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  • 1.
    Alfirevic, Ana
    et al.
    University of Liverpool, UK.
    Gonzalez-Galarza, Faviel
    University of Liverpool, UK.
    Bell, Catherine
    University of Liverpool, UK.
    Martinsson, Klara
    University of Liverpool, UK.
    Platt, Vivien
    University of Liverpool, UK.
    Bretland, Giovanna
    University of Liverpool, UK.
    Evely, Jane
    University of Liverpool, UK.
    Lichtenfels, Maike
    University of Liverpool, UK.
    Cederbrant, Karin
    Safety Assessment, AstraZeneca, Gartuna, Södertälje, Sweden.
    French, Neil
    University of Liverpool, UK.
    Naisbitt, Dean
    University of Liverpool, UK.
    Park, B Kevin
    University of Liverpool, UK.
    Jones, Andrew R
    University of Liverpool, UK.
    Pirmohamed, Munir
    University of Liverpool, UK.
    In silico analysis of HLA associations with drug-induced liver injury: use of a HLA-genotyped DNA archive from healthy volunteers2012In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 4, no 6, article id 51Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Drug-induced liver injury (DILI) is one of the most common adverse reactions leading to product withdrawal post-marketing. Recently, genome-wide association studies have identified a number of human leukocyte antigen (HLA) alleles associated with DILI; however, the cellular and chemical mechanisms are not fully understood.

    METHODS: To study these mechanisms, we established an HLA-typed cell archive from 400 healthy volunteers. In addition, we utilized HLA genotype data from more than four million individuals from publicly accessible repositories such as the Allele Frequency Net Database, Major Histocompatibility Complex Database and Immune Epitope Database to study the HLA alleles associated with DILI. We utilized novel in silico strategies to examine HLA haplotype relationships among the alleles associated with DILI by using bioinformatics tools such as NetMHCpan, PyPop, GraphViz, PHYLIP and TreeView.

    RESULTS: We demonstrated that many of the alleles that have been associated with liver injury induced by structurally diverse drugs (flucloxacillin, co-amoxiclav, ximelagatran, lapatinib, lumiracoxib) reside on common HLA haplotypes, which were present in populations of diverse ethnicity.

    CONCLUSIONS: Our bioinformatic analysis indicates that there may be a connection between the different HLA alleles associated with DILI caused by therapeutically and structurally different drugs, possibly through peptide binding of one of the HLA alleles that defines the causal haplotype. Further functional work, together with next-generation sequencing techniques, will be needed to define the causal alleles associated with DILI.

  • 2.
    Cederbrant, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    The Primary Lymphocyte Culture in the Diagnosis of Drug- and Metal-Induced Allergy2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Drugs and metals are examples of xenobiotics that can induce hypersensitivity in humans. These adverse reactions are classified as allergy if repeated exposure leads to the same type of clinical manifestation. Together with the clinical history, the skin test is the most commonly used test for the diagnosis of allergic disease. However, in vivo testing per se has drawbacks such as the risk of potentiation of the allergy or even sensitisation to a given test substance. For this reason in vitro testing is an attractive diagnostic alternative since it does not involve any exposure of the test subject to the allergen.

    The lymphocyte transformation test (LTT) has been used to complement the diagnosis of allergy to drugs and metals for more than thirty years. The principle behind this test is to show the presence of allergen-specific memory lymphocytes in peripheral blood, which is a sine qua non of a true allergy. LTT reveals the proliferation of such cells by showing DNA synthesis as the uptake of 3H-thymidine in primary PBMC (peripheral blood mononuclear cell) cultures treated with the allergen. However, LIT has not yet been generally accepted as a stand-alone test in the diagnosis of allergy. One reason for this is that different chemical properties of the allergens may lead to either false positive or false negative LTT responses.

    In the present study we investigated allergy to the drug bacampicillin and to the metals Au, Pd, Ni and Hg. Three different protocols for LTT: LIT in micro cultures (LTT-micro), LTT in macro cultures (LTT-macro) and memory lymphocyte immunostimulation assay (MELISA) were compared using a skin test or clinical history as reference methods. LTI showed a sensitivity of 87% and a specificity of 90% when used in the diagnosis of allergy to bacampicillin. When allergy to Au, Pd, Ni and Hg was investigated, the sensitivity was 33- 95% and the specificity 0-79%. There were no significant differences between the test protocols, except that MELISA showed a significantly higher specificity than LTT-micro and LTT-macro when Hg2+ was used as antigen. Even so, this specificity was only 70%, which would result in 30 of 100 healthy subjects receiving a false diagnosis of Hg allergy when using the MELISA protocol. Ni2+ also induced high numbers of false-positive LTI responses, 77-85% patch-test negative subjects showed positive results to these metals. However, group comparisons showed a significantly higher proliferation intensity in allergic than in nonallergic groups for all allergens except Hg2+. Furthermore, only 56% of patients with verified allergy to mercury showed a positive MELISA, a sensitivity that is unacceptably low.

