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  • 1. Aeinehband, Shahin
    et al.
    Brenner, Philip
    Stahl, Sara
    Bhat, Maria
    Fidock, Mark D.
    Khademi, Mohsen
    Olsson, Tomas
    Engberg, Goran
    Jokinen, Jussi
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Psychiatry.
    Erhardt, Sophie
    Piehl, Fredrik
    Cerebrospinal fluid kynurenines in multiple sclerosis: relation to disease course and neurocognitive symptoms2016In: Brain, behavior, and immunity, ISSN 0889-1591, E-ISSN 1090-2139, Vol. 51, p. 47-55Article in journal (Refereed)
    Abstract [en]

    Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system, with a high rate of neurocognitive symptoms for which the molecular background is still uncertain. There is accumulating evidence for dysregulation of the kynurenine pathway (KP) in different psychiatric and neurodegenerative conditions. We here report the first comprehensive analysis of cerebrospinal fluid (CSF) kynurenine metabolites in MS patients of different disease stages and in relation to neurocognitive symptoms. Levels of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYNA) and quinolinic acid (QUIN) were determined with liquid chromatography mass spectrometry in cell-free CSF. At the group level MS patients (cohort 1; n = 71) did not differ in absolute levels of TRP, KYN, KYNA or QUIN as compared to non-inflammatory neurological disease controls (n = 20). Stratification of patients into different disease courses revealed that both absolute QUIN levels and the QUIN/KYN ratio were increased in relapsing-remitting MS (RRMS) patients in relapse. Interestingly, secondary progressive MS (SPMS) displayed a trend for lower TRP and KYNA, while primary progressive (PPMS) patients displayed increased levels of all metabolites, similar to a group of inflammatory neurological disease controls (n = 13). In the second cohort (n = 48), MS patients with active disease and short disease duration were prospectively evaluated for neuropsychiatric symptoms. In a supervised multivariate analysis using orthogonal projection to latent structures (OPLS-DA) depressed patients displayed higher KYNA/TRP and KYN/TRP ratios, mainly due to low TRP levels. Still, this model had low predictive value and could not completely separate the clinically depressed patients from the non-depressed MS patients. No correlation was evident for other neurocognitive measures. Taken together these results demonstrate that clinical disease activity and differences in disease courses are reflected by changes in KP metabolites. Increased QUIN levels of RRMS patients in relapse and generally decreased levels of TRP in SPMS may relate to neurotoxicity and failure of remyelination, respectively. In contrast, PPMS patients displayed a more divergent pattern more resembling inflammatory conditions such as systemic lupus erythematosus. The pattern of KP metabolites in RRMS patients could not predict neurocognitive symptoms.

  • 2. Alcocer, M. J. C.
    et al.
    Murtagh, G. J.
    Mirotti, L.
    Brans, A.
    Harnett, W.
    Rundqvist, Louise
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The allergenicity of 2S plant albumins2011In: Clinical and Experimental Allergy, ISSN 0954-7894, E-ISSN 1365-2222, Vol. 41, no 12, p. 1827-1827Article in journal (Refereed)
  • 3.
    Anderl, Ines
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Activation of the Cellular Immune Response in Drosophila melanogaster Larvae2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors.

    In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes.

    In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation.

    In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction.

    In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1.

    Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis.

  • 4.
    Anderl, Ines
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Infection-induced proliferation is a central hallmark of the activation of the cellular immune response in Drosophila larvae.Manuscript (preprint) (Other academic)
    Abstract [en]

    Blood cells have important roles in immune reactions in all metazoan species. In Drosophila melanogaster larvae, phagocytic plasmatocytes are the main blood cell (hemocyte) type. Lamellocytes participate in encapsulating foreign objects and are formed in response to parasitoid wasps laying their eggs into the hemocoel of the larvae. The immune reaction against wasps requires controlled recruitment and action of hemocytes from the lymph glands, sessile islets and circulation. However, the contribution of these different hematopoietic compartments to the immune-induced hemocyte pool remains unclear. We used eater-GFP and MSNF9MO-mCherry to fluorescently tag plasmatocytes and lamellocytes, respectively, and utilized flow cytometry and in vivo imaging to assess the hemocyte numbers and types in circulation and in sessile compartments after infection by three wasp species of the genus Leptopilina. We detected five different hemocyte types based on fluorescence, and a population of non-fluorescent cells. While non-infected larvae generally had only one, eaterGFP-high plasmatocyte population, early after wasp infection a new, eaterGFP-low cell population appeared in circulation. EaterGFP-high and –low cells both accumulated msnCherry during the immune response, and formed two cell lineages. Whereas the eaterGFP-low cells gradually lost GFP, the eaterGFP-high cells retained it at high levels. We suggest that eaterGFP-low cells represent an immune-induced hemocyte precursor cell pool, which, via a prelamellocyte stage, gives rise to lamellocytes. EaterGFP-high plasmatocytes also differentiated into large, msnCherry-positive hemocytes on wasp eggs, but these cells retain plasmatocyte identity. Importantly, all hemocyte types, except for lamellocytes, were able to divide after wasp infection, contributing to the increased hemocyte numbers after infection. We conclude that orchestrated differentiation and division of different hemocyte types in circulation and in sessile compartment is key to a successful immune response against parasitoid wasps.

