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  • 1.
    AbdelMageed, Manar
    et al.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt.
    Ali, Haytham
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44511, Egypt.
    Ohlsson, Lina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lindmark, Gudrun
    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.
    Sitohy, Basel
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    The Chemokine CXCL16 Is a New Biomarker for Lymph Node Analysis of Colon Cancer Outcome2019In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 20, no 22, article id 5793Article in journal (Refereed)
    Abstract [en]

    hemokines are important in the development and progression of tumors. We investigated the expression of CXCL14 and CXCL16 in colon cancer. Expression of mRNA was assessed in primary tumors and lymph nodes and CXCL16 mRNA levels were correlated to patient’s survival. Protein expression was investigated by two-color immunofluorescence and immunomorphometry. CXCL14 and CXCL16 mRNA levels and protein expression were significantly higher in colon cancer primary tumors compared to apparently normal colon tissue. Positive cells were tumor cells, as revealed by anti-CEA and anti-EpCAM staining. CXCL16, but not CXCL14, mRNA levels were significantly higher in hematoxylin and eosin positive (H&E(+)) compared to H&E(−) colon cancer lymph nodes or control nodes (P < 0.0001). CXCL16 mRNA was expressed in 5/5 colon cancer cell lines while CXCL14 was expressed significantly in only one. Kaplan-Meier analysis revealed that colon cancer patients with lymph nodes expressing high or very high levels (7.2 and 11.4 copies/18S rRNA unit, respectively) of CXCL16 mRNA had a decreased mean survival time of 30 and 46 months at the 12-year follow-up (P = 0.04, P = 0.005, respectively). In conclusion, high expression of CXCL16 mRNA in regional lymph nodes of colon cancer patients is a sign of a poor prognosis.

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  • 2.
    AbdelMageed, Manar
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    Ismail, Hager
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    Ohlsson, Lina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lindmark, Gudrun
    Institution of Clinical Sciences, Lund University, Helsingborg, Sweden.
    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.
    Sitohy, Basel
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Clinical significance of stem cell biomarkers epcam, lgr5 and lgr4 mrna levels in lymph nodes of colon cancer patients2022In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 1, article id 403Article in journal (Refereed)
    Abstract [en]

    The significance of cancer stem cells (CSCs) in initiation and progression of colon cancer (CC) has been established. In this study, we investigated the utility of measuring mRNA expression levels of CSC markers EpCAM, LGR5 and LGR4 for predicting survival outcome in surgically treated CC patients. Expression levels were determined in 5 CC cell lines, 66 primary CC tumors and 382 regional lymph nodes of 121 CC patients. Prognostic relevance was determined using Kaplan‐Meier survival and Cox regression analyses. CC patients with lymph nodes expressing high levels of EpCAM, LGR5 or LGR4 (higher than a clinical cutoff of 0.07, 0.06 and 2.558 mRNA cop-ies/18S rRNA unit, respectively) had a decreased mean survival time of 32 months for EpCAM and 42 months for both LGR5 and LGR4 at a 12‐year follow‐up (p = 0.022, p = 0.005 and p = 0.011, respec-tively). Additional patients at risk for recurrence were detected when LGR5 was combined with the biomarkers CXCL17 or CEA plus CXCL16. In conclusion, the study underscores LGR5 as a particularly useful prognostic biomarker and illustrates the strength of combining biomarkers detecting different subpopulations of cancer cells and/or cells in the tumor microenvironment for predicting recurrence.

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  • 3.
    Alhouayek, Mireille
    et al.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology. Catholic Univ Louvain, Brussels, Belgium.
    Gouveia-Figueira, Sandra
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Swedish Univ Agr Sci, Umea, Sweden.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Fowler, Christopher J.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Pharmacology.
    Involvement of CYP1B1 in interferon gamma-induced alterations of epithelial barrier integrity2018In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 175, no 6, p. 877-890Article in journal (Refereed)
    Abstract [en]

