'-sialyl-lactose both antagonized the AGP-induced response and caused an immediate increase in [Ca2+]i in anti-L-selectin-treated neutrophils, which indicates a signaling capacity of Siglec-5. We used modified forms of AGP (treated with mild periodate or neuraminidase) to establish the importance of sialic acid residues. The modified forms of AGP caused a much smaller rise in [Ca2+]i than did unaltered AGP. Affinity chromatography confirmed that unchanged AGP, but not neuraminidase-treated AGP, bound to Siglec-5. Our report provides the first evidence for a signaling capacity by AGP through a defined receptor. Pre-engagement of L-selectin significantly enhanced this signaling capacity.

  • 13.
    Gunnarsson, Peter
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Levander, Louise
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Grenegård, Magnus
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    α1-acid glycoprotein (AGP)-induced platelet shape change involvesthe Rho/Rho kinase signalling pathway2009Ingår i: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 102, nr 4, s. 694-703Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    α1-acid glycoprotein (AGP) is an acute-phase protein that contributes to inflammation processes. The role of AGP in platelet activation and thrombosis is, however, largely unknown. Therefore, we thoroughly investigated the effects of AGP on human platelets. Platelets were isolated from healthy volunteers and subsequently exposed to AGP. Platelet responses were monitored as change in light transmission, intracellular calcium concentration, light microscopy and protein phosphorylation by Western blot. We found that AGP induced platelet shape change independently of a second release of adenine nucleotides or thromboxane A2, and that effect was abolished by endotheliumderived platelet inhibitors such as nitric oxide (NO) and adenosine. Furthermore, AGP triggered a minor calcium response and a pronounced Rho/Rho-kinase-dependent increase in Thr696 phosphorylation of myosin phosphatase target subunit 1 (MYPT1). Moreover, the Rho/Rho-kinase inhibitor Y-27632 significantly decreased the AGP-induced shape change. The results also showed that the AGP-elicited shape change was antagonised by pretreatment with low doses of collagen and thrombospondin- 1. Our results describe a novel mechanism by which AGP stimulates platelet shape change via activation of the Rho/Rhokinase signalling pathway. Physiological important platelet inhibitors, such as NO, completely counterbalance the effect of AGP. Hence, the present study indicates that AGP directly contributes to platelet activation, which in turn might have an impact in physiological haemostasis and/or pathological thrombosis.

  • 14. Hedlund, M
    et al.
    Bengtson, Per
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi.
    Påhlsson, Peter
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi.
    Leffler, H
    Galectin mediated tethering and arrest of neutrophils under shear flow conditions2003Ingår i: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 17, nr 5, s. A990-A990Konferensbidrag (Övrigt vetenskapligt)
  • 15.
    Hedlund, Maria
    et al.
    Department of Laboratory Medicine, Division of Microbiology, Immunology and Glycobiologi, Lund University, Lund, Sweden.
    Bengtson, Per
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Leffler, Hakon
    Department of Laboratory Medicine, Division of Microbiology, Immunology and Glycobiologi, Lund University, Lund, Sweden.
    Galectin mediated tethering and arrest of neutrophils under shear flow conditionsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    According to the conventional wisdom leukocyte recruitment to an inflammatory site is initiated by selectin mediated capture and rolling along the activated endothelium. The galectins are members of another family oflectins with affinity for ß-galactosides. Expressed on endothelial cells, galectin-1 and galectin-3 have been proposed to mediate cell-cell interactions during inflammation and tumor cell metastasis, and hence act as an alternative to selectins/integrins under certain circumstances. To begin testing this hypothesis, we examined the interaction of neutrophils -with a galectin-1 or galectin-3 coated surface under shear flow conditions. Both galectins were found to trigger neutrophil arrest in a dose and carbohydrate dependent manner at coating concentrations of ≥ 200 nM, and 1 dynes/cm2 wall shear stress. While, galectin-3 mediated neutrophil arrest was immediate, galectin-1 triggered a brief period of tethering before arrest. Rapidly following arrest neutrophils spread onto the galectin-coated surface. Cell spreading was accompanied by a redistribution of actin filaments, from an initial even staining with FITC-phalloidin to a more peripheral distribution in spread cells. These data suggest that galectin-1 and galectin-3 may act as adhesion molecules capturing and arresting neutrophils at sites knovvn to be less dependent on selectins and ß2integrins. They behave in part like selectins in capturing the neutrophils, but also like the integrins in triggering firm adhesion and cell spreading.