  • 5. Andersen, G. Neumann
    et al.
    Andersen, M.
    Nagaeva, Olga
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Wikberg, J. E. S.
    Mincheva-Nilsson, Lucia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Dermal Melanocortin Receptor Rebound in Diffuse Systemic Sclerosis after Anti-TGF ss 1 Antibody Therapy2012In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 76, no 5, p. 478-482Article in journal (Refereed)
    Abstract [en]

    Disturbed transforming growth factor beta (TGF beta) signalling leads to enhanced synthesis of extracellular matrix (ECM), which is manifested as systemic sclerosis (SSc), but this may be attenuated by the melanocortin system. Here, we report of rebound reaction in the gene expression of melanocortin receptor (MCR) subtypes and of the precursor of these receptors ligands, the pro-opio-melanocortin protein (POMC), in the acute skin lesion of diffuse systemic sclerosis (dSSc) after treatment with a recombinant human anti-TGF beta 1 antibody. Biopsies, taken from the leading edge of the skin lesion, before and after treatment of a patient with recent onset dSSc, were examined. Before treatment, increased levels of TGF beta mRNA and suppressed levels of POMC mRNA and MCR subtypes MC1-3, 5R mRNAs were seen in the lesion, compared with healthy controls. After treatment, there was a rebound expression of POMC, MC2, 3, 5R mRNAs. As the melanocortin system regulates collagen and melanin production, our findings add a new understanding to the pathogenetic mechanisms involved in the acute skin lesion of dSSc, which is characterized by enhanced ECM formation and changes in skin pigmentation.

  • 6.
    Avican, Ummehan
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Doruk, Tugrul
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Östberg, Yngve
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    The Tat substrate SufI is critical for the ability of Yersinia pseudotuberculosis to cause systemic infection2017In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 85, no 4, article id e00867-16Article in journal (Refereed)
    Abstract [en]

    The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important humanpathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a.sufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosis. In vivo bioluminescent imaging of orally infected mice revealed that both the.sufI and Delta tatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the Delta tatC mutant nor the Delta sufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the Delta tatC and Delta sufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the Delta tatC mutant.

  • 7.
    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.

  • 8.
    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)
  • 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.
    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.

  • 11.
    Bas, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Forsberg, Göte
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Utility of the housekeeping genes 18S rRNA, β-actin, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) for normalisation in real-time quantitative RT-PCR analysis of gene expression in human T lymphocytes2004In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 59, no 6, p. 566-573Article in journal (Refereed)
    Abstract [en]

    The accuracy of 18S rRNA, β-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, β-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 β-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated γδTCR+, 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.

  • 12. 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.

  • 13. Bassères, Eugénie
    et al.
    Coppotelli, Giuseppe
    Pfirrmann, Thorsten
    Andersen, Jens B
    Masucci, Maria
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton2010In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 12, no 11, p. 1622-1633Article in journal (Refereed)
    Abstract [en]

    Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH-L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH-L1-negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH-L1 controls the early membrane-associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c-cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH-L1(C90S). These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH-L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria-based drug delivery systems.

  • 14. Bergman, Emma Ahlen
    et al.
    Hartana, Ciputra Adijaya
    Linton, Ludvig
    Winerdal, Malin E.
    Krantz, David
    Rosenblatt, Robert
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Urology and Andrology.
    Lundgren, Christian
    Marits, Per
    Sherif, Amir
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Urology and Andrology.
    Winqvist, Ola
    Epigenetic methylation profiles of CD4 T cell signature loci from patients with urinary bladder cancer2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 264-264Article in journal (Other academic)
  • 15. Bergström, Joakim H
    et al.
    Birchenough, George M H
    Katona, Gergely
    Schröder, Björn
    Schütte, André
    Ermund, Anna
    Johansson, Malin E V
    Hansson, Gunnar C
    Gram-positive bacteria are held at a distance in the colon mucus by the lectin-like protein ZG162016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 48, p. 13833-13838Article in journal (Refereed)
  • 16.
    Bianchi, Matteo
    et al.
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Niemiec, Maria J.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Siler, Ulrich
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Reichenbach, Janine
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Redundant ability of phagocytes to kill Aspergillus species: Reply2011In: Journal of Allergy and Clinical Immunology, ISSN 0091-6749, E-ISSN 1097-6825, Vol. 128, no 3, p. 687-688Article in journal (Refereed)
  • 17. Bielig, Harald
    et al.
    Dongre, Mitesh
    Zurek, Birte
    Wai, Sun N
    Kufer, Thomas A
    A role for quorum sensing in regulating innate immune responses mediated by Vibrio cholerae outer membrane vesicles (OMVs).2011In: Gut microbes, ISSN 1949-0976, E-ISSN 1949-0984, Vol. 2, no 5, p. 274-9Article in journal (Refereed)
    Abstract [en]

