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  • 1.
    Dahle, Charlotte
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Kvarnström, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Ekerfelt, Christina
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Samuelsson, Margareta
    Neurology Unit, Örebro University Hospital, Sweden.
    Ernerudh, Jan
    Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Elevated number of cells secreting transforming growth factor β in Guillain-Barré syndrome2003In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 111, no 12, p. 1095-1104Article in journal (Refereed)
    Abstract [en]

    We used ELISPOT and cell ELISA to study secretion of IL-4, IFN-γ, TGF-β, IL-6, and TNF-α by circulating mononuclear cells during the course of Guillain-Barré syndrome (GBS). Compared to healthy controls, patients with GBS had higher numbers of TGF-β-secreting cells and the number of individuals with myelin-peptide-induced IL-4 and TGF-β secretion was higher in the GBS group. No significant differences were seen concerning the predominantly pro-inflammatory cytokines IFN-γ, IL-6 or TNF-α. Our findings indicate a down-regulatory role for TGF-β and IL-4 in GBS.

  • 2.
    Ekerfelt, Christina
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Faculty of Health Sciences.
    Dahle, Charlotte
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Faculty of Health Sciences.
    Weissert, R.
    Department of Medicine, Division of Neuroimmunology, Karolinska Institute, Stockholm, Sweden.
    Kvarnström, Maria
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Faculty of Health Sciences.
    Olsson, T.
    Department of Medicine, Division of Neuroimmunology, Karolinska Institute, Stockholm, Sweden.
    Ernerudh, Jan
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Faculty of Health Sciences.
    Transfer of myelin-specific cells deviated in vitro towards IL-4 production ameliorates ongoing experimental allergic neuritis2001In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 123, no 1, p. 112-118Article in journal (Refereed)
    Abstract [en]

    A causal role of IL-4 (Th2) production for recovery in experimental allergic neuritis (EAN) was indicated by experiments where Th1-like autoreactive cell populations, taken from the induction phase of the disease, were deviated to extensive secretion of IL-4 in a selective fashion, by ex vivo stimulation with autoantigen in the presence of IL-4. The deviated cells were adoptively transferred to EAN rats at a time just prior to the onset of clinical signs. This treatment ameliorated EAN compared with sham treatment. This therapeutic approach, with generation of autoreactive IL-4-secreting cells ex vivo followed by subsequent adoptive transfer, may become a new selective treatment of organ-specific autoimmune diseases since, in contrast to previous attempts, it is done in a physiological and technically easy way.

  • 3.
    Hellqvist, Eva
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kvarnström, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Söderberg, Anita
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Wrethem, Magnus
    Linköping University, Department of Clinical and Experimental Medicine, Neurology. Linköping University, Faculty of Health Sciences.
    Ernerudh, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Immunology and Transfusion Medicine.
    Rosén, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Myelin protein zero is naturally processed in IgM MGUS B cells: Aberrant triggering of patient T cells2010In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 95, no 4, p. 627-636Article in journal (Refereed)
    Abstract [en]

    Background and Objectives: Monoclonal gammopathy of undetermined significance (MGUS) of IgM isotype is a condition with clonally expanded B cells, recently suggested having an infectious origin. MGUS is frequently associated with polyneuropathy and antibodies against myelin protein zero (P0), whereas the role of the T cells remains largely unknown. Here we have analyzed P0-specific B cells, as antigen-presenting cells, and their capacity to activate T helper cells.

    Design and Methods: We used a well-characterized MGUS-derived B cell line, TJ2, expressing anti-P0 IgM. The ability of TJ2 cells to bind, endocytose, process, and present P0 was investigated by receptor-clustering and immunofluorescence. The activation of P0-specific autologous T cells was studied by measuring IL2 and IFNγ with flow cytometry, immunobeads, and ELISPOT.

    Results: Surface-receptor clustering and endocytosis of receptor-ligand (IgM/P0) complexes were pronounced after P0 exposure. Naturally processed or synthetic P0 peptide (194-208)-pulsed TJ2 cells significantly induced IL2 secretion from autologous T cells compared to control antigen pulsed cells (p<0 .001). The numbers of IFNγ producing T helper cells, including CD4+/CD8+ cells, were also significantly increased (p=0.0152). Affinity-isolated naturally processed myelin peptides were potent IFNγ stimulators for autologous PBMCs, but not for control PBMCs.