  • 16.
    Kloster Smerud, Hilde
    et al.
    Uppsala University.
    Barany, Peter
    Karolinska Institutet.
    Lindström, Karin
    Karolinska Institutet.
    Fernström, Anders
    Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Hjärt- och Medicincentrum, Njurmedicinska kliniken US. Linköpings universitet, Institutionen för medicin och hälsa, Njurmedicin.
    Sandell, Anna
    Östergötlands Läns Landsting, Hjärt- och Medicincentrum, Njurmedicinska kliniken US.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Fellström, Bengt
    Uppsala University.
    New treatment for IgA nephropathy:  enteric budesonide targeted to the ileocecal region ameliorates proteinuria2011Ingår i: Nephrology, Dialysis and Transplantation, ISSN 0931-0509, E-ISSN 1460-2385, Vol. 26, nr 10, s. 3237-3241Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background. Systemic corticosteroid treatment has been shown to exert some protection against renal deterioration in IgA nephropathy (IgAN) but is not commonly recommended for long-term use due to the well-known systemic side effects. In this study, we investigated the efficacy and safety of a new enteric formulation of the locally acting glucocorticoid budesonide (Nefecon (R)), designed to release the active compound in the ileocecal region. The primary objective was to evaluate the efficacy of targeted release budesonide on albuminuria. less thanbrgreater than less thanbrgreater thanMethods. Budesonide 8 mg/day was given to 16 patients with IgAN for 6 months, followed by a 3-month follow-up period. The efficacy was measured as change in 24-h urine albumin excretion, serum creatinine and estimated glomerular filtration rate (eGFR). less thanbrgreater than less thanbrgreater thanResults. The median relative reduction in urinary albumin excretion was 23% during the treatment period (interquartile range: -0.36 to -0.04, P = 0.04) with pretreatment values ranging from 0.3 to 6 g/24 h (median: 1.5 g/24 h). The median reduction in urine albumin peaked at 40% (interquartile range: -0.58 to -0.15) 2 months after treatment discontinuation. Serum creatinine was reduced by 6% (interquartile range: -0.12 to -0.02; P = 0.003), and eGFR [Modification of Diet in Renal Disease (MDRD)] increased similar to 8% (interquartile range: 0.02-0.16, P = 0.003) during treatment. No major corticosteroid-related side effects were observed. less thanbrgreater than less thanbrgreater thanConclusions. In the present pilot study, enteric budesonide targeted to the ileocecal region had a significant effect on urine albumin excretion, accompanied by a minor reduction of serum creatinine and a modest increase of eGFR calculated by the MDRD equation, while eGFR calculated from Cockcroft-Gault equation and cystatin C was not changed. Enteric budesonide may represent a new treatment of IgAN warranting further investigation.

  • 17.
    Landberg, Eva
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Huang, Yunping
    Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
    Strömqvist, Mats
    AstraZeneca R&D, Umeå, Sweden.
    Mechref, Yehia
    Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
    Hansson, Lennart
    AstraZeneca R&D, Umeå, Sweden.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Novotny, Milos
    Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Changes in Glycosylation of Human Bile-Salt-Stimulated Lipase during Lactation2000Ingår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 377, nr 2, s. 246-254Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 18.
    Landberg, Eva
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Changes in the relative amounts of α2-3 and α2-6 linked sialic acid on free and protein-bound milk oligosaccharides during lactationManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Human milk contains large amounts of oligosaccharides, both in free and protein-bound forms. Sialic acid is a common constituent of milk oligosaccharides and is present α2-3 or α2-6 linked to galactose, α2-6 to N-acetyl glucosamine or α2-6 to N-acetyl galactosamine. High-performance anionexchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used to quantify four major sialylated milk oligosaccharides. The concentrations of the individual oligosaccharides were analyzed in milk from five donors, followed separately during six to nine months of lactation. The oligosaccharides containing sialic acid a2-6 linked to galactose ( 6-sialyl lactose and LSTc) decreased more than tenfold during lactation. In contrast, the concentration of 3-sialyl lactose (3-SL) containing sialic acid a2-3 linked to galactose remained constant during nine months of lactation. Disialyl lacto-Ntetraose (DSLNT) which contain sialic acid linked α2-3 to Gal and α2-6 to GlcNAc decreased approximately threefold during lactation. Lectin ELISA was used to analyze sialic acid on the secreted milk glycoprotein bile-salt-stimulated lipase (BSSL ). There was a gradual decrease in the binding of Sambucus Nigra lectin to BSSL, indicating decreased amount of α2-6 linked sialic acid during lactation. In contrast, binding of Maackia Amurensis lectin remained constant, indicating a constant expression of α2-3 linked sialic acid on BSSL during lactation. This suggests a shift from preferentially 6-linked to 3-linked sialic acid on free and protein-bound oligosaccharides during lactation. The shift may reflect changes in sialyltransferase activities and, on a higher level, changes in the population of mammary epithelial cells. This finding may be of importance for the development of a correct immune response to environmental challenges.