    BACKGROUND AND PURPOSE CYP1B1 and CYP1A1 are important extra-hepatic cytochromes, expressed in the colon and involved in the metabolism of dietary constituents and exogenous compounds. CYP1B1 expression is increased by pro-inflammatory cytokines, and it has been recently implicated in regulation of blood brain barrier function. We investigated its involvement in the increased permeability of the intestinal epithelial barrier observed in inflammatory conditions. EXPERIMENTAL APPROACH Epithelial monolayers formed by human T84 colon carcinoma cells cultured on transwells, were disrupted by incubation with IFN gamma (10 ng.mL(-1)). Monolayer integrity was measured using transepithelial electrical resistance. CYP1A1 and CYP1B1 inhibitors or inducers were applied apically. Potential mechanisms of action were investigated using RT-qPCR. KEY RESULTS IFN gamma disrupts the barrier integrity of the T84 monolayers and increases CYP1B1 and HIF1 alpha mRNA expression. CYP1B1 induction is inhibited by the NF-kappa B inhibitor ammonium pyrrolidinedithiocarbamate (100 mu M) but not by the HIF1 alpha inhibitor 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (50 mu M). Inhibition of CYP1B1 with the selective inhibitor 2,4,3,5-tetramethoxystilbene (100 nM) partly reverses the effects of IFN gamma on epithelial permeability. CONCLUSIONS AND IMPLICATIONS These data suggest that increased expression of CYP1B1 is involved in the effects of IFN gamma on epithelial permeability. Inhibition of CYP1B1 counteracts the alterations of epithelial barrier integrity induced by IFN gamma and could thus have a therapeutic potential in disorders of intestinal permeability associated with inflammation.

  • 4.
    Ali, Haytham
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Department of Animal and Veterinary Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Oman.
    AbdelMageed, Manar
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    Ohlsson, Lina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lindmark, Gudrun
    Institution of Clinical Sciences, Lund University, Lund, Sweden.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Sitohy, Basel
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Detection of lymph node metastasis in colon cancer by ectopically expressed fibroblast markers FOXQ1 and THBS22023In: Frontiers in Oncology, E-ISSN 2234-943X, Vol. 13, article id 1297324Article in journal (Refereed)
    Abstract [en]

    Introduction: Approximately 25% of colon cancer (CC) patients having curative surgery will relapse. Therefore, it is crucial to identify patients with increased recurrence risk to offer them adjuvant chemotherapy. Three markers with prominent expression in fibroblasts: forkhead box Q1 (FOXQ1), matrix metalloproteinase-11 (MMP11), and thrombospondin-2 (THBS2), and the fibroblast expressed chemokine CXCL12 were selected for studies because of the critical role of fibroblasts in the microenvironment of the tumor.

    Methods: The expression levels of the biomarkers were assessed in primary CC tumors, lymph nodes of CC patients and controls, and CC cell lines at mRNA and protein levels by real-time qRT-PCR and immunohistochemistry, respectively.

    Results: FOXQ1, MMP11, and THBS2 mRNAs were expressed at significantly higher levels in primary tumors compared to normal colon (P=0.002, P<0.0001, and P<0.0001, respectively). In contrast, CXCL12 mRNA levels were higher in normal colon tissue. FOXQ1, MMP11, and THBS2 levels were also expressed at significantly higher levels in metastasis-positive lymph nodes compared to both metastasis-negative- and control nodes (P<0.0001/P=0.002, P<0.0001/P<0.0001, and P<0.0001/P<0.0001, respectively). Immuno-morphometry revealed that 30–40% of the tumor cells expressed FOXQ1, MMP11, and THBS2. FOXQ1 and THBS2 were barely detected in normal colon epithelium (P<0.0001), while MMP11 was expressed in normal colon epithelium at high levels.

    Discussion: We conclude that CC tumor cells show ectopic expression of FOXQ1 and THBS2 possibly making these tumor cells independent of fibroblast cell support. The high expression levels of these two biomarkers in metastatic lymph nodes suggest that they are potential indicators of patients at risk for recurrence.

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  • 5.
    Ali, Haytham
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    AbdelMageed, Manar
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    Olsson, Lina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Israelsson, Anne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lindmark, Gudrun
    Department of Clinical Sciences, Lund University, Helsingborg, Sweden.
    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.
    Sitohy, Basel
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Utility of G protein-coupled receptor 35 expression for predicting outcome in colon cancer2019In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 41, no 6, article id 1010428319858885Article in journal (Refereed)
    Abstract [en]