    Following these findings, we investigated whether other endpoints than DNA synthesis could be used to discriminate allergic from healthy subjects, using primary PBMC cultures with Hg2+ or Ni2+ as a model system. Analysis of the T-cell receptor Vß profiles of lymphoblasts induced by these metal ions showed individual patterns, and there was no difference between healthy and allergic groups. However, the fraction of CD4+/Vß2+ cells correlated significantly with the proliferation intensity induced by Hg2+ in patients with a verified Hg allergy but not in non-allergic controls. Interestingly, such a correlation was not seen with CD8+/Nß2+ cells. This indicates that Hg2+ does not function as a superantigen, since classical superantigens also stimulate CD8+ lymphocytes. When Ni2+ was used as antigen we found significantly higher IL-10 production in allergic than in non-allergic subjects, despite no significant difference in proliferation intensity between these two groups.

    In conclusion, the LTT test is useful for the diagnosis of allergy to bacampicillin. Regarding Au, Pd and Ni the LIT has low validity and can only be used to discriminate groups of allergic from non-allergic individuals. LTT with Hg2+ and Ni2+ is not useful for the diagnosis of allergy to these metals since a high fraction of non-allergic individuals show positive results, irrespective of the test protocol used. This thesis calls for further studies on the usefulness of in vitro IL-10 production for the diagnosis of Ni allergy as well as on the specificity of in vitro induced CD4+N~2+ lymphoblasts from Hg allergic subjects.

    List of papers
    1. Characterization of primary recall in vitro lymphocyte responses to bacampicillin in allergic subjects
    Open this publication in new window or tab >>Characterization of primary recall in vitro lymphocyte responses to bacampicillin in allergic subjects
    2000 (English)In: Clinical and Experimental Allergy, ISSN 0954-7894, E-ISSN 1365-2222, Vol. 30, no 10, p. 1450-1459Article in journal (Refereed) Published
    Abstract [en]

    Background

    Antigen-specific cell lines or clones are often used as models of drug-specific allergy. However, cloning procedures are time consuming, and the repeated antigen stimulation cycles as well as the addition of various growth enhancers may affect the in vivo relevance of these systems.

    Objective

    Using bacampicillin-allergic subjects, we wanted to investigate the applicability of primary recall in vitro lymphocyte responses to characterize type I and type IV allergy. The sensitivity and specificity of LTT (Lymphocyte transformation test), when used as an in vitro diagnostic tool, were also assessed.

    Methods

    A total of 39 patients with symptoms of type I (rhinitis) or type IV (allergic contact dermatitis, ACD) allergy following occupational exposure to bacampicillin, were included. Ten individuals without penicillin allergy or occupational exposure to bacampicillin served as controls. All subjects were LTT tested. Four patients with rhinitis and two patients with ACD were available for studying the immunophenotype and the TCR-Vβ repertoire of bacampicillin induced lymphoblasts as well as the cytokine profiles and expression of the activation markers CD23 and CD134 in primary PBMC cultures.

    Results

    LTT was positive in 87% and at least one of the skin tests was positive in 85% of the patients with allergic symptoms. 69% of the patients with type I allergies were patch test-positive. Results from LTT and skin test correlated in 87% of the cases. The combined sensitivity of LTT and skin tests was 92%. The specificity of LTT was 90% in healthy controls. Bacampicillin induced lymphoblasts were mainly CD4 + in both ACD and rhinitis patients. The TCR-Vβ profiles of the predominant CD4 + lymphoblasts were heterogeneous with individual skewing towards Vβ2, Vβ3, Vβ5.1 and/or Vβ14. An increased expression of IFNγ was detected in bacampicillin treated PBMC cultures from the ACD but not from rhinitis patients. IL-5 was detected in bacampicillin exposed PBMC cultures from all patients but not from healthy controls. This Th2 environment could also be verified by CD23 and CD134 expression.

    Conclusion

    LTT and skin tests are equally sensitive in identifying bacampicillin allergic subjects. When the two tests are combined, the sensitivity increases. The patch test is useful not only for detection of type IV but also for the identification of type I allergies. When using primary PBMC cultures, IFNγ is the most suitable cytokine to discriminate between type I and type IV allergy. IL-5 can possibly be used as a general marker for bacampicillin induced allergy. Thus, primary cell cultures may be considered as an alternative to T-cell lines or clones for the study of drug induced allergy.