    Interpretation and conclusions: We show for the first time that myelin P0 is naturally processed in IgM MGUS B cells, acting as aberrant antigen-presenting cells in activation of patients T helper cells. Our findings cast new light on the important role of autoreactive P0-specific B cells in he induction of the pathogenic T cell responses found in nerve lesions of MGUS patients with peripheral neuropathy.

  • 4.
    Jansson, A.
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Ernerudh, Jan
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Kvarnström, Maria
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Ekerfelt, Christina
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Vrethem, Magnus
    Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Department of Neuroscience and Locomotion, Neurophysiology.
    Elispot assay detection of cytokine secretion in multiple sclerosis patients treated with interferon-β1a or glatiramer acetate compared with untreated patients2003In: Multiple Sclerosis, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 9, no 5, p. 440-445Article in journal (Refereed)
    Abstract [en]

    The mechanisms behind the beneficial effects of interferon-β1a (IFN-β1a) and glatiramer acetate (GA) in the treatment of multiple sclerosis (MS) are still uncertain. Altered cytokine patterns have been suggested including inhibition of proinflammatory cytokines like interferon-γ (IFN-γ) and enhancement of anti-inflammatory cytokines such as interleukin-4 (IL-4). Twenty-nine patients with MS (10 untreated, nine treated with IFN-β1a and 10 with GA) were investigated with elispot of peripheral blood mononuclear cells. Spontaneous and myelin induced (myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG)-14-39 and MOG 63-87) IFN-γ, IL-4, IL-5 and IL-10 secretion was studied. We found a significant reduction of spontaneous IFN-γ, IL-4 and IL-5, but no difference in IL-10 secreting cells in both groups of treated patients compared with the untreated patients. Myelin-specific responses showed a significant decrease of IFN-γ and an increase of IL-5, but no change in IL-4 and IL-10 secreting cells in treated compared with untreated patients. Both treatment groups revealed similar cytokine secretion patterns except for a more pronounced decrease of both spontaneous and MOG 14-39 induced IL-4 secretion in the IFN-β1a treated group. Thus, immunological effects of IFN-β1a and GA were similar showing that disease promoting Th1 (IFN-γ) cells were reduced while the potentially beneficial Th2 response (IL-4) was maintained.

  • 5.
    Kvarnström, Maria
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Mechanisms in inflammatory demyelinating diseases of the nervous system: immunological and methodological aspects2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The diseases studied in this thesis, Guillain-Barré Syndrome (GBS), Multiple Sclerosis (MS) and Polyneuropathy associated with monoclonal gammopathy of uncertain significance (PNMGUS), are of autoimmune origin with myelin components as putative auto antigens. T cells are important for the pathogenesis, as well as the cytokine network and autoantibodies. For all of these diseases, the immunopathogenisis is not fully understood and even if there are treatments available, none of them are curative and there are side effects. Thus there is a need for further clues in the immune mechanisms. Contrary to PNMGUS and MS, GBS is generally self-limiting. The mechanisms of the beneficial effect of interferon-beta (IFN-ß) treatment in MS are not fully understood, (although alterations in the cytokine levels are subject to many reports). In PNMGUS, the proliferation of a monoclonal B cell clone and its antibody production are of great significance, however additional immune mechanisms are also of interest like the role of T cells and the role of B cells as antigen presenting cells.

    In studies of cytokines, frozen cells are often used, sometimes for practical reasons, so also in this thesis. Therefore effects of cryopreservation on cellular expression/secretion of cytokines were studied. The expression before compared to after cryopreservation of IFN-γ, IL-4, IL-5, IL-9, IL-10 and IL-13 was analysed with ELISA, ELISPOT and/or real time RT-PCR We found that the process of cryopreservation and thawing does affect the expression of cytokines, both at the protein and the mRNA level. The most consistent fmding was that expression of IL-4 was generally decreased in spontaneous and auto-antigen/allergen induced expression in cryopreserved cells. Thus, this study points out the importance of investigation of the effects of freezing for each cytokine, stimuli and patient group before using frozen cells in studies of in vitro cytokine secretion.