    Outer membrane vesicles (OMVs) are released from many Gram-negative bacteria. OMVs interact with and are taken up by human cells. We and others have now showed that OMVs contain peptidoglycan, which is sensed mainly by the pattern-recognition receptor NOD1 in the cytoplasm of host cells. Vibrio cholerae is clinically important as one of the causative agents of severe dehydrating diarrhea in humans. We showed that non-O1 non-O139 V. cholerae (NOVC) strains of V. cholera produce OMVs. Of note, we revealed that NOVC can evade NOD1-mediated immune surveillance by the quorum sensing machinery. Here we review these recent findings and discuss the relevance for our understanding of bacterial infections and innate immune responses.

  • 18.
    Binesse, Johan
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lindgren, Helena
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lindgren, Lena
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Conlan, Wayne
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Roles of Reactive Oxygen Species-Degrading Enzymes of Francisella tularensis SCHU S42015In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 83, no 6, p. 2255-2263Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis is a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ROS), potent mediators of the bactericidal activity of macrophages. We investigated the roles of enzymes presumed to be important for protection against ROS. Four mutants of the highly virulent SCHU S4 strain with deletions of the genes encoding catalase (katG), glutathione peroxidase (gpx), a DyP-type peroxidase (FTT0086), or double deletion of FTT0086 and katG showed much increased susceptibility to hydrogen peroxide (H2O2) and slightly increased susceptibility to paraquat but not to peroxynitrite (ONOO-) and displayed intact intramacrophage replication. Nevertheless, mice infected with the double deletion mutant showed significantly longer survival than SCHU S4-infected mice. Unlike the aforementioned mutants, deletion of the gene coding for alkyl-hydroperoxide reductase subunit C (ahpC) generated a mutant much more susceptible to paraquat and ONOO- but not to H2O2. It showed intact replication in J774 cells but impaired replication in bone marrow-derived macrophages and in internal organs of mice. The live vaccine strain, LVS, is more susceptible than virulent strains to ROS-mediated killing and possesses a truncated form of FTT0086. Expression of the SCHU S4 FTT0086 gene rendered LVS more resistant to H2O2, which demonstrates that the SCHU S4 strain possesses additional detoxifying mechanisms. Collectively, the results demonstrate that SCHU S4 ROS-detoxifying enzymes have overlapping functions, and therefore, deletion of one or the other does not critically impair the intracellular replication or virulence, although AhpC appears to have a unique function.

  • 19. Birchenough, George
    et al.
    Schröder, Björn
    Bäckhed, Fredrik
    Hansson, Gunnar C
    Dietary destabilisation of the balance between the microbiota and the colonic mucus barrier.2019In: Gut microbes, ISSN 1949-0976, E-ISSN 1949-0984, Vol. 10, no 2, p. 246-250Article in journal (Refereed)
    Abstract [en]

    It has long been acknowledged that dietary fibres are important to maintain a healthy gut. Over the past decade, several studies have shown that loss of complex polysaccharides from the Western diet has resulted in alterations to our colonic microbiota. The concurrent increase in the incidence of inflammatory bowel disease in the Western world has driven us to explore the potential mechanistic link between diet, the microbiota and the host defence systems that normally prevent inflammation. Using mice fed a low fibre Western-style diet and robust live tissue analytical methods we have now provided evidence that this diet impairs the colonic inner mucus layer that normally separates bacteria from host cells. Western societies urgently need to develop their understanding of the molecular mechanisms of the diet-microbiota-mucus axis and its implications for inflammatory diseases.

  • 20.
    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.

  • 21. Blazkova, Hana
    et al.
    Krejcikova, Katerina
    Moudry, Pavel
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Hodny, Zdenek
    Bartek, Jiri
    Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling2010In: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 14, no 1-2, p. 357-367Article in journal (Refereed)
    Abstract [en]

    Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram‐negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type‐dependent cell‐cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR‐90 and WI‐38 fibroblasts, HeLa and U2‐OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX+ foci), enhanced senescence‐associated β‐galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL‐6, IL‐8 and IL‐24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the ‘distended’ morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.