    Keywords
    activation markers, allergic contact dermatitis, bacampicillin, cytokines, flow cytometry, human lymphocytes, lymphocyte transformation test, rhinitis, skin test, T-cell receptor Vβ
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25872 (URN)10.1046/j.1365-2222.2000.00905.x (DOI)10309 (Local ID)10309 (Archive number)10309 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
    2. In vitro Lymphocyte Proliferation as Compared to Patch Test Using Gold, Palladium and Nickel
    Open this publication in new window or tab >>In vitro Lymphocyte Proliferation as Compared to Patch Test Using Gold, Palladium and Nickel
    1997 (English)In: International Archives of Allergy and Immunology, ISSN 1018-2438, E-ISSN 1423-0097, Vol. 112, no 3, p. 212-217Article in journal (Refereed) Published
    Abstract [en]

    Background: A conventional lymphocyte transformation test (LTT) was compared to the commercially available MELISA® (memory lymphocyte immuno-stimulation assay), a lymphoproliferative assay that has been suggested to be a valuable instrument for the diagnosis of metal allergy. Sensitivity and specificity of the two assays were calculated using a patch test as a reference method.

    Methods: 34 patients were patch-tested for gold sodium thiosulfate, palladium chloride and nickel sulfate, and the lymphocyte proliferation to these metals was tested in vitro using mononuclear cells from peripheral blood.

    Results: No significant differences regarding sensitivity and specificity were found between MELISA and conventional LTT. The sensitivity varied between 55 and 95% and the specificity between 17 and 79%.

    Conclusions: The low specificity of the two in vitro assays suggests that they are not useful for diagnosis of contact allergy to the metals gold, palladium and nickel, since a large number of false-positive results will be obtained.

    Keywords
    Lymphocytes human, Skin test, Metals
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-79573 (URN)10.1159/000237456 (DOI)
    Available from: 2012-08-09 Created: 2012-08-09 Last updated: 2017-12-07Bibliographically approved
    3. IL-10 production in primary PBMC cultures: an in vitro marker for nickel allergy?
    Open this publication in new window or tab >>IL-10 production in primary PBMC cultures: an in vitro marker for nickel allergy?
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Nickel (Ni) is one of the most known contact allergens and at present, patch test and clinical history constitute the two cornerstones in the diagnostic procedure. Since the patch test is inherited with in vivo provocation and subjective interpretation of the test result, a non-invasive in vitro method with objective interpretation of the test result has long been searched for. Unfortunately, in vitro diagnosis of Ni- allergy is hampered by the fact that Ni2+ is able to trigger in vitro proliferative responses in lymphocytes from both Ni-allergic and non-allergic subjects. This constitutes a problem when LTT (lymphocyte transformation test), the most frequently used in vitro test as a complement in the diagnosis of contact allergy, is considered for Ni allergy. However, other parameters in the in vitro response might be more useful. In this study, Ni2+-stimulated primary PBMC-cultures derived from Ni-allergic and non-allergic subjects were assessed for IFN-γ, IL-4, IL-10 and IL-17. Also, Ni2+ induced lymphoblasts from such cultures were characterized by their immunophenotype and T-cell receptor Vß-affiliation.

    We found a significantly higher release of IL-10 in Ni2+ treated cultures from allergic than from non-allergic subjects. The Ni2+-induced lymphoblasts from both groups were predominantly CD4+. Two of the allergic patients (n=5) showed a skewing towards TCR-Vß17, a Vß family earlier associated with Ni-allergy. A significant increase in CD134 and CD23 expression indicated that Ni2+ activates B-cells in vitro. In conclusion, IL-10 seems to be a promising marker for Ni-allergy using primary PBMC cultures. Further, flow cytometric screening of Ni2+ induced lymphoblasts can detect expanded TCR-Vß families that may be used for preparation of Ni-specific T cell clones.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-79574 (URN)
    Available from: 2012-08-09 Created: 2012-08-09 Last updated: 2012-08-09Bibliographically approved
    4. In vitro Lymphoproliferative Assays with HgCl2 Cannot Identify Patients with Systemic Symptoms Attributed to Dental Amalgam
    Open this publication in new window or tab >>In vitro Lymphoproliferative Assays with HgCl2 Cannot Identify Patients with Systemic Symptoms Attributed to Dental Amalgam
    Show others...
    1999 (English)In: Journal of Dental Research, ISSN 0022-0345, E-ISSN 1544-0591, Vol. 78, no 8, p. 1450-1458Article in journal (Refereed) Published
    Abstract [en]

