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  • 1.
    Andersson, Yvonne
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lönnerdal, Bo
    Department of Nutrition, University of California, Davis, CA 95616.
    Graverholt, Gitte
    Arla Foods Ingredients, Aarhus, Denmark.
    Fält, Helen
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Formula feeding skews immune cell composition toward adaptive immunity compared to breastfeeding2009In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 183, no 7, p. 4322-4328Article in journal (Refereed)
    Abstract [en]

    The ontogeny of the immune system and the effect thereon by type of infant feeding is incompletely understood. We analyzed frequencies and composition of immune cells in blood of breastfed (BF) and formula-fed (FF) infants at 1.5, 4, and 6 mo of age. Three formulas with the same protein concentration but with varying levels of alpha-lactalbumin and caseinoglycomacropeptide were compared. Twenty-nine exclusively BF infants served as reference, and 17 infants in each formula group completed the study. Whole blood and PBMCs were analyzed by flow cytometry and immunoflow cytometry, respectively. Leukocyte count of BF infants increased with time due to increased frequency of neutrophils. Lymphocyte count was high at 1.5 mo and was unchanged over time, as were the relative proportions of CD4+ alphabetaT cells, CD8+ alphabetaT cells, B cells, NK cells, and gammadeltaT cells. Most CD45R0+CD3+ cells were HLA-DR- and hence memory cells. Compared with breastfeeding, formula feeding resulted in a significant decrease in proportion of NK cells, but a significant increase in naive CD4+ alphabetaT cells and an elevated CD4-to-CD8 ratio, that is, 3.3 in the combined FF groups compared with 2.6 in the BF group. No significant differences were found between the three groups of FF infants. In conclusion, blood cells of lymphoid lineage did not change significantly in frequencies or composition from 1.5 to 6 mo of age in BF infants. In contrast, FF infants displayed an ongoing maturation of adaptive immunity cells and a delayed recruitment of innate immunity cells as compared with BF infants.

  • 2.
    Banday, Viqar
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Metab-Immune analysis of the non-obese diabetic mouse2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Type 1A diabetes mellitus or T1D is a chronic disease characterized by T cell mediated destruction of the insulin producing β cells in the islets of Langerhans. The classical symptoms include high glucose levels in urine and blood, polyuria, and polydipsia. Complications associated with T1D include blindness, amputations, and end-stage renal disease, and premature death. The non-obese diabetic (NOD) mouse, first described in 1980, is widely used as a model organism for T1D. T1D disease in the NOD mouse shares a number of similarities to human T1D including dependence on genetic and environmental factors. More than 30 disease associated gene regions or loci (termed insulin dependent diabetes, or Idd, loci) have been associated with T1D development in NOD. For some of these Idds, the corresponding region in human has been linked to the development of T1D in human.

    T1D, both in humans and mice, is recognized as a T cell mediated disease. However, many studies have shown the importance of both the metabolome and the immune system in the pathogenesis of the disease. Appearance of autoantibodies in the serum of patients is the first sign of pathogenesis. However, molecular and cellular events precede the immune attack on the β-cell immunity. It has been shown that patients who developed T1D have an altered metabolome prior to the appearance of autoantibodies. Although much is known about the pathogenesis of T1D, the contribution of the environment/immune factors triggering the disease is still to be revealed. 

    In the present study both metabolic and immune deviations observed in the NOD mouse was analyzed. Serum metabolome analysis of the NOD mouse revealed striking resemblance to the human metabolic profile, with many metabolites in the TCA cycle significantly different from the non-diabetic control B6 mice. In addition, an increased level of glutamic acid was of the most distinguishing metabolite. A detailed bioinformatics analysis revealed various genes/enzymes to be present in the Idd regions. Compared to B6 mice, many of the genes correlated to the metabolic pathways, showed single nucleotide polymorphism (SNP), which can eventually affect the functionality of the protein. A genetic analysis of the increased glutamic acid revealed several Idd regions to be involved in this phenotype. The regions mapped in the genetic analysis harbor important enzymes and transporters related to glutamic acid. In-vitro islet culture with glutamic acid led to increased beta cell death indicating a toxic role of glutamic acid specifically towards insulin producing beta cells.

    In the analysis of the immune system, B cells from NOD mice, which are known to express high levels of TACI, were stimulated with APRIL, a TACI ligand. This resulted in enhanced plasma cell differentiation accompanied with increased class switching and IgG production. NOD mice have previously been shown to react vigorously to T-dependent antigens upon immunization. In this study we confirmed this as NOD mice showed an enhanced and prolonged immune response to hen egg lysozyme. Thus, serum IgG levels were significantly increased in the NOD mice and were predominantly of the IgG1 subtype. Immunofluorescence analysis revealed increased number of germinal centers in the NOD mice. Transfer of purified B and T cells from NOD to an immune deficient mouse could reproduce the original phenotype as seen in the NOD mice.    

    Collectively, this thesis has analyzed the metabolomics and immune deviations observed in the NOD mice.

  • 3.
    Banday, Viqar Showkat
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lejon, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Elevated Systemic Glutamic Acid Level in the Non-Obese Diabetic Mouse is Idd Linked and Induces Beta Cell Apoptosis2017In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 150, no 2, p. 162-171Article in journal (Refereed)
    Abstract [en]

    Although type 1 diabetes (T1D) is a T-cell-mediated disease in the effector stage, the mechanism behind the initial beta cell assault is less understood. Metabolomic differences, including elevated levels of glutamic acid, have been observed in patients with T1D before disease onset, as well as in pre-diabetic non-obese diabetic (NOD) mice. Increased levels of glutamic acid damage both neurons and beta cells, implying that this could contribute to the initial events of T1D pathogenesis. We investigated the underlying genetic factors and consequences of the increased levels of glutamic acid in NOD mice. Serum glutamic acid levels from a (NODxB6) F-2 cohort (n = 182) were measured. By genome-wide and Idd region targeted microsatellite mapping, genetic association was detected for six regions including Idd2, Idd4 and Idd22. In silico analysis of potential enzymes and transporters located in and around the mapped regions that are involved in glutamic acid metabolism consisted of alanine aminotransferase, glutamic-oxaloacetic transaminase, aldehyde dehydrogenase 18 family, alutamyl-prolyl-tRNA synthetase, glutamic acid transporters GLAST and EAAC1. Increased EAAC1 protein expression was observed in lysates from livers of NOD mice compared with B6 mice. Functional consequence of the elevated glutamic acid level in NOD mice was tested by culturing NOD. Rag2(-/-) Langerhans' islets with glutamic acid. Induction of apoptosis of the islets was detected upon glutamic acid challenge using TUNEL assay. Our results support the notion that a dysregulated metabolome could contribute to the initiation of T1D. We suggest that targeting of the increased glutamic acid in pre-diabetic patients could be used as a potential therapy.

  • 4.
    Banday, Viqar Showkat
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Thyagarajan, Radha
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lejon, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    B cell intrinsic defects lead to enhanced immune response in the NOD miceManuscript (preprint) (Other academic)
  • 5.
    Banday, Viqar Showkat
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Thyagarajan, Radha
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lejon, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Contribution of both B cell intrinsic alterations as well as non-hematopoietic derived factors in the enhanced immune response of the NOD mouse2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 252-252Article in journal (Other academic)
  • 6.
    Banday, Viqar Showkat
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Thyagarajan, Radha
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lejon, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Contribution of both B-cell intrinsic alterations as well as non-hematopoietic-derived factors in the enhanced immune response of the NOD mouse2017In: Autoimmunity, ISSN 0891-6934, E-ISSN 1607-842X, Vol. 50, no 6, p. 363-369Article in journal (Refereed)
    Abstract [en]

    The underlying cellular and molecular mechanism for the development of Type 1 diabetes is still to be fully revealed. We have previously demonstrated that the NOD mouse, a model for Type 1 diabetes, display a prolonged and enhanced immune response to both self and non-self-antigens. The molecular explanation for this defect however, has not been determined. In this study we immunized NOD and C57BL/6 (B6) with the conventional antigen i.e. hen egg lysozyme (HEL) and analyzed B cell activation, germinal center reaction and antibody clearance. Corroborating our previous observations NOD mice responded robustly to a single immunization of HEL. Immunofluorescence analysis of the spleen revealed an increased number of germinal centers in unimmunized NOD compared to B6. However, post immunization germinal center numbers were similar in NOD and B6. NOD mice showed lower response to BCR stimulation with anti-IgM, in particular at lower concentrations of anti-IgM. Antibody clearance in vivo did not differ between the strains. To determine the cell type that is responsible for the prolonged and enhance immune response, we reconstituted NOD-RAGs with cells from primed donors in different combinations. NOD B cells were required to reproduce the phenotype; however the non-lymphoid compartment of NOD origin also played a role. Based on our results we propose that preexisting GCs in the NOD promote the robust response and alteration in the BCR signaling could promote survival of stimulated cells. Overall, this mechanism could in turn also contribute to the activation and maintenance of autoreactive B cells in the NOD mouse.