  • 22.
    Boal, Frédéric
    et al.
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Puhar, Andrea
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). INSERM U1202, Unite´ de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France.
    Xuereb, Jean-Marie
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Kunduzova, Oksana
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Sansonetti, Philippe J.
    INSERM U1202, Unite´ de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France.
    Payrastre, Bernard
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France; .
    Tronchére, Héléne
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    PI5P Triggers ICAM-1 Degradation in Shigella Infected Cells, Thus Dampening Immune Cell Recruitment2016In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 14, no 4, p. 750-759Article in journal (Refereed)
    Abstract [en]

    Shigella flexneri, the pathogen responsible for bacillary dysentery, has evolved multiple strategies to control the inflammatory response. Here, we show that Shigella subverts the subcellular trafficking of the intercellular adhesion molecule-1 (ICAM-1), a key molecule in immune cell recruitment, in a mechanism dependent on the injected bacterial enzyme IpgD and its product, the lipid mediator PI5P. Overexpression of IpgD, but not a phosphatase dead mutant, induced the internalization and the degradation of ICAM-1 in intestinal epithelial cells. Remarkably, addition of permeant PI5P reproduced IpgD effects and led to the inhibition of neutrophil recruitment. Finally, these results were confirmed in an in vivo model of Shigella infection where IpgD-dependent ICAM-1 internalization reduced neutrophil adhesion. In conclusion, we describe here an immune evasion mechanism used by the pathogen Shigella to divert the host cell trafficking machinery in order to reduce immune cell recruitment.

  • 23. Brauner, Annelie
    et al.
    Brandt, Lena
    Frisan, Teresa
    Department of Clinical Microbiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
    Thelestam, Monica
    Ekbom, Anders
    Is there a risk of cancer development after Campylobacter infection?2010In: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 45, no 7-8, p. 893-897Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: All Campylobacter jejuni species produce a genotoxin, which induce DNA double strand breaks, could lead to an increased risk of cancer especially in the gastro-intestinal tract.

    MATERIAL AND METHODS: All individuals in Stockholm County who tested positive with C. jejuni between 1989 and 2006 were included. The cohort was followed-up until December 31, 2007 for the occurrence of cancer, overall and site specific. Standard incidence ratios (SIR) with 95% confidence intervals (CI) were calculated by comparisons with the background population.

    RESULTS: There were 16,276 individuals who tested positive for C. jejuni generating 124,387 person years. Excluding the first year of follow-up the overall risk for cancer did neither differ from that expected SIR = 0.95 (95% CI 0.82-1.09) nor after 10 years or more of follow-up; SIR = 0.91 (95% CI 0.71-1.16). There was no increased risk for cancer in the gastro-intestinal tract, but there were significantly increased risks for melanomas SIR = 1.84 (95% CI 1.27-2.57) and squamous cell skin cancer SIR = 1.52 (95% CI 1.01-2.19) while a significantly decreased risk of respiratory cancers among males SIR = 0.32 (95% CI 0.12-0.70) was observed.

    CONCLUSIONS: Our results indicate no excess risks of malignancies following an infection by C. jejuni at least during the first decade. Furthermore, the finding of a decreased risk of respiratory cancers could be of interest, if the results are reproduced in future studies in other populations.

  • 24. Del Bel Belluz, Lisa
    et al.
    Guidi, Riccardo
    Pateras, Ioannis S
    Levi, Laura
    Mihaljevic, Boris
    Rouf, Syed Fazle
    Wrande, Marie
    Candela, Marco
    Turroni, Silvia
    Nastasi, Claudia
    Consolandi, Clarissa
    Peano, Clelia
    Tebaldi, Toma
    Viero, Gabriella
    Gorgoulis, Vassilis G
    Krejsgaard, Thorbjørn
    Rhen, Mikael
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection2016In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 12, no 4, article id e1005528Article in journal (Refereed)
    Abstract [en]

    Bacterial genotoxins, produced by several Gram-negative bacteria, induce DNA damage in the target cells. While the responses induced in the host cells have been extensively studied in vitro, the role of these effectors during the course of infection remains poorly characterized. To address this issue, we assessed the effects of the Salmonella enterica genotoxin, known as typhoid toxin, in in vivo models of murine infection. Immunocompetent mice were infected with isogenic S. enterica, serovar Typhimurium (S. Typhimurium) strains, encoding either a functional or an inactive typhoid toxin. The presence of the genotoxic subunit was detected 10 days post-infection in the liver of infected mice. Unexpectedly, its expression promoted the survival of the host, and was associated with a significant reduction of severe enteritis in the early phases of infection. Immunohistochemical and transcriptomic analysis confirmed the toxin-mediated suppression of the intestinal inflammatory response. The presence of a functional typhoid toxin further induced an increased frequency of asymptomatic carriers. Our data indicate that the typhoid toxin DNA damaging activity increases host survival and favours long-term colonization, highlighting a complex cross-talk between infection, DNA damage response and host immune response. These findings may contribute to understand why such effectors have been evolutionary conserved and horizontally transferred among Gram-negative bacteria.

  • 25.
    Eneslätt, Kjell
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Golovliov, Igor
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Rydén, Patrik
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Vaccine-mediated mechanisms controlling replication of Francisella tularensis in human peripheral blood mononuclear cells using a co-culture system2018In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 8, article id 27Article in journal (Refereed)
    Abstract [en]

    Cell-mediated immunity (CMI) is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs) were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P < 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to F. tularensis.

  • 26.
    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.

  • 27.
    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.