  • 19.
    Landberg, Eva
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Quantitative changes of fucosylated human milk oligosaccharides during lactation in comparison to milk fucosyltransferase activityManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Human milk contains 7-20 g/L of free oligosaccharides. The oligosaccharides show large variations in size and structure. It has been suggested that milk oligosaccharides can prevent pathogenic microorganisms from attaching to the gastrointestinal epithelium by blocking bacterial adhesins. However, the biological role of milk oligosaccharides is far from understood. In this study, the major fucosylated oligosaccharides in milk were followed during six to nine months of lactation. Individual oligosaccharides were separated and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The fucosylated oligosaccharides 2-fucosyl lactose, lacto-N-fucopentaose I and lacto-N-di-fucohexaose I showed decreasing concentrations in milk during lactation. The concentrations of lacto-difucotetraose, lacto-N-fucopentaose II and Ill remained constant, while 3-fucosyl lactose (3-FL) showed increasing concentrations during lactation. The increase of 3-FL was found for all individuals independent of secretor status, but did not correlate to milk fucosyltransferase activity when the product 3-FL was measured separately. Instead all individuals showed a decrease in fucosyltransferase activity during lactation, indicating that fucosyltransferase activity in milk does not reflect the biosynthetic activity in the mammary gland. This study shows that the composition of fucosylated oligosaccharides vary considerably during the first six months of lactation. This may reflect unique biological roles of certain oligosaccharides in the infant's adaptation to the environment.

  • 20.
    Landberg, Eva
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Temperature effects in high-performance anion-exchange chromatography of oligosaccharides1998Ingår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 814, nr 1-2, s. 97-104Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection has been widely used for analysis of mono-, oligo- and polysaccharides. Many factors that affect separation of carbohydrates by HPAEC have been evaluated, however effect of temperature has not been carefully studied. In the present study, neutral and sialylated oligosaccharides from human milk and different types of N-linked oligosaccharides were analysed by HPAEC at temperatures ranging from 13 to 30°C. N-Acetyl neuraminic acid, galacturonic acid and stachyose were also analysed since they have been used as internal standards when analysing various oligosaccharides by HPAEC. All oligosaccharides showed decreased retention times with increased temperature. Even small differences in temperature (i.e. ±5°) resulted in considerable changes in retention times. In addition, individual oligosaccharides showed different relative changes in retention time with increased temperature. By changing the temperature, a switch in elution order of individual oligosaccharides were sometimes found. These results show that retention times relative to an internal standard cannot be used for oligosaccharide identification unless temperature is carefully controlled. Regulation of temperature is also a valuable tool in achieving optimal separation of oligosaccharides by HPAEC.

  • 21.
    Landberg, Eva
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Krotkiewski, Hubert
    Institute of Immunology & Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
    Strömqvist, Mats
    Astra Hässle, Preclinical Research and Development, Umeå, Sweden.
    Hansson, Lennart
    Astra Hässle, Preclinical Research and Development, Umeå, Sweden.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Glycosylation of Bile-Salt-Stimulated Lipase from Human Milk: Comparison of Native and Recombinant Forms1997Ingår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 344, nr 1, s. 94-102Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bile-salt-stimulated lipase (BSSL) is an enzyme present in human milk. BSSL is important for fat digestion in infants. It contains one site for N-glycosylation and a serine/threonine-rich domain which is highly O-glycosylated. Both N- and O-linked sugar chains were studied on native BSSL from three donors and compared to the glycosylation of recombinant BSSL produced in Chinese hamster ovary or mouse fibroblast (C-127) cell lines. The carbohydrate composition of oligosaccharides was mapped using sugar and methylation analyses, enzyme-linked immunosorbant assay, and different separation techniques. Native BSSL was found to be highly glycosylated (19–26%). It contained a high amount of fucosylated oligosaccharides and expressed both Lewis a and Lewis b blood group antigens. None of the recombinant BSSL forms contained fucose. N-linked structures on native BSSL were identified as mainly mono- and disialylated biantennary complex type structures with or without fucose substitution. High-pH anion-exchange chromatography analysis indicated that the recombinant forms of BSSL contained similar types ofN-glycan structures differing mainly in their content of sialic acid and by the absence of fucose residues. Native BSSL contained predominantly large O-linked oligosaccharides. This was in contrast to the recombinant forms of BSSL which contained mainly short typeO-glycans with a high content of sialic acid. Interestingly, the estimated number of O-glycans attached to native BSSL was lower than that for the recombinant forms.