  • 7.
    Banday, Viqar Showkat
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Thyagarajan, Radha
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Sundström, Mia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lejon, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Increased expression of TACI on NOD B cells results in germinal centre reaction anomalies, enhanced plasma cell differentiation and immunoglobulin production2016In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 149, no 3, p. 297-305Article in journal (Refereed)
    Abstract [en]

    B cells have an important pathogenic role in the development of type 1 diabetes in the non-obese diabetic (NOD) mouse. We have previously reported that NOD mice display an increased percentage of TACIhigh-expressing B cells compared with C57BL/6 mice and this trait is linked to chromosomes 1 and 8. In this paper the genetic association of the transmembrane activator, calcium modulator and cyclophilin ligand interactor (TACI) trait was confirmed using double congenic NOD.B6C1/Idd22 mice. TACI ligation by a proliferation-inducing ligand (APRIL) has been shown to influence plasma cell differentiation, immunoglobulin production and isotype switch. Hence, the functional consequence of the up-regulation of TACI on NOD B cells was analysed both in vitro and in vivo. NOD B cells stimulated with APRIL showed an enhanced plasma cell differentiation and class switch to IgG and IgA compared with B cells from C57BL/6 mice. Moreover, flow cytometry analyses revealed that germinal centre B cells in NOD failed to down-regulate TACI. Availability of the TACI ligand B-cell activating factor (BAFF) has been shown to be a limiting factor in the germinal centre reaction. In line with this, upon immunization with 4-hydroxy-3-nitrophenylacetyl hapten-conjugated hen egg lysozyme, NOD mice produced higher titres of low-affinity antibodies compared with C57BL/6 mice. This observation was supported by the detection of increased levels of BAFF in NOD germinal centres after immunization compared with C57BL/6 by immunofluorescence. Our results support the hypothesis that increased TACI expression on NOD B cells contributes to the pathogenesis of type 1 diabetes in the NOD mouse.

  • 8. Baptista, Marisa A. P.
    et al.
    Keszei, Marton
    Oliveira, Mariana
    Sunahara, Karen K. S.
    Andersson, John
    Dahlberg, Carin I. M.
    Worth, Austen J.
    Lieden, Agne
    Kuo, I-Chun
    Wallin, Robert P. A.
    Snapper, Scott B.
    Eidsmo, Liv
    Scheynius, Annika
    Karlsson, Mikael C. I.
    Bouma, Gerben
    Burns, Siobhan O.
    Forsell, Mattias N. E.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Thrasher, Adrian J.
    Nylén, Susanne
    Westerberg, Lisa S.
    Deletion of Wiskott-Aldrich syndrome protein triggers Rac2 activity and increased cross-presentation by dendritic cells2016In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, article id 12175Article in journal (Refereed)
    Abstract [en]

    Wiskott-Aldrich syndrome (WAS) is caused by loss-of-function mutations in the WASp gene. Decreased cellular responses in WASp-deficient cells have been interpreted to mean that WASp directly regulates these responses in WASp-sufficient cells. Here, we identify an exception to this concept and show that WASp-deficient dendritic cells have increased activation of Rac2 that support cross-presentation to CD8(+) T cells. Using two different skin pathology models, WASp-deficient mice show an accumulation of dendritic cells in the skin and increased expansion of IFN gamma-producing CD8(+) T cells in the draining lymph node and spleen. Specific deletion of WASp in dendritic cells leads to marked expansion of CD8(+) T cells at the expense of CD4(+) T cells. WASp-deficient dendritic cells induce increased cross-presentation to CD8(+) T cells by activating Rac2 that maintains a near neutral pH of phagosomes. Our data reveals an intricate balance between activation of WASp and Rac2 signalling pathways in dendritic cells.

  • 9.
    Baranov, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1), apically expressed on human colonic M cells, are potential receptors for microbial adhesion.2004In: Histochemistry and Cell Biology, ISSN 0948-6143, E-ISSN 1432-119X, Vol. 121, no 2, p. 83-9Article in journal (Refereed)
    Abstract [en]

    In the human gut mucosa, specialized M cells deliver intact foreign macromolecules and commensal bacteria from the lumen to organized mucosal lymphoid tissues triggering immune responses. M cells are also major sites of adhesion and invasion for enteric pathogens. The molecular features of M cell apical surfaces that promote microbial normal attachment are still largely unknown. We have demonstrated previously that in the human colonic epithelium, carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1) are integral components of the apical glycocalyx which participate in epithelial-microbial interactions. In this study, based on the reactivity of specific monoclonal antibodies and on immunoelectron microscopy, we show that M cells of human colonic solitary lymphoid follicles express CEA and CEACAM1 on the apical surface. Recently these highly glycosylated molecules have been characterized as protein receptors for different bacteria. This leads us to propose a role for CEA and CEACAM1 in the adherence of enteric bacteria to the apical membrane of colonic M cells. We also hypothesize that, unlike colonic enterocytes, M cells lack the defense mechanism that eliminates CEA and CEACAM1 upon microbial binding and which is based on vesiculation of microvillus plasma membrane.

  • 10.
    Baranov, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Nagaeva, Olga
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Mincheva-Nilsson, Lucia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Lipids are a constitutive component of cytolytic granules.2000In: Histochemistry and Cell Biology, ISSN 0948-6143, E-ISSN 1432-119X, Vol. 114, no 2, p. 167-71Article in journal (Refereed)
    Abstract [en]

    Cytolytic granules are specific organelles of activated cytotoxic lymphocytes mediating storage and regulated excretion of lytic molecules for killing of target cells. A variety of the other granule components may also participate in granule-mediated cytotoxicity. In this study, the subcellular localization of lipids in the granules of human decidual CD56+ natural killer-like cells was determined by staining with malachite green aldehyde and imidazole-buffered osmium tetroxide. Lipids were shown, for the first time, to be a constitutive component of cytolytic granules. Lipids formed an additional structural microdomain, located between the granule-limiting membrane and the granule core. Images of the granules on serial sections suggested that intragranular lipids wrap the core. We speculate that granule lipids participate in packing of lytic molecules inside the granules, in autocrine signaling ending granule secretion, and in the killing process.

  • 11. Baranov, Vladimir
    et al.
    Yeung, Moorix Mo-Wai
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Expression of carcinoembryonic antigen and nonspecific cross-reacting 50-kDa antigen in human normal and cancerous colon mucosa: comparative ultrastructural study with monoclonal antibodies1994In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 54, no 12, p. 3305-3314Article in journal (Refereed)
    Abstract [en]

    The precise localization of carcinoembryonic antigen (CEA) and non-specific cross-reacting 50-kDa antigen (NCA 50) in normal colon mucosa and colon adenocarcinoma was investigated by using an indirect immunoperoxidase electron microscopic technique with specific monoclonal antibodies. In normal adult colon both antigens were localized to microvesicles and filaments of the "fuzzy coat" on the apical surface of the epithelial cells. In addition, NCA 50 was found in the narrow spaces between adjoining microvilli. Mature columnar cells at the free luminal surface contained most of the antigen positive material. CEA and NCA 50 were also detected as intracellular components of goblet cells. In multilayered tumor glands, the cell surface expression of the antigens was dependent on the position of the tumor cell in the gland. The neoplastic cells showed either a predominant apical labeling or a positive staining of almost the entire cell surface. Some of the neoplastic cells contained CEA in so-called "intracellular lumina." In contrast to normal colon epithelial cells most tumor cells synthesized NCA 50 actively. In normal colonic mucosa, unlike in cancerous tissue, CEA and NCA 50 appear to be released via vesicles formed from the microvillous membrane of mature columnar cells. These results are consistent with the hypothesis that CEA and NCA play a role in the nonspecific defense against microorganisms in the large intestine.

  • 12. Barrera, Daniel Iván
    et al.
    Matheus, Luisa Marina
    Stigbrand, Torgny
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    Arbeláez, Luis Fernando
    Proteolytic hydrolysis and purification of the LRP/alfa-2-macroglobulin receptor domain from alpha-macroglobulins.2007In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 53, no 1, p. 112-8Article in journal (Refereed)
    Abstract [en]

    A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.

  • 13. Bas, A
    et al.
    Forsberg, G
    Hammarström, S
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, M-L
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes.2004In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 59, no 6, p. 566-73Article in journal (Refereed)
    Abstract [en]

    The accuracy of 18S rRNA, beta-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, beta-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of beta-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gammadeltaTCR(+), CD4(+) and CD8(+) subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30-70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.