    The utility of mRNA and protein determinations of G protein-coupled receptor 35, that is, GPR35a (GPR35 V1) and GPR35b (GPR35 V2/3), as indicators of outcome for colon cancer patients after curative surgery was investigated. Expression levels of V1 and V2/3 GPR35, carcinoembryonic antigen and CXCL17 mRNAs were assessed in primary tumours and regional lymph nodes of 121 colon cancer patients (stage I–IV), colon cancer cell lines and control colon epithelial cells using real-time quantitative reverse transcriptase-polymerase chain reaction. Expression of G protein-coupled receptor 35 was investigated by two-colour immunohistochemistry and immunomorphometry. GPR35 V2/3 mRNA, but not V1 mRNA, was expressed in colon cancer cell lines, primary colon tumours and control colon epithelial cells. Haematoxylin and eosin positive (H&E(+)), but not H&E(–), lymph nodes expressed high levels of GPR35 V2/3 mRNA (P<0.0001). GPR35b and carcinoembryonic antigen proteins were simultaneously expressed in many colon cancer tumour cells. Kaplan–Meier and hazard ratio analysis revealed that patients with lymph nodes expressing high levels of GPR35 V2/3 mRNA and, in particular, in the group of patients with lymph nodes also expressing carcinoembryonic antigen mRNA, had a short disease-free survival time, 67 months versus 122 months at 12-year follow-up (difference: 55 months, P = 0.001; hazard ratio: 3.6, P = 0.002). In conclusion, high level expression of G protein-coupled receptor 35 V2/3 mRNA in regional lymph nodes of colon cancer patients is a sign of poor prognosis.

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  • 6.
    Ali, Haytham
    et al.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    Ohlsson, Lina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Lindmark, Gudrun
    Institution of Clinical Sciences, Lund University, SE, Lund, Sweden.
    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.
    Sitohy, Basel
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    The myeloid cell biomarker EMR1 is ectopically expressed in colon cancer2021In: Tumor Biology, ISSN 1010-4283, E-ISSN 1423-0380, Vol. 43, no 1, p. 209-223Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: The microenvironment of colon cancer (CC) is heterogeneous including cells of myeloid lineage affecting tumor growth and metastasis. Two functional subtypes of myeloid cells have been identified; one (M1) is tumor-inhibitory and the other one (M2) is tumor-promoting. Whether the three myeloid markers EMR1, CD206 and CD86 are expressed only in the infiltrating myeloid cells or also in the tumor cells was investigated.

    METHODS: Expression of the myeloid markers was investigated in CC at the mRNA and protein levels in primary tumors and lymph nodes. mRNA expression was also determined in 5 CC cell lines. Protein expression was investigated by two-color immunofluorescence and consecutive-sections-immune-staining combined with morphometry using specific antibodies for the myeloid cell markers and the epithelial cell markers CEACAM5 and EpCAM.

    RESULTS: EMR1 and CD86, but not CD206, mRNA levels were significantly higher in CC primary tumors compared to apparently normal colon tissue (P <  0.0001). EMR1 mRNA levels were significantly higher in both hematoxylin-eosin positive (H&E(+)) and H&E(-) lymph nodes of CC patients compared to control nodes (P = 0.03 and P = 0.01, respectively). EMR1 and CD206 mRNAs were expressed in 4/5 and 5/5 CC cell lines, respectively, while CD86 mRNA was not expressed. Immuno-morphometry revealed that about 20% of the tumor cells expressed EMR1 and CD206. Positive cells were tumor cells as revealed by anti-CEACAM5 and anti-EpCAM staining. The number of EMR1, CD206 and CD86 positive cells were significantly increased in CC primary tumors compared to normal colon tissue (P <  0.0001). However, CD206 was also expressed in normal colonocytes. Only EMR1 showed significantly increased numbers of positive tumor cells in H&E(+) nodes compared to H&E(-) nodes (P = 0.001). EMR1 expression in CC tumor cells correlated with CXCL17 expressing tumor cells.

    CONCLUSION: EMR1, like the chemokine CXCL17, is ectopically expressed in colon cancer possibly in the same cancer cells.

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

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

  • 8.
    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.

  • 9.
    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.

  • 10. 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.

  • 11.
    Bitar, Aziz
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Aung, Kyaw Min
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Vibrio cholerae derived outer membrane vesicles modulate the inflammatory response of human intestinal epithelial cells by inducing microRNA-146a2019In: Scientific Reports, E-ISSN 2045-2322, Vol. 9, article id 7212Article in journal (Refereed)
    Abstract [en]