  • 22.
    Landberg, Eva
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk kemi.
    Åström, Eva
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Kågedal, Bertil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Klinisk kemi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk kemi.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Disialo–trisialo bridging of transferrin is due to increased branching and fucosylation of the carbohydrate moiety2012Ingår i: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 414, s. 58-64Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background

    Carbohydrate deficient transferrin (CDT) is used for detection of alcohol abuse and follow-up. High performance liquid chromatography (HPLC) of transferrin glycoforms is highly specific for identification of alcohol abuse, but unresolved disialo- and trisialotransferrin glycoforms sometimes makes interpretation difficult. The cause of this phenomenon is unknown, cannot be explained by genetic variants of transferrin, but seems to be associated with liver disease.

    Methods

    Nineteen serum samples showing di–tri bridging when analyzed by HPLC were collected. Transferrin was purified by affinity chromatography, and N-linked oligosaccharides were released enzymatically. The N-glycans were further analyzed by high performance anion-exchange chromatography with pulsed amperometric detection and MALDI-TOF mass spectrometry.

    Results

    The HPLC-analysis showed three different types of glycoform patterns. The N-glycans from fifteen samples showed patterns with increased number of triantennary structures containing one or two fucose residues. One sample contained an increased amount of triantennary glycans without fucose. Three samples showed a glycosylation pattern similar to normal transferrin.

    Conclusions

    The di–tri bridging phenomenon was associated with alterations in transferrin glycosylation in the majority of cases. Transferrin contained a higher extent of triantennary and often fucosylated N-linked oligosaccharides. These results may be important in future diagnostic approaches to liver diseases.

  • 23.
    Levander, Louise
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Grenegård, Magnus
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Ryden, I
    Kalmar County Hospital.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Effects of alpha 1-acid Glycoprotein Fucosylation on its Ca2+ Mobilizing Capacity in Neutrophils2009Ingår i: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 69, nr 5, s. 412-420Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We recently showed that the acute-phase protein alpha(1)-acid glycoprotein (AGP) induces rises in cytosolic calcium concentration, [Ca2+](i,) in neutrophils through sialic acid dependent interactions with the neutrophil receptors siglec-5 and/or siglec-14. Whereas both siglec-5 and siglec-14 have a relatively broad specificity for sialylated oligosaccharide structures, including both structures with terminal alpha 2-3 or alpha 2-6 linked sialic acid, there is a markedly reduced affinity to the fucosylated epitope sialyl Lewis x (SLe(x)). Increased fucosylation, leading to increased expression of SLe(x) on AGP is commonly associated with inflammatory conditions. In the present study, we investigated whether an increased SLe(x) expression would affect the Ca2+-mobilizing effect of AGP. AGP with elevated fucose content isolated from patients with untreated chronic joint inflammation showed a decreased [Ca2+](i) modulatory effect on neutrophils compared to normally fucosylated AGP. Furthermore a hyperfucosylated AGP form produced by in vitro fucosylation, that consequently had an elevated expression of SLe(x), could not elicit a [Ca2+](i) increase in neutrophils. The role of the carbohydrate portion of AGP in modulating neutrophil responses was further strengthened by showing that synthetic glycoconjugates carrying oligosaccharides with terminal alpha 2-3 or alpha 2-6 linked sialic acid were able to mimic the Ca2+-mobilizing effect of AGP whereas a synthetic glycoconjugate carrying SLe(x) was not. Based on these data, we conclude that increased fucosylation can alter the ability of AGP to induce neutrophil signalling and further supports an important role of the oligosaccharide chains of AGP in the modulation of leukocyte functions during an inflammatory process.

  • 24.
    Levander, Louise
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Gunnarsson, Peter
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Grenegård, Magnus
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Alpha-1-acid glycoprotein interacts with the neutrophilproteins S100A8 and S100A9Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The acute phase protein α1-acid glycoprotein (AGP) has been indicated tobind to neutrophils and to affect neutrophil functions. In the presentinvestigation neutrophil proteins interacting with AGP was studied. Twolow molecular weight proteins were isolated from neutrophil lysates byaffinity chromatography on a column with immobilized AGP. The proteinswere identified as the calcium-binding, myeloid related proteins S100A8and S100A9 using mass spectrometry and Western blot analyses. Theinteraction was further confirmed using a biotin affinity-tagging procedure.The interaction between AGP and S100A8/A9 was sensitive to EDTAindicating a calcium-dependent binding. Desialylation andhyperfucosylation of AGP did not affect its binding to S100A8/A9.