    The secretion of IL-4, IFN-γ, TGF-ß, IL-6, and TNF-α during the course of GBS was analysed with ELISPOT and cell-ELISA. Our findings indicate a down-regulatory role for TGF-ß and IL-4 in GBS.

    The longitudinal effects over one year of IFN-ß treatment on secretion of IL-4, IFN-γ and IL-10 was analysed with the ELISPOT technique and IL-13 and IL-17 was analysed in cell supernatants with ELISA. A general finding was that surprisingly few changes occurred, and that most changes occurred early (6 weeks - 3 months). However, we found a shift in the cytokine balance towards more IL-4 and IL-10 secretion and/or less IFN-γ secretion during the treatment as the ratios of IL-4/IFN-γ as well as of IL-10/IFN-γ were increased. The interesting pro-inflammatory cytokine IL-17, associated with T cell mediated autoimmunity, has not been previously investigated during IFN-ß treatment in MS and our findings of decreased IL-17 levels after one year of treatment could be a beneficial result of the IFN-ß treatment.

    B cell clones from a patient with PNMGUS were successfully established by isolating B cells with myelin protein zero (P0) coated magnetic beads and subsequently transforming with Epstein-Barr virus (EBV). The clones were characterised and for instance they strongly expressed HLA-DR and CD80, compatible with antigen-presenting properties. The cell lines may provide useful tools in studies of PNMGUS.

    List of papers
    1. Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4: An overall decrease of interleukin-4
    Open this publication in new window or tab >>Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4: An overall decrease of interleukin-4
    2004 (English)In: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 49, no 2, p. 157-168Article in journal (Refereed) Published
    Abstract [en]

    Studies on cytokine expression in blood cells are commonly performed on cryopreserved cells. Previous studies show that cryopreservation affects cytokine expression, but the findings are not consistent. This may be due to divergent effects of freezing on different cytokines, different stimuli, and different patient groups or to the use of different assays in the studies. This study was designed to investigate the effect of freezing on spontaneous, auto-antigen, allergen, and mitogen induced cytokine secretion from peripheral blood mononuclear cells from several groups of patients expressing different cytokine profiles; multiple sclerosis, atopic children, non-atopic children, and pregnant women. The expression of IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13 was analysed with ELISA, ELISPOT and/or real time RT-PCR. Our data provide evidence that the process of cryopreservation and thawing does affect the expression of cytokines, both at the protein and the mRNA level. Moreover, the effect varied among different cytokines, different stimuli, and different patient groups, which partly may be explained by differences in optimal freezing conditions for non-activated and activated cells. An increase of allergen and PHA stimulated IFN-γ secretion in atopic children was found following cryopreservation, but no such increase in auto-antigen induced IFN-γ was seen in MS-patients. The most consistent finding was that expression of IL-4 was generally decreased in spontaneous and auto-antigen/allergen induced expression in cryopreserved cells. In conclusion, this study points out the importance of investigation of the effects of freezing for each cytokine, stimuli and patient group before using frozen cells in studies of in vitro cytokine secretion.

    Keywords
    Cryopreservation, cytokine, IL-4, ELISA, ELISPOT, Real time RT-PCR
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-22345 (URN)10.1016/j.cryobiol.2004.06.003 (DOI)1546 (Local ID)1546 (Archive number)1546 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
    2. Elevated number of cells secreting transforming growth factor β in Guillain-Barré syndrome
    Open this publication in new window or tab >>Elevated number of cells secreting transforming growth factor β in Guillain-Barré syndrome
    Show others...
    2003 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 111, no 12, p. 1095-1104Article in journal (Refereed) Published
    Abstract [en]

    We used ELISPOT and cell ELISA to study secretion of IL-4, IFN-γ, TGF-β, IL-6, and TNF-α by circulating mononuclear cells during the course of Guillain-Barré syndrome (GBS). Compared to healthy controls, patients with GBS had higher numbers of TGF-β-secreting cells and the number of individuals with myelin-peptide-induced IL-4 and TGF-β secretion was higher in the GBS group. No significant differences were seen concerning the predominantly pro-inflammatory cytokines IFN-γ, IL-6 or TNF-α. Our findings indicate a down-regulatory role for TGF-β and IL-4 in GBS.