    Dental amalgam is suspected, by some exposed individuals, to cause various systemic psychological, sensory, and neurological symptoms. Since not all amalgam-bearers experience such reactions, an individual characteristic—for example, a susceptible immune system—might explain these conditions. In vitro lymphocyte proliferation is a valuable tool in the diagnosis of allergy. With HgCl2 as the antigen, however, the test is hampered, because Hg2+ can cause unspecific lymphocyte proliferation, optimal at 1.4 to 9.5 μg HgCl2/mL. Recently, the use of suboptimal HgCl2 concentrations (≤ 0.5 μg/mL) has been suggested to circumvent these problems. The main aim of this study was to investigate whether patients with systemic symptoms alleged to result from the presence of dental amalgam differ from healthy controls, with reference to in vitro lymphoproliferative responses to HgCl2 ≤ 0.5 μg/mL. Three different test protocols—lymphocyte transformation test (LTT) in micro- and macro-cultures, and the memory lymphocyte immunostimulation assay (MELISA®)—were used. Other immune parameters—such as a standard patch test for dental materials, the number of T- and B-lymphocytes, monocytes, granulocytes, and NK cells in peripheral blood, allergic symptoms, and predisposition-were also investigated. Twenty-three amalgam patients, 30 healthy blood donors with amalgam, ten healthy subjects without amalgam, and nine patients with oral lichen planus (OLP) adjacent to dental amalgam and a positive patch test to Hg0 were tested. None of the investigated immune parameters revealed any significant differences between amalgam patients and controls. The sensitivity of in vitro lymphocyte proliferation ranged from 33 to 67%, with the OLP patients as a positive control group, and the specificity from 0 to 70% for healthy controls with a negative patch test to Hg°. Thus, despite the use of HgCl2 ≤ 0.5 μg/mL, a high frequency of positive results was obtained among healthy subjects with or without dental amalgam. Consequently, in vitro lymphocyte proliferation with HgCl2 cannot be used as an objective marker for mercury allergy in dental amalgam-bearers.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25875 (URN)10.1177/00220345990780081101 (DOI)10312 (Local ID)10312 (Archive number)10312 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
    5. Characterization of mercuric mercury (Hg2+)-induced lymphoblasts from patients with mercury allergy and from healthy subjects
    Open this publication in new window or tab >>Characterization of mercuric mercury (Hg2+)-induced lymphoblasts from patients with mercury allergy and from healthy subjects
    2000 (English)In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 121, no 1, p. 23-30Article in journal (Refereed) Published
    Abstract [en]

    Hg2+ induces lymphocyte proliferation when added to cell cultures from both healthy and mercury-allergic subjects. Consequently, when measuring DNA synthesis a possible Hg2+-specific response, resulting from proliferating memory cells, cannot be discriminated from a non-allergic response. The mechanism behind this non-allergic response is unknown but a superantigenic effect of Hg2+ has been suggested. In this study, five mercury-allergic patients, with oral lichen planus (OLP) lesions adjacent to dental amalgam and a positive patch test to Hg0, and five healthy subjects without amalgam were examined. The immunophenotype and the T cell receptor Vβ (TCR Vβ) repertoire of Hg2+-induced lymphoblasts as well as the expression of the lymphocyte activation markers CD23 and CD134 were analysed for possible differences between healthy and allergic subjects. The mechanism of Hg2+-induced proliferation was examined by comparing the TCR Vβ expression of Hg- and staphylococcal enterotoxin B (SEB)-activated lymphoblasts, the latter used as a positive superantigen control. It was not possible to discriminate between mercury-allergic and healthy subjects using the immunophenotype or the TCR Vβ profile of the Hg2+-induced lymphoblasts or the expression of CD23 and CD134. However, Hg2+-induced CD4+ lymphoblasts showed a skewing towards Vβ2. This relative increase in Vβ2 was only detected in the CD4+ but not in the CD8+ lymphoblast population. In conclusion, Hg2+ induced a proliferation-dependent skewing towards CD4+ but not CD8+ lymphocytes expressing Vβ2. In this respect Hg2+ differs from the classical bacterial superantigen SEB, which also stimulates unique TCR Vβ families among CD8+ cells.

    Keywords
    Hg allergy, oral lichen planus, human lymphocytes, in vitro
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25873 (URN)10.1046/j.1365-2249.2000.01268.x (DOI)10310 (Local ID)10310 (Archive number)10310 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
  • 3.
    Cederbrant, Karin
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Andersson, C.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Andersson, T.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Marcusson-Ståhl, Maritha
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Hultman, Per
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    IL-10 production in primary PBMC cultures: an in vitro marker for nickel allergy?Manuscript (preprint) (Other academic)
    Abstract [en]

    Nickel (Ni) is one of the most known contact allergens and at present, patch test and clinical history constitute the two cornerstones in the diagnostic procedure. Since the patch test is inherited with in vivo provocation and subjective interpretation of the test result, a non-invasive in vitro method with objective interpretation of the test result has long been searched for. Unfortunately, in vitro diagnosis of Ni- allergy is hampered by the fact that Ni2+ is able to trigger in vitro proliferative responses in lymphocytes from both Ni-allergic and non-allergic subjects. This constitutes a problem when LTT (lymphocyte transformation test), the most frequently used in vitro test as a complement in the diagnosis of contact allergy, is considered for Ni allergy. However, other parameters in the in vitro response might be more useful. In this study, Ni2+-stimulated primary PBMC-cultures derived from Ni-allergic and non-allergic subjects were assessed for IFN-γ, IL-4, IL-10 and IL-17. Also, Ni2+ induced lymphoblasts from such cultures were characterized by their immunophenotype and T-cell receptor Vß-affiliation.