  • 14.
    Bas, A
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Forsberg, G
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Sjöberg, Veronika
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Aberrant extrathymic T cell receptor gene rearrangement in the small intestinal mucosa: a risk factor for coeliac disease?2009In: Gut, ISSN 0017-5749, E-ISSN 1468-3288, Vol. 58, no 2, p. 189-195Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Coeliac disease is a small intestine enteropathy caused by permanent intolerance to wheat gluten. Gluten intake by patients with coeliac disease provokes a strong reaction by intestinal intraepithelial lymphocytes (IELs), which normalises on a gluten-free diet. AIM: To investigate whether impaired extrathymic T cell maturation and/or secondary T cell receptor (TCR) gene recombination in IELs are features of coeliac disease which could contribute to the failure of establishing tolerance to gluten.

    METHODS: Expression levels of the four splice-forms of recombination activating gene-1 (RAG1) mRNA and preT alpha-chain (preTalpha) mRNA were determined in IEL-subsets of children with coeliac disease and controls. Frequencies of RAG1 expressing IELs were determined by immunomorphometry.

    RESULTS: In controls, the RAG1-1A/2 splice-form selectively expressed outside the thymus, was dominant and expressed in both mature (TCR(+)) and immature (CD2(+)CD7(+)TCR(-)) IELs ( approximately 8 mRNA copies/18S rRNA U). PreTalpha was expressed almost exclusively in CD2(+)CD7(+)TCR(-) IELs ( approximately 40 mRNA copies/18S rRNA U). By contrast, RAG1 and preTalpha mRNA levels were low in patients with coeliac disease compared to controls, both with active disease and with inactive, symptom-free disease on a gluten-free diet (p values <0.01 for mature and <0.05 for immature IELs). Similarly, the frequencies of RAG1+ IELs were significantly lower in patients with coeliac disease compared to controls (p<0.001).

    CONCLUSIONS: Patients with coeliac disease appear to have an impaired capacity for extrathymic TCR gene rearrangement. This is an inherent feature, which probably plays a pivotal role in the failure to efficiently downregulate the T cell response to gluten.

  • 15. Bas, Anna
    et al.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression.2003In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 171, no 7, p. 3359-71Article in journal (Refereed)
    Abstract [en]

    Two new 5'-untranslated region (5'UTR) exons were identified in the human gene for the lymphocyte-specific endonuclease recombination activating gene-1 (RAG1) required for the somatic recombination yielding functional Ag receptors. These 5'UTR exons were used in three different splice forms by jejunal lymphocytes of the T cell lineage. RAG1 mRNA containing the previously described 5'UTR exon was not expressed in these cells. Conversely, one of the new 5'UTR exons was not expressed in thymus. The new RAG1 mRNA splice forms were all expressed in immature T cells (CD2(+)CD7(+)CD3(-)). This cell population also expressed high levels of mRNA for the pre-T alpha-chain. In situ hybridization demonstrated jejunal cells expressing the new splice forms of RAG1 mRNA, both intraepithelially and in lamina propria. Pre-T alpha-chain mRNA-expressing cells were detected at the same sites. These results strongly suggest ongoing TCR gene rearrangement in human small intestinal mucosa, yielding T cells specially adapted for this environment. This seems to be achieved by two parallel processes, extrathymic T cell development and peripheral Ag-driven TCR editing.

  • 16. Bazarian, Jeffrey J
    et al.
    Zemlan, Frank P
    Mookerjee, Sohug
    Stigbrand, Torgny
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    Serum S-100B and cleaved-tau are poor predictors of long-term outcome after mild traumatic brain injury.2006In: Brain Injury, ISSN 0269-9052, E-ISSN 1362-301X, Vol. 20, no 7, p. 759-65Article in journal (Refereed)
    Abstract [en]

    PRIMARY OBJECTIVE: To determine the relationship of serum S-100B and C-tau levels to long-term outcome after mild traumatic brain injury (mild TBI). RESEARCH DESIGN: A prospective study of 35 mild TBI subjects presenting to the emergency department. METHODS AND PROCEDURES: Six hour serum S-100B and C-tau levels compared to 3-month Rivermead Post Concussion Questionnaire (RPCQ) scores and post-concussive syndrome (PCS). MAIN OUTCOMES AND RESULTS: The linear correlation between marker levels and RPCQ scores was weak (S-100B: r = 0.071, C-tau: r = -0.21). There was no statistically significant correlation between marker levels and 3-month PCS (S-100B: AUC = 0.589, 95%CI. 038, 0.80; C-tau: AUC = 0.634, 95%CI 0.43, 0.84). The sensitivity of these markers ranged from 43.8-56.3% and the specificity from 35.7-71.4%. CONCLUSIONS: Initial serum S-100B and C-tau levels appear to be poor predictors of 3-month outcome after mild TBI.

  • 17.
    Bitar, Aziz
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    De, Rituparna
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Melgar, Silvia
    Aung, Kyaw Min
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rahman, Arman
    Qadri, Firdausi
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Shirin, Tahmina
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Induction of immunomodulatory miR-146a and miR-155 in small intestinal epithelium of Vibrio cholerae infected patients at acute stage of cholera2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 3, article id 0173817Article in journal (Refereed)
    Abstract [en]

    The potential immunomodulatory role of microRNAs in small intestine of patients with acute watery diarrhea caused by Vibrio cholerae O1 or enterotoxigenic Escherichia coli (ETEC) infection was investigated. Duodenal biopsies were obtained from study-participants at the acute (day 2) and convalescent (day 21) stages of disease, and from healthy individuals. Levels of miR-146a, miR-155 and miR-375 and target gene (IRAK1, TRAF6, CARD10) and 11 cytokine mRNAs were determined by qRT-PCR. The cellular source of microRNAs in biopsies was analyzed by in situ hybridization. The ability of V. cholerae bacteria and their secreted products to cause changes in microRNA- and mRNA levels in polarized tight monolayers of intestinal epithelial cells was investigated. miR-146a and miR-155 were expressed at significantly elevated levels at acute stage of V. cholerae infection and declined to normal at convalescent stage (P<0.009 versus controls; P = 0.03 versus convalescent stage, pairwise). Both microRNAs were mainly expressed in the epithelium. Only marginal down-regulation of target genes IRAK1 and CARD10 was seen and a weak cytokine-profile was identified in the acute infected mucosa. No elevation of microRNA levels was seen in ETEC infection. Challenge of tight monolayers with the wild type V. cholerae O1 strain C6706 and clinical isolates from two study-participants, caused significant increase in miR-155 and miR-146a by the strain C6706 (P<0.01). One clinical isolate caused reduction in IRAK1 levels (P<0.05) and none of the strains induced inflammatory cytokines. In contrast, secreted factors from these strains caused markedly increased levels of IL-8, IL-1β, and CARD10 (P<0.001), without inducing microRNA expression. Thus, miR-146a and miR-155 are expressed in the duodenal epithelium at the acute stage of cholera. The inducer is probably the V. cholerae bacterium. By inducing microRNAs the bacterium can limit the innate immune response of the host, including inflammation evoked by its own secreted factors, thereby decreasing the risk of being eliminated.

  • 18.
    Bitar, Aziz
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Kyaw, Min Aung
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Marie-Louise, Hammarström
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Wai, Sun Nynt
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Vibrio cholerae derived outer membrane vesicles modulate the inflammatory response ofhuman intestinal epithelial cells by inducing microRNA-146aIn: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322Article in journal (Refereed)
    Abstract [en]

    The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulencefactors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wild-type and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α(TNF-α), CCL20, IL-1β, and IRAK2 mRNA levels while challenge with OMVs did not causeany changes. Notably, OMVs from the VCC deficient mutant caused significant decreases in CCL20 and IL-18 mRNA levels. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing thestrength of an epithelial innate immune defence reaction and also preventing inflammation inthe mucosa that factors like VCC can evoke.

  • 19. Bjerner, J
    et al.
    Lebedin, Y
    Bellanger, L
    Kuroki, M
    Shively, J E
    Varaas, T
    Nustad, K
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Børmer, O P
    Protein epitopes in carcinoembryonic antigen. Report of the ISOBM TD8 workshop.2002In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 23, no 4, p. 249-62Article in journal (Refereed)
    Abstract [en]

    To characterize antigenic sites in carcinoembryonic antigen (CEA) further and to investigate whether there are differences between colon tumor CEA and meconium CEA (NCA-2) that can be detected by anti-CEA monoclonal antibodies (MAb), 19 new anti-CEA MAb were analyzed with respect to specificity, epitope reactivity and affinity. Their reactivities were compared with 10 anti-CEA MAb with known CEA-domain binding specificity that have previously been classified into five nonoverlapping epitope groups, GOLD 1-5. Cross-inhibition assays with antigen-coated microtiter plates and immunoradiometric assays were performed in almost all combinations of MAbs, using conventionally purified CEA (domain structure: N-A1B1-A2B2-A3B3-C) from liver metastasis of colorectal carcinomas, recombinant CEA, meconium CEA (NCA-2), truncated forms of CEA and NCA (CEACAM6) as the antigens. The affinity of the MAbs for CEA was also determined. The new MAbs were generally of high affinity and suitable for immunoassays. Three new MAbs were assigned to GOLD epitope group 5 (N-domain binding), 3 MAbs to group 4 (A1B1 domain), 1 to group 3 (A3B3 domain), 3 to group 2 (A2B2 domain) and 3 to group 1 (also the A3B3 domain). Three MAbs formed a separate group related to group 4, they were classified as GOLD 4' (A1B1 domain binding). The remaining 3 MAbs appear to represent new subspecificities with some relationship to GOLD groups 1, 2 or 4, respectively. Five MAbs, all belonging to epitope group 1 and 3, reacted strongly with tumor CEA but only weakly or not at all with meconium CEA, demonstrating that the two products of the CEA gene differ from each other, probably due to different posttranslational modifications.