  • 28.
    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)
  • 29.
    Fahlgren, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Danielsson, Åke
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Hammarstrom, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    β-Defensin-3 and -4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis2004In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 137, no 2, p. 379-385Article in journal (Refereed)
    Abstract [en]

    mRNA expression of two recently described human beta-defensins (hBD-3 and hBD-4) in epithelial cells of normal small and large intestine and the impact of chronic intestinal inflammation on their expression levels was investigated. Intestinal specimens from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls with no history of inflammatory bowel disease were studied. hBD-3 and hBD-4 mRNAs were determined in freshly isolated epithelial cells by real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and by in situ hybridization. The effect of proinflammatory cytokines on hBD-3 and hBD-4 mRNA expression in colon carcinoma cells was also investigated. Purified epithelial cells of normal small and large intestine expressed both hBD-3 and hBD-4 mRNA, with higher expression levels of hBD-3 mRNA. In situ hybridization revealed higher levels of mRNA expression in the crypt- compared to the villus/luminal-compartment. Interferon (IFN)-gamma, but not tumour necrosis factor (TNF)-alpha or IL-1beta, augmented hBD-3 mRNA expression. None of these agents stimulated hBD-4 expression. Colonic epithelial cells from patients with UC displayed a significant increase in hBD-3 and hBD-4 mRNA compared to epithelial cells of controls. In contrast, small intestinal epithelial cells from CD patients did not show increased expression levels compared to the corresponding control cells. Moreover, Crohn's colitis did not show increased expression of hBD-4 mRNA, while the data are inconclusive for hBD-3 mRNA. We conclude that the chronic inflammatory reaction induced in the colon of UC patients enhances hBD-3 and hBD-4 mRNA expression in the epithelium, whereas in CD this is less evident.

  • 30.
    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)
  • 31.
    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.

  • 32.
    Frisan, Teresa
    Dept. Cell and Molecular Biology, Karolinska Institutet.
    Bacterial genotoxins: the long journey to the nucleus of mammalian cells2016In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1858, no 3, p. 567-575, article id S0005-2736(15)00267-9Article, review/survey (Refereed)
    Abstract [en]

    Bacterial protein genotoxins target the DNA of eukaryotic cells, causing DNA single and double strand breaks. The final outcome of the intoxication is induction of DNA damage responses and activation of DNA repair pathways. When the damage is beyond repair, the target cell either undergoes apoptosis or enters a permanent quiescent stage, known as cellular senescence. In certain instances, intoxicated cells can survive and proliferate. This event leads to accumulation of genomic instability and acquisition of malignant traits, underlining the carcinogenic potential of these toxins. The toxicity is dependent on the toxins' internalization and trafficking from the extracellular environment to the nucleus, and requires a complex interaction with several cellular membrane compartments: the plasma membrane, the endosomes, the trans Golgi network and the endoplasmic reticulum, and finally the nucleus. This review will discuss the current knowledge of the bacterial genotoxins internalization pathways and will highlight the issues that still remain unanswered. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  • 33.
    Frisan, Teresa
    et al.
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Sebo, Peter
    Editorial: Why still study bacterial toxins in the third millennium?2016In: Pathogens and Disease, E-ISSN 2049-632X, Vol. 74, no 3, article id ftw009Article in journal (Refereed)
  • 34.
    Gokumakulapalle, Madhuri
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Mei, Ya-Fang
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Replication-competent human adenovirus 11p vectors can propagate in Vero cells2016In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 495, p. 42-51Article in journal (Refereed)
    Abstract [en]

    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus lip (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields.

  • 35.
    Golovliov, Igor
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Lindgren, Helena
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Eneslätt, Kjell
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Conlan, Wayne
    Mosnier, Amandine
    Henry, Thomas
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy2016In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 152Article in journal (Refereed)
    Abstract [en]

    Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naive, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, Delta clpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naive splenocytes, Delta clpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the Delta clpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the Delta clpB-immune splenocytes also contained higher levels of IFN-gamma, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-gamma, TNF-alpha, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS(-/-) mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis.

  • 36. 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.

  • 37. Grasso, Francesca
    et al.
    Frisan, Teresa
    Department Cell and Molecular Biology, Karolinska Institutet.
    Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology2015In: Biomolecules, E-ISSN 2218-273X, Vol. 5, no 3, p. 1762-1782Article, review/survey (Refereed)
    Abstract [en]

    Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence) or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT) family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins.

  • 38. Guerra, Lina
    et al.
    Albihn, Ami
    Tronnersjö, Susanna
    Yan, Qinzi
    Guidi, Riccardo
    Stenerlöw, Bo
    Sterzenbach, Torsten
    Josenhans, Christine
    Fox, James G
    Schauer, David B
    Thelestam, Monica
    Larsson, Lars-Gunnar
    Henriksson, Marie
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Myc is required for activation of the ATM-dependent checkpoints in response to DNA damage2010In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 5, no 1, article id e8924Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown.

    PRINCIPAL FINDINGS: In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status.

    CONCLUSION: These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.