    We found a significantly higher release of IL-10 in Ni2+ treated cultures from allergic than from non-allergic subjects. The Ni2+-induced lymphoblasts from both groups were predominantly CD4+. Two of the allergic patients (n=5) showed a skewing towards TCR-Vß17, a Vß family earlier associated with Ni-allergy. A significant increase in CD134 and CD23 expression indicated that Ni2+ activates B-cells in vitro. In conclusion, IL-10 seems to be a promising marker for Ni-allergy using primary PBMC cultures. Further, flow cytometric screening of Ni2+ induced lymphoblasts can detect expanded TCR-Vß families that may be used for preparation of Ni-specific T cell clones.

  • 4.
    Cederbrant, Karin
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, L-G
    Department of Neurology and Neurophysiology and Centre for Environmental Sensitivity, Örebro Medical Centre Hospital, Örebro, Sweden.
    Hultman, Per
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Norda, R.
    Department of Transfusion Medicine and Immunohaemotherapy, Örebro Medical Centre Hospital, Örebro, Sweden.
    Tibbling-Grahn, L.
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    In vitro Lymphoproliferative Assays with HgCl2 Cannot Identify Patients with Systemic Symptoms Attributed to Dental Amalgam1999In: Journal of Dental Research, ISSN 0022-0345, E-ISSN 1544-0591, Vol. 78, no 8, p. 1450-1458Article in journal (Refereed)
    Abstract [en]

    Dental amalgam is suspected, by some exposed individuals, to cause various systemic psychological, sensory, and neurological symptoms. Since not all amalgam-bearers experience such reactions, an individual characteristic—for example, a susceptible immune system—might explain these conditions. In vitro lymphocyte proliferation is a valuable tool in the diagnosis of allergy. With HgCl2 as the antigen, however, the test is hampered, because Hg2+ can cause unspecific lymphocyte proliferation, optimal at 1.4 to 9.5 μg HgCl2/mL. Recently, the use of suboptimal HgCl2 concentrations (≤ 0.5 μg/mL) has been suggested to circumvent these problems. The main aim of this study was to investigate whether patients with systemic symptoms alleged to result from the presence of dental amalgam differ from healthy controls, with reference to in vitro lymphoproliferative responses to HgCl2 ≤ 0.5 μg/mL. Three different test protocols—lymphocyte transformation test (LTT) in micro- and macro-cultures, and the memory lymphocyte immunostimulation assay (MELISA®)—were used. Other immune parameters—such as a standard patch test for dental materials, the number of T- and B-lymphocytes, monocytes, granulocytes, and NK cells in peripheral blood, allergic symptoms, and predisposition-were also investigated. Twenty-three amalgam patients, 30 healthy blood donors with amalgam, ten healthy subjects without amalgam, and nine patients with oral lichen planus (OLP) adjacent to dental amalgam and a positive patch test to Hg0 were tested. None of the investigated immune parameters revealed any significant differences between amalgam patients and controls. The sensitivity of in vitro lymphocyte proliferation ranged from 33 to 67%, with the OLP patients as a positive control group, and the specificity from 0 to 70% for healthy controls with a negative patch test to Hg°. Thus, despite the use of HgCl2 ≤ 0.5 μg/mL, a high frequency of positive results was obtained among healthy subjects with or without dental amalgam. Consequently, in vitro lymphocyte proliferation with HgCl2 cannot be used as an objective marker for mercury allergy in dental amalgam-bearers.

  • 5.
    Cederbrant, Karin
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Hultman, Per
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Characterization of mercuric mercury (Hg2+)-induced lymphoblasts from patients with mercury allergy and from healthy subjects2000In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 121, no 1, p. 23-30Article in journal (Refereed)
    Abstract [en]