    Keywords
    Cytokines, ELISPOT, Guillain-Barré, IL-4, Syndrome, TGF-ß
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-46369 (URN)10.1111/j.1600-0463.2003.apm1111204.x (DOI)
    Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
    3. IFN-ß treatment in multiple sclerosis: Longitudinal effects on secretion of IFN-γ, IL-4, IL-10, IL-13 and IL-17
    Open this publication in new window or tab >>IFN-ß treatment in multiple sclerosis: Longitudinal effects on secretion of IFN-γ, IL-4, IL-10, IL-13 and IL-17
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Proinflammatory cytokines like IFN-γ and TNF-α seem to have disease-promoting roles in multiple sclerosis (MS) whereas anti-inflammatory cytokines like IL-10 and TGF-ß may downregulate the disease. IFN-ß treatment reduces the frequency and severity of relapses, however, the mechanisms of action for IFN-ß are only partly understood and modulation of cytokine secretion could be one possible explanation for the therapeutic effects. The IFN-ß products approved for the treatment of MS differ in their composition and effects, and recently differences in effects on cytokine secretion were reported. Peripheral blood was collected from 25 patients with MS, both IFN-ß1a and IFN-ß1b treated, before onset of treatment and after 6 weeks, 3 months, 6 months and one year. Spontaneous as well as myelin specific secretion of IL-4, IFN-γ and IL-10 was analysed with the ELISPOT technique. PHA stimulated secretion of IL-13 and IL-17 was analysed in cell supernatants with ELISA. A general finding was that surprisingly few changes occurred, and that most changes occurred early (6 weeks - 3 months). We found a shift in the cytokine balance towards more IL-4 and IL-10 secretion and/or less IFN-γ secretion during the treatment as the ratios of IL-4!IFN-y as well as of IL-10/IFN-γ were increased. The interesting pro-inflammatory cytokine IL-17, that has been associated with T-cell mediated autoimmunity, has not been previously investigated during IFN-ß treatment in MS. Our findings of decreased IL-17 levels after one year of treatment, following an increase in early treatment, could be a beneficial result of the IFN-ß treatment. Further we noticed differences in effects on cytokines of IFN-ß1a and IFN-ß1b respectively; the latter seemed to have more effects on cytokine secretion.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84278 (URN)
    Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2013-08-29Bibliographically approved
    4. Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)
    Open this publication in new window or tab >>Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)
    Show others...
    2002 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 127, no 2, p. 255-262Article in journal (Refereed) Published
    Abstract [en]

    Monoclonal expansion of B cells and plasma cells, producing antibodies against ‘self’ molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström’s macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P0, using beads coated with P0. P0-coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein–Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5+ positive, although the expression declined in vitro over time. The anti-P0 antibodies were of IgM-λ type. The antibodies belonged to the VH3 gene family with presence of somatic mutations. The IgM reacted with P0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5+ clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P0-specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25117 (URN)10.1046/j.1365-2249.2002.01739.x (DOI)9550 (Local ID)9550 (Archive number)9550 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
  • 6.
    Kvarnström, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Ekerfelt, Christina
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Vrethem, Magnus
    Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Department of Neuroscience and Locomotion, Neurophysiology. Linköping University, Faculty of Health Sciences.
    Ernerudh, Jan
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    IFN-ß treatment in multiple sclerosis: Longitudinal effects on secretion of IFN-γ, IL-4, IL-10, IL-13 and IL-17Manuscript (preprint) (Other academic)
    Abstract [en]

    Proinflammatory cytokines like IFN-γ and TNF-α seem to have disease-promoting roles in multiple sclerosis (MS) whereas anti-inflammatory cytokines like IL-10 and TGF-ß may downregulate the disease. IFN-ß treatment reduces the frequency and severity of relapses, however, the mechanisms of action for IFN-ß are only partly understood and modulation of cytokine secretion could be one possible explanation for the therapeutic effects. The IFN-ß products approved for the treatment of MS differ in their composition and effects, and recently differences in effects on cytokine secretion were reported. Peripheral blood was collected from 25 patients with MS, both IFN-ß1a and IFN-ß1b treated, before onset of treatment and after 6 weeks, 3 months, 6 months and one year. Spontaneous as well as myelin specific secretion of IL-4, IFN-γ and IL-10 was analysed with the ELISPOT technique. PHA stimulated secretion of IL-13 and IL-17 was analysed in cell supernatants with ELISA. A general finding was that surprisingly few changes occurred, and that most changes occurred early (6 weeks - 3 months). We found a shift in the cytokine balance towards more IL-4 and IL-10 secretion and/or less IFN-γ secretion during the treatment as the ratios of IL-4!IFN-y as well as of IL-10/IFN-γ were increased. The interesting pro-inflammatory cytokine IL-17, that has been associated with T-cell mediated autoimmunity, has not been previously investigated during IFN-ß treatment in MS. Our findings of decreased IL-17 levels after one year of treatment, following an increase in early treatment, could be a beneficial result of the IFN-ß treatment. Further we noticed differences in effects on cytokines of IFN-ß1a and IFN-ß1b respectively; the latter seemed to have more effects on cytokine secretion.