  • 39. Guerra, Lina
    et al.
    Cortes-Bratti, Ximena
    Guidi, Riccardo
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.
    The biology of the cytolethal distending toxins2011In: Toxins, ISSN 2072-6651, E-ISSN 2072-6651, Vol. 3, no 3, p. 172-190Article, review/survey (Refereed)
    Abstract [en]

    The cytolethal distending toxins (CDTs), produced by a variety of Gram-negative pathogenic bacteria, are the first bacterial genotoxins described, since they cause DNA damage in the target cells. CDT is an A-B(2) toxin, where the CdtA and CdtC subunits are required to mediate the binding on the surface of the target cells, allowing internalization of the active CdtB subunit, which is functionally homologous to the mammalian deoxyribonuclease I. The nature of the surface receptor is still poorly characterized, however binding of CDT requires intact lipid rafts, and its internalization occurs via dynamin-dependent endocytosis. The toxin is retrograde transported through the Golgi complex and the endoplasmic reticulum, and subsequently translocated into the nuclear compartment, where it exerts the toxic activity. Cellular intoxication induces DNA damage and activation of the DNA damage responses, which results in arrest of the target cells in the G1 and/or G2 phases of the cell cycle and activation of DNA repair mechanisms. Cells that fail to repair the damage will senesce or undergo apoptosis. This review will focus on the well-characterized aspects of the CDT biology and discuss the questions that still remain unanswered.

  • 40. Guerra, Lina
    et al.
    Guidi, Riccardo
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Do bacterial genotoxins contribute to chronic inflammation, genomic instability and tumor progression?2011In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 278, no 23, p. 4577-4588Article, review/survey (Refereed)
    Abstract [en]

    Cytolethal distending toxin, produced by several Gram-negative bacteria, and colibactin, secreted by several commensal and extraintestinal pathogenic Escherichia coli strains, are the first bacterial genotoxins to be described to date. Exposure to cytolethal distending toxin and colibactin induces DNA damage, and consequently activates the DNA damage response, resulting in cell cycle arrest of the intoxicated cells and DNA repair. Irreversible DNA damage will lead to cell death by apoptosis or to senescence. It is well established that chronic exposure to DNA damaging agents, either endogenous (reactive oxygen species) or exogenous (ionizing radiation), may cause genomic instability as a result of the alteration of genes coordinating the DNA damage response, thus favoring tumor initiation and progression. In this review, we summarize the state of the art of the biology of cytolethal distending toxin and colibactin, focusing on the activation of the DNA damage response and repair pathways, and discuss the cellular responses induced in intoxicated cells, as well as how prolonged intoxication may lead to chronic inflammation, the accumulation of genomic instability, and tumor progression in both in vitro and in vivo models.

  • 41. Guidi, R
    et al.
    Belluz, L Del Bell
    Frisan, Teresa
    Dept. of Cell and Molecular Biology, Karolinska Institute, Stockholm Sweden.
    Bacterial genotoxin functions as immune-modulator and promotes host survival2016In: Microbial cell (Graz, Austria), ISSN 2311-2638, Vol. 3, no 8, p. 355-357Article in journal (Refereed)
    Abstract [en]

    Bacterial genotoxins are effectors that cause DNA damage in target cells. Many aspects of the biology of these toxins have been characterised in vitro, such as structure, cellular internalisation pathways and effects on the target cells. However, little is known about their function in vivo. Salmonella enterica serovar Typhi (S. Typhi) is a Gram-negative, intracellular bacterium that causes typhoid fever, a debilitating disease infecting more than 20 million people every year. S. Typhiproduce a genotoxin named typhoid toxin (TT), but its role in the contest of host infection is poorly characterized. The major obstacle in addressing this issue is that S. Typhi is exclusively a human pathogen. To overcome this limitation, we have used as model bacterium S. Typhimurium, and engineered it to produce endogenous levels of an active and inactive typhoid toxin, hereby named as TT (or genotoxic) and cdtB (or control), respectively. To our surprise, infection with the genotoxin strain strongly suppressed intestinal inflammation, leading to a better survival of the host during the acute phase of infection, suggesting typhoid toxin may exert a protective role. The presence of a functional genotoxin was also associated with an increased frequency of asymptomatic carriers.

  • 42. Guidi, Riccardo
    et al.
    Guerra, Lina
    Levi, Laura
    Stenerlöw, Bo
    Fox, James G
    Josenhans, Christine
    Masucci, Maria G
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Chronic exposure to the cytolethal distending toxins of Gram-negative bacteria promotes genomic instability and altered DNA damage response2013In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, no 1, p. 98-113Article in journal (Refereed)
    Abstract [en]