    Hg2+ induces lymphocyte proliferation when added to cell cultures from both healthy and mercury-allergic subjects. Consequently, when measuring DNA synthesis a possible Hg2+-specific response, resulting from proliferating memory cells, cannot be discriminated from a non-allergic response. The mechanism behind this non-allergic response is unknown but a superantigenic effect of Hg2+ has been suggested. In this study, five mercury-allergic patients, with oral lichen planus (OLP) lesions adjacent to dental amalgam and a positive patch test to Hg0, and five healthy subjects without amalgam were examined. The immunophenotype and the T cell receptor Vβ (TCR Vβ) repertoire of Hg2+-induced lymphoblasts as well as the expression of the lymphocyte activation markers CD23 and CD134 were analysed for possible differences between healthy and allergic subjects. The mechanism of Hg2+-induced proliferation was examined by comparing the TCR Vβ expression of Hg- and staphylococcal enterotoxin B (SEB)-activated lymphoblasts, the latter used as a positive superantigen control. It was not possible to discriminate between mercury-allergic and healthy subjects using the immunophenotype or the TCR Vβ profile of the Hg2+-induced lymphoblasts or the expression of CD23 and CD134. However, Hg2+-induced CD4+ lymphoblasts showed a skewing towards Vβ2. This relative increase in Vβ2 was only detected in the CD4+ but not in the CD8+ lymphoblast population. In conclusion, Hg2+ induced a proliferation-dependent skewing towards CD4+ but not CD8+ lymphocytes expressing Vβ2. In this respect Hg2+ differs from the classical bacterial superantigen SEB, which also stimulates unique TCR Vβ families among CD8+ cells.

  • 6.
    Cederbrant, Karin
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Surgery. Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Hultman, Per
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Marcusson, Jan A.
    Department of Dermatology, Huddinge Hospital, Huddinge.
    Tibbling, Lita
    Linköping University, Department of Clinical and Experimental Medicine, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    In vitro Lymphocyte Proliferation as Compared to Patch Test Using Gold, Palladium and Nickel1997In: International Archives of Allergy and Immunology, ISSN 1018-2438, E-ISSN 1423-0097, Vol. 112, no 3, p. 212-217Article in journal (Refereed)
    Abstract [en]

    Background: A conventional lymphocyte transformation test (LTT) was compared to the commercially available MELISA® (memory lymphocyte immuno-stimulation assay), a lymphoproliferative assay that has been suggested to be a valuable instrument for the diagnosis of metal allergy. Sensitivity and specificity of the two assays were calculated using a patch test as a reference method.

    Methods: 34 patients were patch-tested for gold sodium thiosulfate, palladium chloride and nickel sulfate, and the lymphocyte proliferation to these metals was tested in vitro using mononuclear cells from peripheral blood.

    Results: No significant differences regarding sensitivity and specificity were found between MELISA and conventional LTT. The sensitivity varied between 55 and 95% and the specificity between 17 and 79%.

    Conclusions: The low specificity of the two in vitro assays suggests that they are not useful for diagnosis of contact allergy to the metals gold, palladium and nickel, since a large number of false-positive results will be obtained.

  • 7.
    Cederbrant, Karin
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Marcusson-Ståhl, M.
    AstraZeneca R & D Södertälje, Safety Assessment, Department of Molecular Toxicology and Immunotoxicology, Södertälje, Sweden.
    Hultman, Per
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Characterization of primary recall in vitro lymphocyte responses to bacampicillin in allergic subjects2000In: Clinical and Experimental Allergy, ISSN 0954-7894, E-ISSN 1365-2222, Vol. 30, no 10, p. 1450-1459Article in journal (Refereed)
    Abstract [en]

    Background

    Antigen-specific cell lines or clones are often used as models of drug-specific allergy. However, cloning procedures are time consuming, and the repeated antigen stimulation cycles as well as the addition of various growth enhancers may affect the in vivo relevance of these systems.

    Objective

    Using bacampicillin-allergic subjects, we wanted to investigate the applicability of primary recall in vitro lymphocyte responses to characterize type I and type IV allergy. The sensitivity and specificity of LTT (Lymphocyte transformation test), when used as an in vitro diagnostic tool, were also assessed.

    Methods

    A total of 39 patients with symptoms of type I (rhinitis) or type IV (allergic contact dermatitis, ACD) allergy following occupational exposure to bacampicillin, were included. Ten individuals without penicillin allergy or occupational exposure to bacampicillin served as controls. All subjects were LTT tested. Four patients with rhinitis and two patients with ACD were available for studying the immunophenotype and the TCR-Vβ repertoire of bacampicillin induced lymphoblasts as well as the cytokine profiles and expression of the activation markers CD23 and CD134 in primary PBMC cultures.

    Results

    LTT was positive in 87% and at least one of the skin tests was positive in 85% of the patients with allergic symptoms. 69% of the patients with type I allergies were patch test-positive. Results from LTT and skin test correlated in 87% of the cases. The combined sensitivity of LTT and skin tests was 92%. The specificity of LTT was 90% in healthy controls. Bacampicillin induced lymphoblasts were mainly CD4 + in both ACD and rhinitis patients. The TCR-Vβ profiles of the predominant CD4 + lymphoblasts were heterogeneous with individual skewing towards Vβ2, Vβ3, Vβ5.1 and/or Vβ14. An increased expression of IFNγ was detected in bacampicillin treated PBMC cultures from the ACD but not from rhinitis patients. IL-5 was detected in bacampicillin exposed PBMC cultures from all patients but not from healthy controls. This Th2 environment could also be verified by CD23 and CD134 expression.