  • 20.
    Blomberg, Jeanette
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Höglund, Andreas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Eriksson, David
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Ruuth, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Jacobsson, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nilsson, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lundgren, Erik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Inhibition of cellular FLICE-like inhibitory protein abolishes insensitivity to interferon-α in a resistant variant of the human U937 cell line2011In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 16, no 8, p. 783-794Article in journal (Refereed)
    Abstract [en]

    Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.

  • 21.
    Brink, Mikael
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Reumatology.
    Lejon, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Ärlestig, Lisbeth
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Reumatology.
    Rantapää-Dahlqvist, Solbritt M.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Reumatology.
    B-Regulatory-, CD19(+)CD20(+) CD24(high)CD38(high) -Cells Are Functionally Impaired In Patients With Rheumatoid Arthritis and Healthy First Degree Relatives Compared With Controls2013In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 65, no Special issue, Supplement 10, p. S393-S394, Meeting Abstract: 917Article in journal (Other academic)
  • 22.
    Brink, Mikael
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Reumatology.
    Ärlestig, Lisbeth
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Reumatology.
    Rantapää-Dahlqvist, Solbritt
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Reumatology.
    Lejon, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    B Regulatory Cells are Functionally Impaired in Patients with Rheumatoid Arthritis and in Their First-Degree Relatives Compared with Controls2014In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 79, no 6, p. 450-450Article in journal (Other academic)
  • 23. Colucci, Francesco
    et al.
    Simpson, Elizabeth
    McLaren, Anne
    Hayakawa, Satoshi
    Andersson, Elisabet
    Mincheva-Nilsson, Lucia
    Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Clinical Microbiology.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    "A Japanese gentleman of the Samurai tradition": Takeshi Matsunaga 1945-2003.2003In: Immunogenetics, ISSN 0093-7711, E-ISSN 1432-1211, Vol. 55, no 8, p. 515-20Article in journal (Refereed)
  • 24. Damoiseaux, Jan
    et al.
    Heijnen, Ingmar
    Van Campenhout, Christel
    Eriksson, Catharina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Fabien, Nicole
    Herold, Manfred
    van der Molen, Renate G.
    Egner, William
    Patel, Dina
    Plaza-Lopez, Aresio
    Radice, Antonella
    Rego de Sousa, Marie Jose
    Viander, Markku
    Shoenfeld, Yehuda
    An international survey on anti-neutrophil cytoplasmic antibodies (ANCA) testing in daily clinical practice2018In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 56, no 10, p. 1759-1770Article in journal (Refereed)
    Abstract [en]

    Background: Detection of anti-neutrophil cytoplasmic antibodies (ANCA) is important for the diagnosis of the ANCA-associated vasculitides (AAV). For AAV, especially ANCA directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are most relevant. ANCA with less well-defined specificities may, however, also be detected in other inflammatory and non-inflammatory conditions.

    Methods: A questionnaire, initiated by the European Autoimmunity Standardisation Initiative (EASI), was used to gather information on methods and testing algorithms used for ANCA in clinical laboratories of 12 European countries (EASI survey).

    Results: Four hundred and twenty-nine responses were included in the EASI survey analysis which revealed differences within countries and between countries. Laboratories overall were poor in adherence to international consensus on ANCA testing. Substantial variation was observed with respect to the use of ANCA indirect immunofluorescence (IIF) in the algorithm, application of distinct methods for MPO- and PR3-ANCA, the daily availability of new ANCA results, and interpretation of test results.

    Conclusions: Awareness of these differences may stimulate further harmonization and standardization of ANCA testing. This may be promoted by an update of the international ANCA consensus and the introduction of international standards.

  • 25.
    Ekici, Rifat
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    B cells in Type 1 diabetes: studies on cell surface antibody binding2010Licentiate thesis, monograph (Other academic)
  • 26.
    Ekici, Rifat
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Sundström, Mia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Thay, Bernard
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology.
    Lejon, Kristina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Biomedical Laboratory Science.
    Enhanced capture of extramembranous IgM and IgG on B cells in the NOD mouse: implications for immune complex trapping2009In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 21, no 5, p. 533-541Article in journal (Refereed)
    Abstract [en]

    Binding of various antibody isotypes to B cells through either FcgammaRs or complement receptors has been attributed to play several roles, e.g. in immune complex (IC) transportation and regulation of B cell receptor signaling. We have revealed a novel B cell intrinsic receptor for IgM and IgG which is present in C57BL/6 (B6) mice and is more abundant in non-obese diabetic (NOD) mice. As a consequence, the level of extramembranous IgG monomers and IgM pentamers on peripheral blood B cells from NOD mice was significantly higher compared with B6 mice. The effect of this aberration was that all B cells in peripheral blood of (NOD.IgH(a) x B6(IgH(b)))F(1) mice carried both IgM allotypes on their surface. In addition, analysis of IC binding using IgG- or IgM-opsonized bacterial particles revealed a higher degree of binding in NOD mice compared with B6. We hypothesize that this novel Ig-binding receptor is part of the normal immune system function. The aberrant function in the NOD mouse could contribute to the development of Type 1 diabetes by altering normal B cell functions such as activation, IC transportation and B cell homeostasis.

  • 27.
    Eriksson, David
    Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Radiation Sciences, Diagnostic Radiology. Umeå University, Faculty of Medicine, Radiation Sciences, Radiation Physics.
    Experimental radioimmunotherapy and effector mechanisms2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Radioimmunotherapy is becoming important as a new therapeutic strategy for treatment of tumour diseases. Lately monoclonal antibodies tagged with radionuclides have demonstrated encouraging results in treatment of hematological malignancies. The progress in treatment of solid tumours using radioimmunotherapy, however, has been slow. New strategies to improve the treatment response need to be evaluated. Such new strategies include the combination of radioimmunotherapy with other treatment modalities but also elucidation and exploration of the death effector mechanisms involved in tumour eradication.

    As the combination of radioimmunotherapy and radiotherapy provides several potential synergistic effects, we started out by optimising a treatment schedule to detect benefits combining these treatment modalities. An anti-cytokeratin antibody labelled with 125I administered before, after, or simultaneously with radiotherapy, indicated that the highest dose to the tumour was delivered when radiotherapy was given prior to the antibody administration. The optimised treatment schedule was then applied therapeutically in an experimental study on HeLa Hep2 tumour bearing nude mice given radiotherapy prior to administration of 131I-labelled monoclonal antibodies. Combining these treatment regimes enhanced the effect of either of the treatment modalities given alone, and a significant reduction in tumour volumes could be demonstrated. This treatment caused a dramatic change in tumour morphology, with increased amounts of connective tissue, giant cells and cysts. Furthermore cellular alterations like heterogeneity of nuclear and cytoplasmic size and shape were observed, and at least a fraction of the tumour cells presented some characteristics of apoptosis.

    The induced sequential events in Hela Hep2 cells exposed to 2.5-10 Gy of ionizing radiation were studied further, with special emphasis on cell cycle arrest, mitotic aberrations and finally cell death. Following radiation HeLa Hep2 cells initiated a transient G2/M arrest trying to repair cellular damage. This arrest was followed by a sequence of disturbed mitoses with anaphase bridges, lagging chromosomal material, hyperamplification of centrosomes and multipolar mitotic spindles. These mitotic disturbances produced multinuclear polyploid cells and cells with multiple micronuclei, cells that were destined to die via mitotic catastrophes and delayed apoptosis.

    Induction of apoptosis in HeLa Hep2 cells following radiation doses and dose-rates equivalent to those delivered at radioimmunotherapy was concurrently studied in vitro. Significant induction of apoptosis was obtained and found to be induced relatively slowly, peaking 72-168 hours post irradiation. Caspases from the intrinsic pathway as well as the extrinsic pathway were found to be activated in response to ionizing radiation. Furthermore caspase-2, which has recently been acknowledged for its role as an initiator caspase was found to be activated following radiation and seems to play an important role in this delayed apoptosis.