  • 7.
    Kvarnström, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Jenmalm, Maria
    Linköping University, Department of Molecular and Clinical Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
    Ekerfelt, Christina
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4: An overall decrease of interleukin-42004In: Cryobiology, ISSN 0011-2240, E-ISSN 1090-2392, Vol. 49, no 2, p. 157-168Article in journal (Refereed)
    Abstract [en]

    Studies on cytokine expression in blood cells are commonly performed on cryopreserved cells. Previous studies show that cryopreservation affects cytokine expression, but the findings are not consistent. This may be due to divergent effects of freezing on different cytokines, different stimuli, and different patient groups or to the use of different assays in the studies. This study was designed to investigate the effect of freezing on spontaneous, auto-antigen, allergen, and mitogen induced cytokine secretion from peripheral blood mononuclear cells from several groups of patients expressing different cytokine profiles; multiple sclerosis, atopic children, non-atopic children, and pregnant women. The expression of IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13 was analysed with ELISA, ELISPOT and/or real time RT-PCR. Our data provide evidence that the process of cryopreservation and thawing does affect the expression of cytokines, both at the protein and the mRNA level. Moreover, the effect varied among different cytokines, different stimuli, and different patient groups, which partly may be explained by differences in optimal freezing conditions for non-activated and activated cells. An increase of allergen and PHA stimulated IFN-γ secretion in atopic children was found following cryopreservation, but no such increase in auto-antigen induced IFN-γ was seen in MS-patients. The most consistent finding was that expression of IL-4 was generally decreased in spontaneous and auto-antigen/allergen induced expression in cryopreserved cells. In conclusion, this study points out the importance of investigation of the effects of freezing for each cytokine, stimuli and patient group before using frozen cells in studies of in vitro cytokine secretion.

  • 8.
    Kvarnström, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Sidorova, E.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Nilsson, Joakim
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ekerfelt, Christina
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Vrethem, Magnus
    Linköping University, Department of Neuroscience and Locomotion, Neurology. Linköping University, Faculty of Health Sciences.
    Söderberg, O.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Johansson, Malin
    Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Rosén, Anders
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ernerudh, Jan
    Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)2002In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 127, no 2, p. 255-262Article in journal (Refereed)
    Abstract [en]

    Monoclonal expansion of B cells and plasma cells, producing antibodies against ‘self’ molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström’s macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P0, using beads coated with P0. P0-coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein–Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5+ positive, although the expression declined in vitro over time. The anti-P0 antibodies were of IgM-λ type. The antibodies belonged to the VH3 gene family with presence of somatic mutations. The IgM reacted with P0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5+ clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P0-specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation.

  • 9.
    Kvarnström, Maria
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Ydrefors, J
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Ekerfelt, Christina
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Vrethem, Magnus
    Linköping University, Department of Clinical and Experimental Medicine, Neurology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland, Department of Neurology.
    Ernerudh, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Immunology and Transfusion Medicine.
    Longitudinal interferon-β effects in multiple sclerosis: differential regulation of IL-10 and IL-17A, while no sustained effects on IFN-γ, IL-4 or IL-132013In: Journal of the Neurological Sciences, ISSN 0022-510X, E-ISSN 1878-5883, Vol. 325, no 1-2, p. 79-85Article in journal (Refereed)
    Abstract [en]

    Background:

    Recent studies in experimental models and in vitro indicate lowering of IL-17/Th17 as an important mechanism of interferon-beta (IFN-β) treatment in multiple sclerosis (MS).