    Conclusion

    LTT and skin tests are equally sensitive in identifying bacampicillin allergic subjects. When the two tests are combined, the sensitivity increases. The patch test is useful not only for detection of type IV but also for the identification of type I allergies. When using primary PBMC cultures, IFNγ is the most suitable cytokine to discriminate between type I and type IV allergy. IL-5 can possibly be used as a general marker for bacampicillin induced allergy. Thus, primary cell cultures may be considered as an alternative to T-cell lines or clones for the study of drug induced allergy.

  • 8.
    Christiansen Clifford, Jenny
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Färm, Gunilla
    Department of Dermatology, University Hospital, 701 85 Örebro, Sweden.
    Eid-Forest, Ruth
    Department of Dermatology, University Hospital, 701 85 Örebro, Sweden.
    Anderson, Chris
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland, Department of Dermatology and Venerology UHL.
    Cederbrant, Karin
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Hultman, Per
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Cytology.
    Interferon-gamma secreted from peripheral blood mononuclear cells as a possible diagnostic marker for allergic contact dermatitis to gold2006In: Contact Dermatitis, ISSN 0105-1873, E-ISSN 1600-0536, Vol. 55, no 2, p. 101-112Article in journal (Refereed)
    Abstract [en]

    10% of patch-tested patients have a positive reaction to gold. Most lack clinical symptoms, but allergic contact dermatitis (ACD) to gold is increasing. In this study, 77 dermatological outpatients were divided into 3 groups depending on epicutaneous patch test outcomes: a group positive to gold (EPI+), a group negative to gold (EPI-), and a group with irritant reactions to gold (EPI-IR). Lymphocytes were stimulated in vitro with gold sodium thiosulfate. Proliferation was assessed using the lymphocyte transformation test (LTT), and cytokine secretion was assessed using a multibead array (Luminex; Linco Research Inc., St. Charles, MO, USA), in order to evaluate whether an in vitro method with high diagnostic accuracy could be devised. The EPI+ group showed a significantly increased secretion of interferon (IFN)-gamma, interleukin (IL)-2, and IL-13 and also showed a significantly higher stimulation indexes for LTT, compared to the other 2 subject groups. Sensitivity and specificity were calculated for all methods individually and combined, but IFN-gamma assessment alone was the most accurate method for identifying ACD to gold, with sensitivity and specificity of 81.8% and 82.1%, respectively. This method also identified 87.5% of the EPI-IR subjects as non-allergic. Therefore, assessment of secretion of IFN-gamma should be a valuable complement to patch test for diagnosing gold allergy.

  • 9.
    Clifford, Jenny
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology . Linköping University, Faculty of Health Sciences.
    Anderson, Chris
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Local Health Care Services in Central Östergötland, Department of Dermatology and Venerology UHL.
    Karin, Cederbrant
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology . Linköping University, Faculty of Health Sciences.
    Hultman, Per
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology . Linköping University, Faculty of Health Sciences.
    T-cells expressing CD4, CD45RO and CLA from gold-allergic but not healthy subjects react to gold sodium thiosufate in vitroManuscript (preprint) (Other academic)
    Abstract [en]

    Patch test positivity to gold is common in western societies, but in contrast to nickel (Ni) allergy it is uncommon that the patch test positive patient shows any clinical symptoms. In this study we investigated cytotoxic effects of gold sodium thiosulphate (GSTS) on peripheral blood mononuclear cells (PBMC), including different T-cell subsets. We also separated lymphocytes from allergic and non-allergic subjects into CD45RA and CD45R0 cell fractions. We also expressed CLA. The fraction of analyzed the effects of GSTS using lymphocyte transformation test, propidium iodide staining and flow cytometry to determine lymphocyte memory status, expression of chemokine receptors and cutaneous lymphocyte-associated antigen (CLA), and compared the results to what has previously been reported on Ni allergy. We found that only the cells from the allergic subjects proliferated in the lymphocyte transformation test (LTT), and in the CD45R0 fraction there was a dose-dependent increase in the fraction of CD3/CD4 cells. Similar to Ni-allergy, these CD3/CD4/CD45R0 cells also expressed CLA. The fraction of CD3/CD8 in the CD45R0 enriched fraction decreased with GSTS exposure. In contrast to Ni allergy, however, we found no differences between the allergic and non-allergic subjects regarding the chemokine receptors CCR4, CXCR3 and CCR10.