  • 28.
    Eriksson, David
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Blomberg, Jeanette
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lindgren, Theres
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Löfroth, Per-Olov
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Radiation Physics.
    Johansson, Lennart
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Radiation Physics.
    Riklund, Katrine
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Diagnostic Radiology.
    Stigbrand, Torgny
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Iodine-131 induces mitotic catastrophes and activates apoptotic pathways in HeLa Hep2 cells2008In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 23, no 5, p. 541-549Article in journal (Refereed)
    Abstract [en]

    Iodine-131 (131I) has been used both in unconjugated form and conjugated to antibody derivates (i.e., radioimmunotherapy; RIT) to treat malignant diseases. The mechanisms by which 131I-irradiation causes growth retardation are, however, inadequately understood. The aim of this study was to elucidate the sequential molecular and cellular events that initiate cell death in HeLa Hep2 cells exposed to 131I. In this paper, HeLa Hep2 cells were found to display a transient G2-M arrest following irradiation, but then reentered the cell cycle still containing unrepaired cellular damage. An increase of multipolar mitotic spindles, as well as a significant increase in centrosome numbers from 8.8% +/- 1.9% in controls to 54.7% +/- 2.2% in irradiated cells, was observed (p < 0.0001). A subsequent failure of cytokinesis caused the cells to progress into mitotic catastrophe. This was accompanied by the formation of giant cells with multiple nuclei, multilobulated nuclei, and an increased frequency of polyploidy cells. A fraction of the cells also displayed apoptotic features, including the activation of initiator caspases-2, -8, -9, and effector caspase-3, as well as cleavage of poly(ADP-ribose) polymerase, a cell-death substrate for active caspase-3. These findings demonstrate that mitotic catastrophes and the activation of a delayed type of apoptosis might be important mechanisms involved in cell death following the RIT of solid tumors with -emitting radionuclides, such as 131I.

  • 29.
    Eriksson, David
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Löfroth, Per-Olov
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Radiation Physics.
    Johansson, Lennart
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Radiation Physics.
    Riklund, Katrine
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Diagnostic Radiology.
    Stigbrand, Torgny
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Apoptotic signalling in HeLa Hep2 cells following 5 Gy of cobalt-60 gamma radiation2009In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 29, no 11, p. 4361-4366Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The apoptotic signalling pathways involved in the delayed type of apoptosis occurring in HeLa Hep2 cells following radiation were investigated. MATERIALS AND METHODS: HeLa Hep2 cells were exposed to 5 Gy of cobalt-60 radiation. The activation of caspase-2, caspase-8, caspase-9 and effector caspase-3 was investigated by caspase assay plates and Western blots. Cleavage of poly (ADP-ribose) polymerase (PARP) was analysed on Western blots. HeLa Hep2 cells were irradiated with or without preincubation with inhibitors of protein synthesis (cycloheximide, CHX) and caspases, followed by TUNEL staining and caspase assay plate evaluation. RESULTS: Initiator caspases-2, -8, -9, and effector caspase-3, were found to be activated and PARP cleaved following irradiation. CHX completely inhibited the caspase activation and the associated apoptosis. Pretreatment with caspase-2 inhibitor indicated that caspase-2 was involved in the execution of the apoptosis. CONCLUSION: Activation of the apoptotic signalling pathways following irradiation of HeLa Hep2 cells includes components from the intrinsic as well as the extrinsic pathways and seems to require de novo protein synthesis.

  • 30.
    Eriksson, David
    et al.
    Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    Löfroth, Per-Olov
    Umeå University, Faculty of Medicine, Radiation Sciences, Radiation Physics.
    Johansson, Lennart
    Umeå University, Faculty of Medicine, Radiation Sciences, Radiation Physics.
    Åhlström Riklund, Katrine
    Umeå University, Faculty of Medicine, Radiation Sciences, Diagnostic Radiology.
    Stigbrand, Torgny
    Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    Cell cycle disturbances and mitotic catastrophes in HeLa Hep2 cells following 2.5 to 10 Gy of ionizing radiation.2007In: Clin Cancer Res, ISSN 1078-0432, Vol. 13, no 18 Pt 2, p. 5501s-5508sArticle in journal (Refereed)
    Abstract [en]

    PURPOSE: Experimental radioimmunotherapy delivering absorbed doses of 2.5 to 10 Gy has been shown to cause growth retardation of tumors. The purpose of this study was to elucidate the sequential molecular and cellular events occurring in HeLa Hep2 cells exposed to such doses. METHODS: Dose-response curves, activation of cell cycle checkpoints, and mitotic behavior were investigated in HeLa Hep2 cells following 2.5- to 10-Gy irradiation by carrying out 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Western blots, fluorescence-activated cell sorting analysis, and immunofluorescence stainings. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining was used to detect apoptosis. RESULTS: A G2-M arrest was shown by fluorescence-activated cell sorting analysis. p53 and p21 were found to be up-regulated but were not immediately related to the arrest. The G2-M arrest was transient and the cells reentered the cell cycle still containing unrepaired cellular damage. This premature entry caused an increase of anaphase bridges, lagging chromosomal material, and multipolar mitotic spindles as visualized by propidium iodide staining and immunofluorescence staining with alpha-tubulin and gamma-tubulin antibodies. Furthermore, a dose-dependent significant increase in centrosome numbers from 12.6+/-6.6% to 67+/-5.3% was identified as well as a dose-dependent increase of polyploid cells from 2.8+/-1.3% to 17.6+/-2.1% with the highest absorbed dose of 10 Gy. These disturbances caused the cells to progress into mitotic catastrophe and a fraction of these dying cells showed apoptotic features as displayed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining 5 to 7 days after irradiation. CONCLUSION: An absorbed dose of 2.5 to 10 Gy was shown to force HeLa Hep2 cells into mitotic catastrophe and delayed apoptosis. These might be important cell death mechanisms involved in tumor growth retardation following radioimmunotherapy of solid tumors.

  • 31.
    Eriksson, David
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Stigbrand, Torgny
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Radiation-induced cell death mechanisms2010In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 31, no 4, p. 363-372Article in journal (Refereed)
    Abstract [en]

    The main goal when treating malignancies with radiation therapy is to deprive tumor cells of their reproductive potential. One approach to achieve this is by inducing tumor cell apoptosis. Accumulating evidences suggest that induction of apoptosis alone is insufficient to account for the therapeutic effect of radiotherapy. It has become obvious in the last few years that inhibition of the proliferative capacity of malignant cells following irradiation, especially with solid tumors, can occur via alternative cell death modalities or permanent cell cycle arrests, i.e., senescence. In this review, apoptosis and mitotic catastrophe, the two major cell deaths induced by radiation, are described and dissected in terms of activating mechanisms. Furthermore, treatment-induced senescence and its relevance for the outcome of radiotherapy of cancer will be discussed. The importance of p53 for the induction and execution of these different types of cell deaths is highlighted. The efficiency of radiotherapy and radioimmunotherapy has much to gain by understanding the cell death mechanisms that are induced in tumor cells following irradiation. Strategies to use specific inhibitors that will manipulate key molecules in these pathways in combination with radiation might potentiate therapy and enhance tumor cell kill.

  • 32. Erlandsson, Ann
    et al.
    Holm, Patrik
    Jafari, Rozbeh
    Stigbrand, Torgny
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Sundström, Birgitta E
    Functional mapping of the anti-idiotypic antibody anti-TS1 scFv using site-directed mutagenesis and kinetic analysis2010In: mAbs, ISSN 1942-0870, Vol. 2, no 6, p. 662-669Article in journal (Refereed)
    Abstract [en]

    Recombinant antibodies may be engineered to obtain improved functional properties. Functional mapping of the residues in the binding surfaces is of importance for predicting alterations needed to yield the desired properties. In this investigation, 17 single mutation mutant single-chain variable fragments (scFvs) of the anti-idiotypic antibody anti-TS1 were generated in order to functionally map amino acid residues important for the interaction with its idiotype TS1. Residues in anti-TS1 determined to be very important for the interaction were identified, Y32L, K50L, K33H, and Y52H, and they were distributed adjacent to a centrally located hydrophobic area, and contributed extensively to the interaction energy (≥2.5 kcal/mol) in the interaction. Quantitative ELISA assays, BIAcore technologies and three-dimensional surface analysis by modeling were employed to visualize the consequences of the mutations. The expression levels varied between 2 - 1,800 nM as determined by ELISA. All the 17 scFvs displayed higher dissociation rates (60 - 1,300 times) and all but two of them also faster association rates (1.3 - 56 times). The decrease in affinity was determined to be 1.6 - 12,200 times. Two of the mutants displayed almost identical affinity with the wild type anti-TS1, but with a change in both association and dissociation rates. The present investigation demonstrates that it is possible to generate a large panorama of anti-idiotypic antibodies, and single out a few that might be of potential use for future clearing and pre-targeting purposes of idiotypic-anti-idiotypic interactions.