  • 25.
    Levander, Louise
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Gunnarsson, Peter
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Grenegård, Magnus
    Linköpings universitet, Institutionen för medicin och hälsa, Farmakologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Alpha1-acid glycoprotein is a carrier of hydroxyeicosatetraenoic acids – role in calcium mobilization ofpolymorphonuclear granulocytesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    We have previously shown that α1-acid glycoprotein (AGP) induces rises incytosolic calcium concentration, [Ca2+]i, in human polymorphonuclear granulocytes(PMN) and that this effect is enhanced by prior pre-sensitization of PMN with theanti L-selectin antibody DREG-56. AGP is a known carrier of several lipophilicsubstances. This study was designed to determine whether lipids bound to AGP areinvolved in the induction of [Ca2+]i mobilization in PMN. We found that delipidatedAGP elicited a smaller rise in [Ca2+]i in DREG-56 pretreated PMN compared tonative AGP and that lipids extracted from AGP provoked an increase in [Ca2+]i thatwas potentiated by L-selectin pre-engagement with DREG-56. Similarly to whatwas previously found for AGP, the increase in [Ca2+]i produced by the AGP lipidextract involved activation of src-tyrosine kinases and PI3-kinases. The AGP lipidextract was analyzed by high-performance thin layer chromatography. Individualbands were extracted from the plate and their Ca2+ mobilizing activity wasanalyzed. One band contained activity and was further analyzed by electro-spraytandem mass spectrometry. The active band contained a mixture of hydroxyeicosatetraenoic acids (HETEs) with 12-HETE being one of the major components.Pharmacological studies indicated that the AGP lipid extract acted through theleukotriene B4-receptor type II, BLT2. This study supports the hypothesis that someof the immunomodulatory properties that have been attributed to AGP may beconnected to lipids carried by this plasma protein.

  • 26.
    Liljeblad, Mathias
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Ohlson, Sten
    Department of Natural Sciences, University College of Kalmar, Kalmar, Sweden.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Detection of low-molecular-weight heparin oligosaccharides (Fragmin™) using surface plasmon resonance1998Ingår i: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 11, nr 1-6, s. 191-193Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide–protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin™) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore™), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.

  • 27.
    Liljeblad, Mathias
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance2000Ingår i: Glycoconjugate Journal, ISSN 0282-0080, E-ISSN 1573-4986, Vol. 17, nr 5, s. 323-329Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 28.
    Liljeblad, Mathias
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique2002Ingår i: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 17, nr 10, s. 883-891Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 29.
    Liljeblad, Mathias
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Rydén, Ingvar
    Department of Clinical Chemistry, Kalmar County Hospital, Kalmar, Sweden.
    Ohlson, Sten
    Division of Biochemistry/Biotechnology, Department of Chemistry and Biomedical Sciences, University of Kalmar, Kalmar, Sweden.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    A Lectin Immunosensor Technique for Determination of α1-Acid Glycoprotein Fucosylation2001Ingår i: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 288, nr 2, s. 216-224Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 30.
    Olausson, Johan
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Jonsson, Bengt-Harald
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Tibell, Lena
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Detection of a high affinity binding site in recombinantAleuria aurantia lectin2008Ingår i: Glycoconjugate Journal, ISSN 0282-0080, E-ISSN 1573-4986, Vol. 25, nr 8, s. 753-762Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with Kd values in the micromolar range. This would make mushroom lectins ideal candidates to study protein–carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucosecontaining oligosaccharides with Kd values in the nanomolar range. This site could bind to oligosaccharides with fucose linked α1-2, α1-3 or α1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with α1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.