    Material and methods:

    In this longitudinal study of MS patients (n = 25), spontaneous and myelin antigen-induced secretion of IL-4, IFN-γ and IL-10 (ELISPOT), mitogen stimulated secretion of IL-13 and IL-17A (ELISA) and circulating cytokine levels (Luminex) were recorded at inclusion and after 1.5, 3, 6 and 12 months of IFN-β treatment.

    Results:

    Early changes were noted for IL-4, while after one year of treatment the only recorded significant effects were a decrease in secreted IL-17A levels and an increase in IL-10 secreting cells. While IL-17A levels tended to be higher in non-responders (n = 8), the decrease in IL-17A levels seemed to be more pronounced in responders (n = 17) showing significantly lower IL-17A levels after one year as compared with non-responders.

    Conclusion:

    IFN-β treatment seems to mainly affect IL-17/IL-10-associated pathways rather than the IFN-γ/IL-4 axis.

  • 10.
    Schöier, Johan
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Öllinger, Karin
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Kvarnström, Maria
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Söderlund, Gustaf
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Kihlström, Erik
    Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
    Chlamydia trachomatis -induced apoptosis occurs in uninfected McCoy cells late in the developmental cycle and is regulated by the intracellular redox state2001In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 31, no 4, p. 173-184Article in journal (Refereed)
    Abstract [en]

    Infections with the obligate intracellular bacterium Chlamydia trachomatis are characterized by avoidance of fusion between chlamydia-containing endosomes and lysosomes, bacterial persistence and development of post-infectious sequelae. In this report we show that C. trachomatis induces apoptosis in McCoy and HeLa cells. Apoptosis was monitored by three different techniques; enzyme-linked immunoassay (EIA) of fragmented nucleosomes, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and flow cytometry of propidium iodide-stained cells. Apoptosis occurred in uninfected cells, was induced late in the chlamydial developmental cycle, beyond 24 h post-infection and was dependent on bacterial protein synthesis. Apoptosis was not significantly increased in infected, inclusion-containing cells. Treatment of cells with the antioxidants ascorbic acid (10 μM) and α-tocopherol (10 μM) reduced the degree of apoptosis. These results suggest that host cells infected with C. trachomatis generate proapoptotic stimuli that induce apoptosis in uninfected, neighbouring cells and that the redox state of the cell is a regulator in chlamydia-induced apoptosis.

  • 11.
    Vrethem, Magnus
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Neurology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland, Department of Neurology.
    Kvarnström, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Stenstam, J
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Cassel, Petra
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences.
    Gustafsson, M
    Linköping University, Department of Clinical and Experimental Medicine, Neurology. Linköping University, Faculty of Health Sciences.
    Landtblom, Anne-Marie
    Linköping University, Department of Clinical and Experimental Medicine, Neurology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland, Department of Neurology. Östergötlands Läns Landsting, Local Health Care Services in the West of Östergötland, Department of Medical Specialist.
    Ernerudh, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Immunology and Transfusion Medicine.
    Cytokine mapping in cerebrospinal fluid and blood in multiple sclerosis patients without oligoclonal bands2012In: Multiple Sclerosis, ISSN 1352-4585, E-ISSN 1477-0970, Vol. 18, no 5, p. 669-673Article in journal (Refereed)
    Abstract [en]

    Objective: Since there are clinical and genetic differences between MS patients with intrathecal oligoclonal bands (OCB+ ) in the cerebrospinal fluid (CSF) compared with those without (OCB-), the aim was to find out if OCB- patients showed a different pattern of cytokine immune activation compared with OCB+ patients. Methods: The study included 25 MS patients (10 OCB- and 15 OCB+ ) and 13 controls. A panel of cytokines was measured; IL-1β, IL-6, IL-8/CXCL8, IL-10, TNF and GM-CSF in serum, CSF and in supernatants from polyclonally stimulated blood mononuclear cells, where also levels of IL-12p40, IL-13, IL-15, IL-17 and IFN-γ were measured. The concentrations of soluble (s) VCAM-1 and sCD14 were measured in serum and CSF. Results: In general, there were no extensive differences in cytokine concentrations between the OCB- and OCB+ groups. Conclusion: OCB- MS patients do not seem to constitute a separate entity concerning inflammatory parameters measured as cytokine concentrations in CSF and blood.

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