  • 33.
    Fahlgren, A
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, S
    Danielsson, A
    Hammarström, M-L
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.2003In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 131, no 1Article in journal (Refereed)
    Abstract [en]

    The impact of chronic inflammation on the expression of human alpha-defensins 5 and 6 (HD-5, HD-6), beta-defensins 1 and 2 (hBD-1, hBD-2) and lysozyme in epithelial cells of small and large intestine was investigated. Intestinal specimens from 16 patients with ulcerative colitis (UC), 14 patients with Crohn's disease (CD) and 40 controls with no history of inflammatory bowel disease were studied. mRNA expression levels of the five defence molecules were determined in freshly isolated epithelial cells by real-time quantitative RT-PCR. Specific copy standards were used allowing comparison between the expression levels of the different defensins. HD-5 and lysozyme protein expression was also studied by immunohistochemistry. Colonic epithelial cells from patients with UC displayed a significant increase of hBD-2, HD-5, HD-6 and lysozyme mRNA as compared to epithelial cells in controls. Lysozyme mRNA was expressed at very high average copy numbers followed by HD-5, HD-6, hBD-1 and hBD-2 mRNA. HD-5 and lysozyme protein was demonstrated in metaplastic Paneth-like cells in UC colon. There was no correlation between hBD-2 mRNA levels and HD-5 or HD-6 mRNA levels in colon epithelial cells of UC patients. Colonic epithelial cells of Crohn's colitis patients showed increased mRNA levels of HD-5 and lysozyme mRNA whereas ileal epithelial cells of Crohn's patients with ileo-caecal inflammation did not. Chronic inflammation in colon results in induction of hBD-2 and alpha-defensins and increased lysozyme expression. hBD-1 expression levels in colon remain unchanged in colitis. The high antimicrobial activity of epithelial cells in chronic colitis may be a consequence of changes in the epithelial lining, permitting adherence of both pathogenic bacteria and commensals directly to the epithelial cell surface.

  • 34.
    Fahlgren, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Frängsmyr, L
    Zoubir, F
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Interferon-gamma tempers the expression of carcinoembryonic antigen family molecules in human colon cells: a possible role in innate mucosal defence.2003In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 58, no 6, p. 628-41Article in journal (Refereed)
    Abstract [en]

    Four carcinoembryonic antigen-related cell adhesion molecule (CEACAM)s, i.e. CEA, CEACAM1, CEACAM6 and CEACAM7, are localized to the apical glycocalyx of normal colonic epithelium and have been suggested to play a role in innate immunity. The expression of these molecules in colon carcinoma cells was studied at the mRNA and protein levels after treatment with interferon-gamma (IFN-gamma), interleukin-1beta, live bacteria or lipopolysaccharide. The colon carcinoma cell lines LS174T and HT-29 were studied in detail using real-time quantitative reverse transcriptase-polymerase chain reaction, immunoflow cytometry and immunoelectron microscopy. IFN-gamma, but not the other agents, modified expression of CEA, CEACAM1 and CEACAM6. None of the agents upregulated CEACAM7 expression. Two expression patterns were seen. HT-29 cells, which initially showed low quantities of mRNAs and proteins, displayed marked upregulation of both mRNAs and proteins. LS174T cells transcribed stable high levels of mRNA before and after treatment. Additionally, IFN-gamma induced increased cell surface expression of CEA, CEACAM1 and CECAM6. IFN-gamma has two important effects on the expression levels of the CEA family molecules in colon epithelial cells: direct upregulation of CEACAM1 and promotion of cell differentiation resulting in increased expression of CEA and CEACAM6 and decreased expression of CEACAM7.

  • 35.
    Fahlgren, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Baranov, Vladimir
    Frängsmyr, Lars
    Zoubir, Fairouz
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Interferon-γ tempers the expression of carcinoembryonic antigen (CEA) family molecules – a role in innate colonic defence.2003In: Scandinavian Journal of Immunology, Vol. 58, no 6, p. 628-641Article in journal (Refereed)
  • 36.
    Fahlgren, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Danielsson, Åke
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.2003In: Clinical Experimental Immunology, Vol. 131, no 1, p. 90-101Article in journal (Refereed)
  • 37.
    Forsberg, Göte
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Innate and adaptive immunity in childhood celiac disease2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Celiac disease (CD) is an inflammatory small-bowel enteropathy caused by a permanent intolerance to wheat gluten and related proteins in rye and barley. Even though the disease originate from the small intestine the clinical symptoms varies in affected individuals and are often different in small children compared to adolescents and adults. Susceptibility to develop the disease is strongly associated with certain genetic factors i.e. HLADQ2/DQ8 but it is undoubtedly that additional inherited and environmental factors are involved. As specific T lymphocyte reactions are central in the pathogenesis of CD, six key cytokine messenger RNA levels in intestinal intraepithelial and lamina propria T lymphocytes (IEL, LPL), retrieved from small intestinal biopsies, were determined by using quantitative real-time reverse transcription polymerase chain reaction (RTPCR). Levels of cytokines, small secreted proteins which mediate and regulate immunity, in children with active disease were compared with that of treated children and controls. Interferon (IFN)-γ and interleukin (IL)-10 were also determined at the protein level by immunohistochemistry. Active celiac disease was characterized by distortions in cytokine expression, with highly significant increases of IFN-γ and IL-10 but no concomitant increases in tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGFβ1), or IL-2 and no induction of IL-4. A marked shift of IFN-γ and IL-10 production from LPLs to IELs was characteristic of active celiac disease, and as many as one fourth of the IELs expressed IFN-γ. IELs in treated, symptom-free celiac patients still had increased IFN-γ levels compared with controls. In CD, gluten intake seems to cause an overreaction in IELs, with uncontrolled production of IFN-γ and IL-10 which may cause both recruitment of more IELs and a leaky epithelium, leading to a vicious circle with amplified immune activity and establishment of the intestinal lesion. In order to determine different IEL subsets contribution of the produced cytokines, γδIELs, CD4+αβIELs, and CD8+αβIELs as well as CD94+CD8+αβIELs and CD94CD8+αβIELs of children with active CD and children with no food-intolerance were analyzed for cytokine mRNA expression levels by RT-PCR. In active CD, CD8+αβIELs had the highest expression levels of IFN-γ- and IL-10 mRNA and constituted the cellular source for almost all IFN-γ and a large fraction of the IL-10. Expression levels of these two cytokines correlated and were higher in CD94-CD8+αβIELs than CD94+CD8+αβIELs CD4+αβIELs had the highest expression levels of TNF-α and despite the small number of this cell subset they contributed with half of the small amounts of this cytokine. Interestingly, TNF-α levels correlated with IL-10 in CD4+αβIELs. γδIELs had the lowest expression levels of IFN-γ, TNF-α, IL-10, and TGF-β1. Essentially no IL-2 mRNA was detected in the three IEL subpopulations. “Classical” CD8+CD94-αβT cells in the epithelial compartment are responsible for most of the excessive production of proinflammatory IFN-γ. The question whether an impaired extrathymic T cell maturation and/or capacity for secondary T cell receptor (TCR) gene recombination in iIELs is a contributing factor to CD was addressed. Expression levels of recombination activating gene-1 (RAG1) and the pre T α-chain (preTα) mRNAs were determined in IEL T cell lineage subsets of children with CD and controls. In controls, RAG1 was expressed in both mature (TCRγδ+ and TCRαβ+) and immature (CD2+CD7+TCR-) IELs while preTα was expressed preferentially in immature IELs. The RAG1 splice form selectively expressed outside thymus (RAG1 1A/2) as well as preTα were significantly decreased in CD patients both in active and inactive disease suggesting a deteriorated capacity of de novo TCR gene rearrangement in local T cell development and / or of secondary TCR gene rearrangement during editing or antigen-driven revision. This may lead to an imbalance between thymus- and gut derived T lymphocytes in the intestinal mucosa with consequent inefficient regulation of T cell responses against food antigens. Innate or nonspecific immunity is the first line, immediate defense against pathogens mediated by the epithelial cells in the intestine (IECs). As certain adaptive immune reaction in CD mimics that of intestinal infections, aberrant innate immune reaction could be a contributing factor to CD. Therefore jejunal biopsies were screened for bacteria and the innate immune status of the epithelium was investigated. Bacteria were freqently (40%) associated with the mucosa of children with active but also treated disease (20%) compared to controls (2%). Lack of antimicrobial factors such as mucins, proteins forming protective biofilm on the IECs, defensins and lysozym, peptides and enzymes with antibacterial effects, could not explain the presence of bacteria. If anything, mucin-2 (MUC2), α-defensins, HD-5, HD-6, and lysozyme mRNA levels were increased in epithelial cells in active CD, returning to normal levels in treated CD. Their expression levels correlated to the IFN-γ mRNA levels in IELs. Analysis of beta defensins, hBD-1 and hBD-2 as well as carcinoembryonic antigen (CEA) cell adhesion molecule 1a (CECAM1a), glycoproteins in the glycocalyx with ability to bind micro organisms, were not affected by the disease. Lectin staining by histochemistry revealed that goblet cells were stained by UEA1 in CD both active and treated but not in controls. The opposite pattern was seen for the lectin PNA where staining was seen in controls in the glycocalyx layer but not in CD. Thus altered glycocalyx/mucous layer may promote bacterial adhesion in CD.