  • 31.
    Olausson, Johan
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Jonsson, Bengt-Harald
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Tibell, Lena
    Linköpings universitet, Institutionen för teknik och naturvetenskap, Medie- och Informationsteknik. Linköpings universitet, Tekniska högskolan.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Production and characterization of a monomeric form and a single-site form of Aleuria aurantia lectin2011Ingår i: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 21, nr 1, s. 34-44Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Lectins have been widely used in structural and functional studies of complex carbohydrates. Lectins usually bind carbohydrates with relatively low affinity but compensate for this by multivalency. When using lectins in different biological and analytical assays the multivalent nature of lectins can sometimes produce unwanted reactions such as agglutination or precipitation of target glycoproteins. The mushroom lectin Aleuria aurantia binds to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. In the present study two forms of recombinant AAL were produced using site-directed mutagenesis. A monomeric form of AAL was produced by exchange of Tyr6 to Arg6, and a monovalent fragment of AAL was produced by insertion of a NdeI restriction enzyme cleavage site and a stop codon in the coding sequence. The AAL forms were expressed as His-tagged proteins in E.coli and purified by affinity chromatography. Binding properties of the two AAL forms were performed using hemagglutination assay, surface plasmon resonance and enzyme-linked lectin assay analyses. Both the monomeric AAL form (mAAL) and the monovalent AAL form (S2-AAL) retained their capacity to bind fucosylated oligosaccharides. However, both constructs exhibited properties that differed from the intact recombinant AAL (rAAL). Monomeric AAL showed similar binding affinities to fucosylated oligosaccharides compared to rAAL but had less hemagglutinating capacity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and, in contrast to rAAL and mAAL, S2-AAL did not bind to sialylated fuco-oligosaccharides such as sialyl-Lex. The study shows that molecular engineering techniques may be a tool for producing lectins with more defined properties such as decreased valency and defined specificities and affinities. This may be very valuable for development of reliable diagnostic and biological assays for carbohydrate analysis.

  • 32.
    Preechaburana, Pakorn
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Tillämpad Fysik. Linköpings universitet, Tekniska fakulteten.
    Erlandsson, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Ytors Fysik och Kemi. Linköpings universitet, Tekniska högskolan.
    Åström, Eva
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Filippini, Daniel
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Tillämpad Fysik. Linköpings universitet, Tekniska högskolan.
    Robinson, Nathaniel D.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Ytors Fysik och Kemi. Linköpings universitet, Tekniska högskolan.
    Disposable total internal reflection fluorescence lab-on-a-chip for medical diagnosis2012Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Lab-on-a-chip detection of fluorescence transduced chemical stimuli is demonstrated using fluidics and optical coupling disposable elements in a configuration compatible with distributed diagnosis.

    Disposable optical elements are designed to separate excitation by total internal reflection using regular glass slides as optical light guide and fluidics support, while high dynamic range image acquisition with consumer cameras complement the platform to support a broad range of responses with a same configuration. Complementary tone mapping procedures are introduced to systematically double the sensitivity for selected range intervals.

    Chemical sensitization to free fucose, a diagnostic marker for liver cirrhosis and several cancer forms, illustrates the platform capabilities for diagnosis targets.

  • 33.
    Påhlsson, Peter
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk kemi.
    Spitalnik, Steven
    Spitalnik, Patrice
    Fantini, Jacques
    Rakotonirainy, Olivier
    Ghardashkhani, Sohbat
    Lindberg, Jan
    Konradsson, Peter
    Larson, Göran
    Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line2001Ingår i: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 396, nr 2, s. 187-198Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 34.
    Påhlsson, Peter
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Strindhall, Jan
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Srinivas, Uppugunduri
    Hospital Pharmacy, University Hospital, Linköping, Sweden.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Role of N-linked glycosylation in expression of E-selectin on human endothelial cells1995Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 25, nr 9, s. 2452-2459Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.

  • 35.
    Rydén, Ingvar
    et al.
    Department of Clinical Chemistry, Kalmar County Hospital, Kalmar, Sweden.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lectin ELISA for Analysis of α1-Acid Glycoprotein Fucosylation in the Acute Phase Response1999Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 45, nr 11, s. 2010-2012Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    No abtract available.

  • 36. Rydén, Ingvar
    et al.
    Påhlsson, Peter
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi.
    Lindgren, Stefan
    Diagnostic accuracy of a1-acid glycoprotein fucosylation for liver cirrhosis in patients undergoing hepatic biopsy2002Ingår i: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 48, nr 12, s. 2195-2201Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 37.
    Rydén, Ingvar
    et al.
    Department of Clinical Chemistry, Kalmar County Hospital, Sweden.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lindgren, Stefan
    Gastroenterology-Hepatology Division, Department of Medicine, Malmö University Hospital, Sweden.
    Increased α1-acid glycoprotein fucosylation in patients with liver cirrhosisManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Background: Increased fucosylation of serum glycoproteins has previously been reported in patients with liver disease. We analyzed α1-acid glycoprotein (AGP) fucosylation in serum samples from patients investigated for suspected liver disease, in order to evaluate its value as a biochemical marker for liver cirrhosis.

    Methods: We used a novel lectin innnunoassay adapted to the Auto-DELFIA system to analyze AGP fucosylation in 261 consecutive patients admitted for liver biopsy at Malmö university hospital in Southern Sweden. The results were compared with histopathological findings. In addition, AGP fucosylation was compared to other biochemical markers described to be useful in the diagnosis of liver cirrhosis. The different biochemical markers were compared by ROC curve analysis.