    Epidemiological evidence links chronic bacterial infections to the increased incidence of certain types of cancer but the molecular mechanisms by which bacteria contribute to tumour initiation and progression are still poorly characterized. Here we show that chronic exposure to the genotoxin cytolethal distending toxin (CDT) of Gram-negative bacteria promotes genomic instability and acquisition of phenotypic properties of malignancy in fibroblasts and colon epithelial cells. Cells grown for more than 30 weeks in the presence of sublethal doses of CDT showed increased mutation frequency, and accumulation of chromatin and chromosomal aberrations in the absence of significant alterations of cell cycle distribution, decreased viability or senescence. Cell survival was dependent on sustained activity of the p38 MAP kinase. The ongoing genomic instability was associated with impaired activation of the DNA damage response and failure to efficiently activate cell cycle checkpoints upon exposure to genotoxic stress. Independently selected sublines showed enhanced anchorage-independent growth as assessed by the formation of colonies in semisolid agarose. These findings support the notion that chronic infection by CDT-producing bacteria may promote malignant transformation, and point to the impairment of cellular control mechanisms associated with the detection and repair of DNA damage as critical events in the process.

  • 43. Guidi, Riccardo
    et al.
    Levi, Laura
    Rouf, Syed Fazle
    Puiac, Speranta
    Rhen, Mikael
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden..
    Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells2013In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, no 12, p. 2034-2050Article in journal (Refereed)
    Abstract [en]

    Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram-negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella-containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71-CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71-CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71-CDT also secreted OMVs-like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin-loaded OMVs by bystander cells was dependent on dynamin-1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.

  • 44.
    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.

  • 45.
    Hashemian, Sanaz
    et al.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
    Alhouayek, Mireille
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
    Fowler, Christopher J.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
    TLR4 receptor expression and function in F11 dorsal root ganglion x neuroblastoma hybrid cells2017In: Innate Immunity, ISSN 1753-4259, E-ISSN 1753-4267, Vol. 23, no 8, p. 687-696Article in journal (Refereed)
    Abstract [en]

    TLR4 respond to bacterial LPS to produce inflammatory cytokines. TLR4 are expressed in dorsal root ganglia and play a role in pain. F11 dorsal root ganglia x mouse neuroblastoma cells possess many of the properties seen in nociceptive dorsal root ganglia neuronal cells. Here, we investigated the effect of 2h and 6h treatment with LPS upon the expression of inflammatory proteins in undifferentiated and differentiated F11 cells. The cells expressed mRNA for TRL4 (mouse, not rat) and proteins involved in TLR4 signaling. TLR4 expression was confirmed using immunohistochemistry. LPS produced modest increases in mouse and rat IL-6 and in mouse cyclooxygenase-2 levels in undifferentiated cells, but did not significantly affect mouse TNF- expression. This contrasts with the robust effects of LPS upon cyclooxygenase-2 expression in cultured dorsal root ganglia neurons. F11 cells expressed the endocannabinoid metabolizing enzymes fatty acid amide hydrolase and N-acylethanolamine acid amidase (both murine), which were functionally active. These data suggest that F11 cells are not a useful model for the study of LPS-mediated effects but may be useful for the study of endocannabinoid catabolism.

  • 46.
    Hauser, Jannek
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christine
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Allelic Exclusion of IgH through Inhibition of E2A in a VDJ Recombination Complex2014In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 192, no 5, p. 2460-2470Article in journal (Refereed)
    Abstract [en]

    A key feature of the immune system is the paradigm that one lymphocyte has only one Ag specificity that can be selected for or against. This requires that only one of the alleles of genes for AgR chains is made functional. However, the molecular mechanism of this allelic exclusion has been an enigma. In this study, we show that B lymphocytes with E2A that cannot be inhibited by calmodulin are dramatically defective in allelic exclusion of the IgH locus. Furthermore, we provide data supporting that E2A, PAX5, and the RAGs are in a VDJ recombination complex bound to key sequences on the Igh gene. We show that pre-BCR activation releases the VDJ recombination complex through calmodulin binding to E2A. We also show that pre-BCR signaling downregulates several components of the recombination machinery, including RAG1, RAG2, and PAX5, through calmodulin inhibition of E2A.

  • 47. Hein, Kyaw Zaw
    et al.
    Takahashi, Hitoshi
    Tsumori, Toshiko
    Yasui, Yukihiko
    Nanjoh, Yasuko
    Toga, Tetsuo
    Wu, Zhihong
    Grötzinger, Joachim
    Jung, Sascha
    Wehkamp, Jan
    Schröder, Björn
    Schroeder, Jens M
    Morita, Eishin
    Disulphide-reduced psoriasin is a human apoptosis-inducing broad-spectrum fungicide.2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 42, p. 13039-44Article in journal (Refereed)
    Abstract [en]