  • 38.
    Forsberg, Göte
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hörstedt, Per
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease2004In: American Journal of Gastroenterology, ISSN 0002-9270, E-ISSN 1572-0241, Vol. 99, no 5, p. 894-904Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: Exposure to gliadin and related prolamins and appropriate HLA-DQ haplotype are necessary but not sufficient for contracting celiac disease (CD). Aberrant innate immune reactions could be contributing risk factors. Therefore, jejunal biopsies were screened for bacteria and the innate immune status of the epithelium investigated.

    METHODS: Children with untreated, treated, challenged CD, and controls were analyzed. Bacteria were identified by scanning electron microscopy. Glycocalyx composition and mucin and antimicrobial peptide production were studied by quantitative RT-PCR, antibody and lectin immunohistochemistry.

    RESULTS: Rod-shaped bacteria were frequently associated with the mucosa of CD patients, with both active and inactive disease, but not with controls. The lectin Ulex europaeus agglutinin I (UEAI) stained goblet cells in the mucosa of all CD patients but not of controls. The lectin peanut agglutinin (PNA) stained glycocalyx of controls but not of CD patients. mRNA levels of mucin-2 (MUC2), alpha-defensins HD-5 and HD-6, and lysozyme were significantly increased in active CD and returned to normal in treated CD. Their expression levels correlated to the interferon-gamma mRNA levels in intraepithelial lymphocytes. MUC2, HD-5, and lysozyme proteins were seen in absorptive epithelial cells. beta-defensins hBD-1 and hBD-2, carcinoembryonic antigen (CEA), CEA cell adhesion molecule-1a (CEACAM1a), and MUC3 were not affected.

    CONCLUSIONS: Unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients. These glycosylation differences could facilitate bacterial adhesion. Ectopic production of MUC2, HD-5, and lysozyme in active CD is compatible with goblet and Paneth cell metaplasia induced by high interferon-gamma production by intraepithelial lymphocytes.

  • 39.
    Forsberg, Göte
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hernell, O
    Umeå University, Faculty of Medicine, Department of Clinical Sciences.
    Melgar, S
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Israelsson, A
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Paradoxical coexpression of proinflammatory and down-regulatory cytokines in intestinal T cells in childhood celiac disease2002In: Gastroenterology, ISSN 0016-5085, E-ISSN 1528-0012, Vol. 123, no 3, p. 667-678Article in journal (Refereed)
  • 40.
    Forsberg, Göte
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Concomitant increase of IL-10 and pro-inflammatory cytokines in intraepithelial lymphocyte subsets in celiac disease.2007In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 19, no 8, p. 993-1001Article in journal (Refereed)
    Abstract [en]

    Celiac disease (CD) is a small intestinal enteropathy caused by permanent intolerance to wheat gluten. Active disease is characterized by a prominent cytokine response of intraepithelial lymphocytes (IELs) to gluten-containing diet with concomitant increase in expression of pro-inflammatory IFN-gamma and down-regulatory IL-10 without increase in tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). The aim was to understand the local immune reaction by determining which intraepithelial T cell subsets produce the different cytokines. The three major IEL-subsets gammadeltaIELs, CD4(+)alphabetaIELs and CD8(+)alphabetaIELs, as well as CD94(+)CD8(+)alphabetaIELs, selectively expanded in active CD, were retrieved from small intestinal biopsies of children with active CD and controls and analyzed quantitatively for cytokine mRNA expression. In active CD, CD8(+)alphabetaIELs showed a significant increase in expression levels of both IFN-gamma and IL-10. CD8(+)alphabetaIELs were also the IEL subset with highest expression level per cell of both cytokines and constituted the cellular source for almost all IFN-gamma and most IL-10. Expression levels of both cytokines were higher in CD94(-)CD8(+)alphabetaIELs than CD94(+)CD8(+)alphabetaIELs. TNF-alpha levels were only increased in CD4(+)alphabetaIELs, which also showed the highest expression level per cell and constituted the major source of this cytokine. Interestingly, IL-10 was increased also in CD4(+)alphabetaIELs. Cytokine levels were low in gammadeltaIELs. 'Classical' CD94(-)CD8(+)alphabeta T cells within the epithelium are responsible for the excessive production of IFN-gamma, believed to drive the formation of intestinal lesions in active CD. Production of IL-10 may be a common feature of IELs producing pro-inflammatory cytokines, thereby attempting to limit inflammation in an autocrine fashion.

  • 41.
    Forsell, Mattias N. E.
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Kvastad, Linda
    Sedimbi, Saikiran K.
    Andersson, John
    Karlsson, Mikael C. I.
    Regulation of Subunit-Specific Germinal Center B Cell Responses to the HIV-1 Envelope Glycoproteins by Antibody-Mediated Feedback2017In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, article id 738Article in journal (Refereed)
    Abstract [en]

    The regulation of germinal center (GC) B cell responses to single epitopes is well investigated. How monoclonal B cells are regulated within the polyclonal B cell response to protein antigens is less so. Here, we investigate the primary GC B cell response after injection of mice with HIV-1 envelope glycoproteins. We demonstrate that single GCs are seeded by a diverse number of B cell clones shortly after a single immunization and that the presence of Env-specific antibodies can inhibit the development of early GC B cells. Importantly, the suppression was dependent on the GC B cells and the infused antibodies to target the same subunit of the injected HIV-1 envelope glycoproteins. An affinity-dependent antibody feedback has previously been shown to regulate GC B cell development. Here, we propose that this antibody-based feedback acts on GC B cells only if they target the same or overlapping epitopes. This study provides important basic information of GC B cell regulation, and for future vaccine designs with aim to elicit neutralizing antibodies against HIV-1.

  • 42.
    Frängsmyr, Lars
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Prall, F
    Yeung, Moorix Mo-Wai
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Wagener, C
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Cell- and region-specific expression of biliary glycoprotein and its messenger RNA in normal human colonic mucosa1995In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 55, no 14, p. 2963-2967Article in journal (Refereed)
    Abstract [en]

    The localization of biliary glycoprotein (BGP) and its mRNA in normal colonic mucosa was studied by immunohistochemistry and in situ hybridization. BGP mRNA was confined to columnar epithelial cells and expressed abundantly in the superficial mature cells and at low levels in differentiating cells in the upper crypts. Epithelial expression of BGP coincided with that of BGP mRNA. Ultrastructurally, BGP was localized to microfilaments of the fuzzy coat of the columnar cells at the luminal surface and the upper crypts. Additionally, BGP was found in cryptal caveolated cells. The results are consistent with primary transcriptional regulation of BGP production and suggest that BGP synthesis is controlled by the degree of cytodifferentiation. The fuzzy-coat localization of BGP implies a role in nonspecific defense mechanisms against pathogens.

  • 43.
    Frängsmyr, Lars
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Israelsson, Anne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Teglund, S
    Matsunaga, T
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Evolution of the carcinoembryonic antigen family: Structures of CGM9, CGM11 and pregnancy-specific glycoprotein promoters.2000In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 21, no 2, p. 63-81Article in journal (Refereed)
  • 44. Ginley, Brandon
    et al.
    Emmons, Tiffany
    Sasankan, Prabhu
    Urban, Constantin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Segal, Brahm H.
    Sarder, Pinaki
    Identification and characterization of neutrophil extracellular trap shapes in flow cytometry2017In: Medical Imaging 2017: Digital Pathology / [ed] Gurcan, MN Tomaszewski, JE, 2017, article id 101400DConference paper (Refereed)
    Abstract [en]

    Neutrophil extracellular trap (NET) formation is an alternate immunologic weapon used mainly by neutrophils. Chromatin backbones fused with proteins derived from granules are shot like projectiles onto foreign invaders. It is thought that this mechanism is highly anti-microbial, aids in preventing bacterial dissemination, is used to break down structures several sizes larger than neutrophils themselves, and may have several more uses yet unknown. NETs have been implied to be involved in a wide array of systemic host immune defenses, including sepsis, autoimmune diseases, and cancer. Existing methods used to visually quantify NETotic versus non-NETotic shapes are extremely time-consuming and subject to user bias. These limitations are obstacles to developing NETs as prognostic biomarkers and therapeutic targets. We propose an automated pipeline for quantitatively detecting neutrophil and NET shapes captured using a flow cytometry-imaging system. Our method uses contrast limited adaptive histogram equalization to improve signal intensity in dimly illuminated NETs. From the contrast improved image, fixed value thresholding is applied to convert the image to binary. Feature extraction is performed on the resulting binary image, by calculating region properties of the resulting foreground structures. Classification of the resulting features is performed using Support Vector Machine. Our method classifies NETs from neutrophils without traps at 0.97/0.96 sensitivity/specificity on n = 387 images, and is 1500X faster than manual classification, per sample. Our method can be extended to rapidly analyze whole-slide immunofluorescence tissue images for NET classification, and has potential to streamline the quantification of NETs for patients with diseases associated with cancer and autoimmunity.