    Results: AGP fucosylation was significantly higher in patients with liver cirrhosis (n=65) than in nmmal controls (n=72), patients with normal histology (n=29), patients with steatosis only (n=38), patients with viral or chronic hepatitis without ciiThosis (n=71), and patients with other liver diseases without histological signs of cirrhosis (n=58). By calculating an AGP fucosylation index (AGP-FI = AGP fucosylation/AGP serum concentration), a high diagnostic accuracy was obtained. The area under the curve for AGP-FI was 0.83 and 0.74 for men and women respectively, compared to 0.82 for hyaluronic acid, and 0.77 for AST/ALT ratio in both men and women.

    Conclusion: We conclude that 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.

  • 38.
    Rydén, Ingvar
    et al.
    Department of Clinical Chemistry, Kalmar County Hospital, Sweden.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Skogh, Thomas
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Reumatologi. Linköpings universitet, Hälsouniversitetet.
    Fucosylation of α1-acid glycoprotein (orosomucoid) compared with traditional biochemical markers of inflammation in recent onset rheumatoid arthritis2002Ingår i: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 317, nr 1-2, s. 221-229Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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.

  • 39.
    Rydén, Ingvar
    et al.
    Department of Clinical Chemistry, County Hospital, Kalmar, Sweden.
    Skude, Gunnar
    Department of Clinical Chemistry, County Hospital, Kalmar, Sweden.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Glycosylation of α1-acid glycoprotein in inflammatory disease: analysis by high-pH anion-exchange chromatography and concanavalin A crossed affinity immunoelectrophoresis1997Ingår i: Glycoconjugate Journal, ISSN 0282-0080, E-ISSN 1573-4986, Vol. 14, nr 4, s. 481-488Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    High-pH anion-exchange chromatography with pulsed amperometric detection is a highly sensitive technique that can be used for detecting changes in sialylation and fucosylation, as well as different branching patterns of N-linked oligosaccharides in glycoproteins. We examined the N-glycans of α1-acid glycoprotein obtained from twelve patients with various inflammatory conditions with this technique, as well as traditional concanavalin A crossed affinity immunoelectrophoresis. We found the chromatographic profiles of N-glycans in all patients with rheumatoid arthritis to be very similar, but significantly different from normal controls. N-glycans from patients with ulcerative colitis also showed specific alterations in their chromatographic profiles. However, some heterogeneity was found between these patients, perhaps reflecting changes in glycosylation secondary to certain states of the disease, or to medical treatment. We conclude that this technique is useful for detailed mapping of glycosylation changes in α1-acid glycoprotein in clinical samples, and that it may be used to further increase our knowledge about glycosylation changes in response to inflammatory disease.

  • 40.
    Rydén, Ingvar
    et al.
    Department of Clinical Chemistry, Kalmar County Hospital, Sweden.
    Woxler, Per
    Linköpings universitet, Institutionen för hälsa och samhälle. Linköpings universitet, Hälsouniversitetet.
    Bendtsen, Preben
    Linköpings universitet, Institutionen för hälsa och samhälle. Linköpings universitet, Hälsouniversitetet.
    Almer, Sven
    Linköpings universitet, Institutionen för molekylär och klinisk medicin, Gastroenterologi och hepatologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Increased α1-acid glycoprotein fucosylation in patients with high alcohol consumptionManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Background: Changes in glycosylation of senun glycoproteins have been described in different pathologic conditions, and increased fucosylation has previously been reported in patients with liver disease. We analyzed serwn samples from patients admitted to hospital for detoxification of excessive alcohol consumption in order to study α1-acid glycoprotein (AGP) fucosylation and its correlation to other biochemical markers of alcoholism and liver damage.

    Methods: We used a novel lectin innmmoassay to analyze AGP fucosylation (AGP-F) in a prospective study of 21 consecutive patients admitted for treatment at Linköping University Hospital in the Southeast of Sweden. The results were compared with markers conunonly used for detecting alcoholism and liver cirrhosis, such as carbohydrate deficient transferrin, aminotransferase activity, including aspartate aminotransferase/alanine aminotransferase ratio (AST/ALT), and with hyaluronic acid (HA). In addition, ultrasonography of the liver was performed in 16 of the 21 patients.

    Results: AGP-F was significantly higher in the male study patients than in normal controls. In addition, in these patients AGP-F correlated to the levels of AST/ALT-ratio and HA No correlation was found between AGP-F and steatosis of the liver, as indicated by ultrasonography, or between AGP-F and CDT.

    Conclusion: We conclude that AGP-F is increased in men with a high alcohol intake and correlates with AST/ALT ratio and HA, which previously have been found to be indictors of liver cirrhosis. AGP-F should be further evaluated as a potential early indicator ofliver cirrhosis in this patient category.