    The unexpected resistance of psoriasis lesions to fungal infections suggests local production of an antifungal factor. We purified Trichophyton rubrum-inhibiting activity from lesional psoriasis scale extracts and identified the Cys-reduced form of S100A7/psoriasin (redS100A7) as a principal antifungal factor. redS100A7 inhibits various filamentous fungi, including the mold Aspergillus fumigatus, but not Candida albicans. Antifungal activity was inhibited by Zn(2+), suggesting that redS100A7 interferes with fungal zinc homeostasis. Because S100A7-mutants lacking a single cysteine are no longer antifungals, we hypothesized that redS100A7 is acting as a Zn(2+)-chelator. Immunogold electron microscopy studies revealed that it penetrates fungal cells, implicating possible intracellular actions. In support with our hypothesis, the cell-penetrating Zn(2+)-chelator TPEN was found to function as a broad-spectrum antifungal. Ultrastructural analyses of redS100A7-treated T. rubrum revealed marked signs of apoptosis, suggesting that its mode of action is induction of programmed cell death. TUNEL, SYTOX-green analyses, and caspase-inhibition studies supported this for both T. rubrum and A. fumigatus. Whereas redS100A7 can be generated from oxidized S100A7 by action of thioredoxin or glutathione, elevated redS100A7 levels in fungal skin infection indicate induction of both S100A7 and its reducing agent in vivo. To investigate whether redS100A7 and TPEN are antifungals in vivo, we used a guinea pig tinea pedes model for fungal skin infections and a lethal mouse Aspergillus infection model for lung infection and found antifungal activity in both in vivo animal systems. Thus, selective fungal cell-penetrating Zn(2+)-chelators could be useful as an urgently needed novel antifungal therapeutic, which induces programmed cell death in numerous fungi.

  • 48.
    Holm, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Nagaeva, Olga
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Nagaev, Ivan
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Loizou, Christos
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Laurell, Göran
    Mincheva-Nilsson, Lucia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Nylander, Karin
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Olofsson, Katarina
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Otorhinolaryngology.
    Lymphocyte profile and cytokine mRNA expression in peripheral blood mononuclear cells of patients with recurrent respiratory papillomatosis suggest dysregulated cytokine mRNA response and impaired cytotoxic capacity2017In: Immunity, Inflammation and Disease, E-ISSN 2050-4527, Vol. 5, no 4, p. 541-550Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: Recurrent respiratory papillomatosis (RRP) is a relatively rare, chronic disease caused by Human Papilloma Virus (HPV) 6 and 11, and characterized by wart-like lesions in the airway affecting voice and respiratory function. The majority of HPV infections are asymptomatic and resolve spontaneously, however, some individuals are afflicted with persistent HPV infections. Failure to eliminate HPV 6 and 11 due to a defect immune responsiveness to these specific genotypes is proposed to play a major role in the development of RRP.

    METHODS: We performed a phenotypic characterization of peripheral blood mononuclear cells (PBMC) collected from 16 RRP patients and 12 age-matched healthy controls, using immunoflow cytometry, and monoclonal antibodies against differentiation and activation markers. The cytokine mRNA profile of monocytes, T helper-, T cytotoxic-, and NK cells was assessed using RT-qPCR cytokine analysis, differentiating between Th1-, Th2-, Th3/regulatory-, and inflammatory immune responses.

    RESULTS: We found a dominance of cytotoxic T cells, activated NK cells, and high numbers of stressed MIC A/B expressing lymphocytes. There was an overall suppression of cytokine mRNA production and an aberrant cytokine mRNA profile in the activated NK cells.

    CONCLUSION: These findings demonstrate an immune dysregulation with inverted CD4(+) /CD8(+) ratio and aberrant cytokine mRNA production in RRP patients, compared to healthy controls.

  • 49.
    Kerkman, Priscilla
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Tuiskunen-Bäck, Anne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Dernstedt, Andy
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Wigren, Julia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Forsell, Mattias
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    The B cell response towards Puumala virus infection: can B cells be infected?2017In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 260-260Article in journal (Other academic)
  • 50. Khalil, Hussein
    et al.
    Ecke, Frauke
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Magnusson, Magnus
    Hornfeldt, Birger
    Declining ecosystem health and the dilution effect2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 31314Article in journal (Refereed)
    Abstract [en]

    The "dilution effect" implies that where species vary in susceptibility to infection by a pathogen, higher diversity often leads to lower infection prevalence in hosts. For directly transmitted pathogens, non-host species may "dilute" infection directly (1) and indirectly (2). Competitors and predators may (1) alter host behavior to reduce pathogen transmission or (2) reduce host density. In a well-studied system, we tested the dilution of the zoonotic Puumala hantavirus (PUUV) in bank voles (Myodes glareolus) by two competitors and a predator. Our study was based on long-term PUUV infection data (2003-2013) in northern Sweden. The field vole (Microtus agrestis) and the common shrew (Sorex araneus) are bank vole competitors and Tengmalm's owl (Aegolius funereus) is a main predator of bank voles. Infection probability in bank voles decreased when common shrew density increased, suggesting that common shrews reduced PUUV transmission. Field voles suppressed bank vole density in meadows and clear-cuts and indirectly diluted PUUV infection. Further, Tengmalm's owl decline in 1980-2013 may have contributed to higher PUUV infection rates in bank voles in 2003-2013 compared to 1979-1986. Our study provides further evidence for dilution effect and suggests that owls may have an important role in reducing disease risk.

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