  • 45. Ginley, Brandon G
    et al.
    Emmons, Tiffany
    Lutnick, Brendon
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Segal, Brahm H
    Sarder, Pinaki
    Computational detection and quantification of human and mouse neutrophil extracellular traps in flow cytometry and confocal microscopy2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, no 1, article id 17755Article in journal (Refereed)
    Abstract [en]

    Neutrophil extracellular traps (NETs) are extracellular defense mechanisms used by neutrophils, where chromatin is expelled together with histones and granular/cytoplasmic proteins. They have become an immunology hotspot, implicated in infections, but also in a diverse array of diseases such as systemic lupus erythematosus, diabetes, and cancer. However, the precise assessment of in vivo relevance in different disease settings has been hampered by limited tools to quantify occurrence of extracellular traps in experimental models and human samples. To expedite progress towards improved quantitative tools, we have developed computational pipelines to identify extracellular traps from an in vitro human samples visualized using the ImageStream® platform (Millipore Sigma, Darmstadt, Germany), and confocal images of an in vivo mouse disease model of aspergillus fumigatus pneumonia. Our two in vitro methods, tested on n = 363/n =145 images respectively, achieved holdout sensitivity/specificity 0.98/0.93 and 1/0.92. Our unsupervised method for thin lung tissue sections in murine fungal pneumonia achieved sensitivity/specificity 0.99/0.98 in n = 14 images. Our supervised method for thin lung tissue classified NETs with sensitivity/specificity 0.86/0.90. We expect that our approach will be of value for researchers, and have application in infectious and inflammatory diseases.

  • 46. Goodier, M R
    et al.
    Lundqvist, C
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Troye-Blomberg, M
    Langhorne, J
    Cytokine profiles for human V gamma 9+ T cells stimulated by Plasmodium falciparum.1995In: Parasite immunology (Print), ISSN 0141-9838, E-ISSN 1365-3024, Vol. 17, no 8, p. 413-23Article in journal (Refereed)
    Abstract [en]

    V gamma 9+ T cells from malaria non-exposed donors make proliferative responses to Plasmodium falciparum on in vitro stimulation. V gamma 9+ cells are strongly activated by components of the schizont stage of the parasite and by antigens released into the culture upon schizogony, while CD4+V gamma 9- cells are stimulated by the earlier stages of the parasite. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined mRNA expression for 14 cytokines in highly purified V gamma 9+ cells enriched by positive selection after in vitro stimulation with P. falciparum schizont antigens. Interferon-gamma (IFN-gamma) and Tumor Necrosis Factor-alpha (TNF-alpha) were detected in all samples tested. The majority of samples also expressed TNF-beta, transforming growth factor-beta (TGF-beta) and Interleukin-8 (IL-8). Only occasional samples expressed IL-2, IL-5 and IL-10. Using the ELISPOT assay we found that a large fraction of the reactive V gamma 9+ cells produced IFN-gamma and that gamma delta T cells are the major producers of IFN-gamma in cultures stimulated with schizont antigens. The majority of V gamma 9+ cells in these cultures also express the membrane-bound form of TNF-alpha. Expression of these cytokines speaks for a cytolytic and/or inflammatory role of gamma delta cells in the response to malaria in non-exposed individuals.

  • 47.
    Hammarström, Sten
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Is there a role for CEA in innate immunity in the colon?2001In: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 9, no 3, p. 119-25Article in journal (Refereed)
    Abstract [en]

    Carcinoembryonic antigen (CEA) is a well known tumor marker associated with the progression of colorectal tumors. The CEA family of glycoproteins has been fully characterized and the function of some of its members is now beginning to be understood. Here, we advance the hypothesis that, rather than functioning in cell adhesion as has been suggested previously, CEA plays a role in protecting the colonic mucosa from microbial invasion. This hypothesis is based on new microscopic, molecular, phylogenetic and microbiological evidence.

  • 48.
    Hammarström, Sten
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Berzins, Klavs
    Biberfeld, Peter
    Engvall, Eva
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Holm, Göran
    Troye-Blomberg, Marita
    Wahlgren, Mats
    Peter Perlmann 1919-2005.2006In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 63, no 6, p. 487-9Article in journal (Refereed)
  • 49.
    Haraldsson, Katarina
    Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    Defining the genetics of systemic autoimmunity in mouse models of lupus2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Systemic Lupus Erythematosus (SLE) is a chronic multi-organ autoimmune disease considered a prototype for autoantibody and immune complex-mediated tissue injury. Although autoantibodies against a wide diversity of self-antigens are characteristically found in this disease, an important hallmark is the presence of autoantibodies to nuclear antigens. Despite this common clinical feature, individual patients vary widely in the organ systems afflicted, disease severity, disease course, and response to treatment. These characteristics make clinical management of SLE challenging and highlight the need for effective and less toxic therapeutic interventions.

    Susceptibility to lupus has been shown in both human studies and mouse models to be dependent on genetic predisposition. Therefore, it is likely that knowledge of the genetic basis of SLE will be required before full understanding of SLE pathogenesis can be achieved. In this thesis, studies to define the genetic basis of lupus in an induced and two spontaneous models of the disease are presented. These studies encompass mapping, characterization of interval congenic mice, and cloning of the Lmb3 locus gene. In the first study, a genomewide mapping study was performed to define the genetic basis for resistance of the DBA/2 mice to mercury-induced autoimmunity. On chromosome 1, a single quantitative trait was linked with resistance to HgIA. These results linked the locus Hmr1 to a late stage of lupus with GN. Interval congenic mice are important tools to define and characterize the roles of different loci in lupus-like diseases. The second paper identifies the effect of NZB and NZW Lbw2 alleles on lupus susceptibility by using BWF1 mice with none, one or two copies of the lupus-predisposing NZB.Lbw2 locus. The lack of the NZB locus significantly reduced mortality, GN and B cell activation. IgM anti-chromatin levels in genome-wide mapping was linked only to Lmb2 and none of the known B cell hyperactivity-promoting genes were present in this location, which might indicate a novel B cell activation gene. The third study used reciprocal single locus interval-specific congenic mice to characterize the contribution of Lmb1-4 on the MRL-Faslpr and B6-Faslpr backgrounds. The Lmb3 locus on chromosome 7 was found to have the most prominent phenotype with clear effects on lymphoproliferation, GN and mortality. In the fourth paper the Lmb3 was cloned and shown to be a spontaneous nonsense mutation in the Coro1a gene that encodes an actin-binding and -regulatory protein. Upon further characterization, this genetic alteration was discovered to be a new lupus suppressing mutation that reduced T cell migration, activation, and survival. Our findings highlight the complexity of the genetics of lupus, and further suggest that genes involved in controlling the actin cytoskeleton might be potential targets for autoimmune therapeutics.

  • 50.
    Haraldsson, M Katarina
    et al.
    Umeå University, Faculty of Medicine, Clinical Microbiology. Umeå University, Faculty of Medicine, Clinical Microbiology, Immunology/Immunchemistry.
    Louis-Dit-Sully, Christine A
    Lawson, Brian R
    Sternik, Gabriel
    Santiago-Raber, Marie-Laure
    Gascoigne, Nicholas R J
    Theofilopoulos, Argyrios N
    Kono, Dwight H
    The lupus-related Lmb3 locus contains a disease-suppressing Coronin-1A gene mutation.2008In: Immunity, ISSN 1074-7613, E-ISSN 1097-4180, Vol. 28, no 1, p. 40-51Article in journal (Refereed)
    Abstract [en]

    Here, we show that a lupus-suppressing locus is caused by a nonsense mutation of the filamentous actin-inhibiting Coronin-1A gene. This mutation was associated with developmental and functional alterations in T cells including reduced migration, survival, activation, and Ca2+ flux. T-dependent humoral responses were impaired, but no intrinsic B cell defects were detected. By transfer of T cells, it was shown that suppression of autoimmunity could be accounted for by the presence of the Coro1a(Lmb3) mutation in T cells. Our results demonstrate that Coronin-1A is required for the development of systemic lupus and identify actin-cytoskeleton regulatory proteins as potential targets for modulating autoimmune diseases.

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