  • 41.
    Strindhall, Jan
    et al.
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Lundblad, Arne
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för biomedicin och kirurgi, Klinisk kemi. Linköpings universitet, Hälsouniversitetet.
    Interferon-γ enhancement of E-selectin expression on endothelial cells is inhibited by monensin1997Ingår i: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 46, nr 4, s. 338-343Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The expression of E-selectin reaches a maximum 4–6 h after stimulation of human umbilical vein endothelial cells (HUVEC) in vitro with tumour necrosis factor-α (TNF-α) and then declines to basal level within 24 h. If interferon-γ (IFN-γ) is added to the cell culture medium together with TNF-α the surface expression of E-selectin is augmented and prolonged in a synergistic way. The aim of the present study was to investigate if altered protein glycosylation could explain the IFN-γ induced persistent surface expression of E-selectin. SDS–PAGE analysis of HUVEC glycoproteins, metabolically radiolabelled in the carbohydrate portion, indicated that addition of IFN-γ produced an altered protein glycosylation. Lectin blot analysis using the Sambucus nigra agglutinin lectin also indicated differences in protein glycosylation when HUVEC were incubated with IFN-γ/TNF-α compared to TNF-α alone. The kinetics of surface expression of E-selectin were measured using a cell ELISA assay. When HUVEC were incubated with monensin, a potent inhibitor of late Golgi function, together with both TNF-α and IFN-γ, the additive effect of IFN-γ on E-selectin expression was almost abolished. Since monensin is known to affect glycosylation processing, this experiment suggested that the IFN-γ induced change in protein glycosylation might induce the prolonged surface expression of E-selectin. However, when HUVEC were cultured with IFN-γ/TNF-α in the presence of several different inhibitors of N-glycosylation processing, no significant effect of E-selectin expression was observed. Regulation of adhesion molecule expression after activation of endothelial cells is likely to play a pivotal role for the inflammatory response. Further studies are needed to understand the mechanisms underlying this regulation.

  • 42.
    Tengdelius, Mattias
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Gurav, Deepanjali
    Uppsala University, Sweden; Savitri Bai Phule Pune University, India.
    Konradsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Griffith, May
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Medicinska fakulteten.
    Oommen, Oommen P.
    Uppsala University, Sweden.
    Synthesis and anticancer properties of fucoidan-mimetic glycopolymer coated gold nanoparticles2015Ingår i: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 51, nr 40, s. 8532-8535Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Gold nanoparticles coated with fucoidan-mimetic glycopolymers were synthesized that displayed good colloidal stability and promising anti-cancer properties. Fucoidan mimetic glycopolymers on their own were nontoxic, while glycopolymer coated gold nanoparticles displayed selective cytotoxicity to human colon cancer cell lines (HCT116) while it was non-toxic to mouse fibroblast cells (NIH3T3).

  • 43.
    Tengdelius, Mattias
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Lee, Chyan-Jang
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Sensorvetenskap och Molekylfysik. Linköpings universitet, Tekniska högskolan.
    Grenegård, Magnus
    Linköpings universitet, Institutionen för medicin och hälsa, Avdelningen för läkemedelsforskning. Linköpings universitet, Hälsouniversitetet.
    Griffith, May
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Påhlsson, Peter
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för cellbiologi. Linköpings universitet, Hälsouniversitetet.
    Konradsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska högskolan.
    Synthesis and biological evaluation of fucoidan-mimetic glycopolymers through cyanoxyl-mediated free-radical polymerization2014Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 15, nr 7, s. 2359-2368Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The sulfated marine polysaccharide fucoidan has been reported to have health benefits ranging from antivirus and anticancer properties to modulation of high blood pressure. Hence, they could enhance the biological function of materials for biomedical applications. However, the incorporation of fucoidan into biomaterials has been difficult, possibly due to its complex structure and lack of suitable functional groups for covalent anchoring to biomaterials. We have developed an approach for a rapid synthesis of fucoidanmimetic glycopolymer chains through cyanoxyl-mediated free-radical polymerization, a method suitable for chain-end functionalizing and subsequent linkage to biomaterials. The resulting sulfated and nonsulfated methacrylamido alpha-L-fucoside glycopolymers fucoidan-mimetic properties were studied in HSV-1 infection and platelet activation assays. The sulfated glycopolymer showed similar properties to natural fucoidan in inducing platelet activation and inhibiting HSV-1 binding and entry to cells, thus indicating successful syntheses of fucoidan-mimetic glycopolymers.

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