  • 10.
    Lundgren, Hanna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland.
    Martinsson, Klara
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland.
    Cederbrant, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Swetox, Karolinska Institutet, Unit of Toxicology Sciences, Södertälje, Sweden.
    Jirholt, Johan
    AstraZeneca RandD, Sweden.
    Mucs, Daniel
    Swetox, Karolinska Institutet, Unit of Toxicology Sciences, Södertälje, Sweden.
    Madeyski-Bengtson, Katja
    AstraZeneca RandD, Sweden.
    Havarinasab, Said
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland.
    Hultman, Per
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Clinical pathology.
    HLA-DR7 and HLA-DQ2: Transgenic mouse strains tested as a model system for ximelagatran hepatotoxicity2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 9, article id e0184744Article in journal (Refereed)
    Abstract [en]

    The oral thrombin inhibitor ximelagatran was withdrawn in the late clinical trial phase because it adversely affected the liver. In approximately 8% of treated patients, drug-induced liver injury (DILI) was expressed as transient alanine transaminase (ALT) elevations. No evidence of DILI had been revealed in the pre-clinical in vivo studies. A whole genome scan study performed on the clinical study material identified a strong genetic association between the major histocompatibility complex alleles for human leucocyte antigens (HLA) (HLA-DR7 and HLA-DQ2) and elevated ALT levels in treated patients. An immunemediated pathogenesis was suggested. Here, we evaluated whether HLA transgenic mice models could be used to investigate whether the expression of relevant HLA molecules was enough to reproduce the DILI effects in humans. In silico modelling performed in this study revealed association of both ximelagatran (pro-drug) and melagatran (active drug) to the antigen-presenting groove of the homology modelled HLA-DR7 molecule suggesting "altered repertoire" as a key initiating event driving development of DILI in humans. Transgenic mouse strains (tgms) expressing HLA of serotype HLA-DR7 (HLA-DRB1*0701, -DRA*0102), and HLA-DQ2 (HLA-DQB1*0202, -DQA1*0201) were created. These two lines were crossed with a human (h) CD4 transgenic line, generating the two tgms DR7xhCD4 and DQ2xhCD4. To investigate whether the DILI effects observed in humans could be reproduced in tgms, the mice were treated for 28 days with ximelagatran. Results revealed no signs of DILI when biomarkers for liver toxicity were measured and histopathology was evaluated. In the ximelagatran case, presence of relevant HLA-expression in a preclinical model did not fulfil the prerequisite for reproducing DILI observed in patients. Nonetheless, for the first time an HLA-transgenic mouse model has been investigated for use in HLA-associated DILI induced by a low molecular weight compound. This study shows that mimicking of genetic susceptibility, expressed as DILI-associated HLA-types in mice, is not sufficient for reproducing the complex pathogenesis leading to DILI in man.

  • 11.
    Martinsson, Klara
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Cederbrant, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology . Linköping University, Faculty of Health Sciences.
    Hultman, Per
    Linköping University, Department of Clinical and Experimental Medicine, Molecular and Immunological Pathology . Linköping University, Faculty of Health Sciences.
    Cytokines in induction of ANoA and hypergammaglobulinemia in mercury-induced autoimmunity: a lesson from Fc!RIII deficient miceManuscript (Other academic)
    Abstract [en]

    Xenobiotic agents such as metals, drugs, toxic oils and pristane can induce autoimmune diseases. Heavy metal induction of autoimmunity has been observed for mercury (Hg), silver and gold in mice. Mercury-induced autoimmunity (HgIA) in mice is characterised by lymphoproliferation, hypergammaglobulinemia, antinucleolar autoantibodies (ANoA) and immune complex deposits in the renal glomerular mesangium and systemically in vessel walls. HgIA is T-cell dependent, IFNγ is necessary for all manifestations of HgIA, and the activating Fc!RIII enhance development of ANoA. This study focused firstly on exploring the cytokine profile in the genetically susceptible DBA/1 (H-2q) wild type (wt) and DBA/1 FcγRIII-/- mice treated with 15 mg/l Hg, and secondly on the hypothesis that IFN-! producing NK cells are vital for induction of ANoA in the HgIA model. DBA/1 wt mice showed a significantly more marked Th1 profile compared to DBA/1 FcγRIII-/- mice following Hg treatment, whereas the total Th2 and Th17 profile increased in both DBA/1 wt and DBA/1 FcγRIII-/- mice. However, during Hg treatment IL-21 mRNA expression was significantly reduced in DBA/1 FcγRIII-/- mice compared with DBA/1 wt mice. However, we were unable to show that the increased Th1 profile in the DBA/1 wt mice was due to IFN-γsecretion from NK cells. Our findings suggest that the delayed ANoA induction in DBA/1 FcγRIII-/- mice is due to the attenuated Th1 profile. In addition the reduced expression of IL-21 in DBA/1 FcγRIII-/- mice might be responsible for the lack of serum IgG1 response in these mice.

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