    The small intestinal epithelium of Vibrio cholerae infected patients expresses the immunomodulatory microRNAs miR-146a and miR-155 at acute stage of disease. V. cholerae release outer membrane vesicles (OMVs) that serve as vehicles for translocation of virulence factors including V. cholerae cytolysin (VCC). The aim was to investigate whether OMVs, with and/or without VCC-cargo could be responsible for induction of microRNAs in intestinal epithelial cells and thereby contribute to immunomodulation. Polarized tight monolayers of T84 cells were challenged with OMVs of wildtype and a VCC deletion mutant of the non-O1/non-O139 (NOVC) V. cholerae strain V:5/04 and with soluble VCC. OMVs, with and without VCC-cargo, caused significantly increased levels of miR-146a. Increase was seen already after 2 hours challenge with OMVs and persisted after 12 hours. Challenge with soluble VCC caused significant increases in interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), CCL20, IL-1β, and IRAK2 mRNA levels while challenge with OMVs did not cause increases in expression levels of any of these mRNAs. These results suggest that V. cholerae bacteria release OMVs that induce miR-146a in order to pave the way for colonization by reducing the strength of an epithelial innate immune defence reaction and also preventing inflammation in the mucosa that factors like VCC can evoke.

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  • 12.
    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). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    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). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rahman, Arman
    Qadri, Firdausi
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    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, 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.

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  • 13.
    Eltorky, Hagar
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Diagnostics and Intervention. Department of Biochemistry, Faculty of Science, Zagazig University, Zagazig, Egypt.
    AbdelMageed, Manar
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Diagnostics and Intervention. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    Ismail, Hager
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Diagnostics and Intervention. Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    Zahran, Faten
    Department of Biochemistry, Faculty of Science, Zagazig University, Zagazig, Egypt.
    Guirgis, Adel
    Department of Molecular Biology, Genetic Engineering, Biotechnology Research Institute, University of Sadat City, Menoufia, Sadat, Egypt.
    Ohlsson, Lina
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lindmark, Gudrun
    Institution of Clinical Sciences, Lund University, Lund, Sweden.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Sitohy, Basel
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Diagnostics and Intervention.
    LGR6 is a prognostic biomarker for less differentiated tumors in lymph nodes of colon cancer patients2024In: Frontiers in Oncology, E-ISSN 2234-943X, Vol. 14, article id 1393075Article in journal (Refereed)
    Abstract [en]

    Introduction: The aim was to investigate whether the stem cell marker LGR6 has prognostic value in colon cancer, alone or in combination with the prognostic biomarkers CEA and CXCL16.

    Methods: LGR6 mRNA levels were determined in 370 half lymph nodes of 121 colon cancer patients. Ability to predict relapse after curative surgery was estimated by Kaplan-Meier survival model and Cox regression analyses.

    Results: Patients with high LGR6 levels [LGR6(+)] had a decreased mean survival time of 11 months at 5-year follow-up and 47 months at 12-year follow-up, respectively, with hazard ratios of 3.2 and 2.8. LGR6 mRNA analysis added prognostic value to CEA and CXCL16 mRNA analysis. In the poor prognosis groups CEA(+) and CXCL16(+), further division was achieved by LGR6 analysis. LGR6(+) patients had a very poor prognosis. LGR6 also identified a small number of CEA(-), TNM stage I patients who relapsed suggesting stem cell origin of these tumors. LGR6 and LGR5 levels correlated strongly in lymph nodes of stage I and IV patients but not in stage II patients, suggesting that these stem cell markers are differentially regulated.

    Conclusion: This study highlights LGR6 as a useful prognostic biomarker independently and in combination with CEA, CXCL16 or LGR5 identifying different risk groups.

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  • 14.
    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.

  • 15.
    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)
  • 16.
    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.

  • 17.
    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)
  • 18.
    Flood, Peter
    et al.
    University College Cork, Cork, Ireland.
    Fanning, Aine
    University College Cork, Cork, Ireland.
    Woznicki, Jerzy A.
    University College Cork, Cork, Ireland.
    Crowley, Tadhg
    University College Cork, Cork, Ireland.
    Christopher, Andrea
    University College Cork, Cork, Ireland.
    Vaccaro, Alessandra
    University College Cork, Cork, Ireland.
    Houston, Aileen
    University College Cork, Cork, Ireland; Department of Medicine, University College Cork, Cork, Ireland.
    McSweeney, Sheila
    University College Cork, Cork, Ireland.
    Ross, Sarah
    University College Cork, Cork, Ireland.
    Hogan, Aileen
    University College Cork, Cork, Ireland.
    Brint, Elizabeth
    University College Cork, Cork, Ireland; Department of Pathology, Cork University Hospital, University College Cork, Clinical Sciences Building, Cork, Ireland.
    Skowyra, Agnieszka
    University College Cork, Cork, Ireland.
    Bustamante, Milan
    University College Cork, Cork, Ireland.
    Ambrose, Monica
    University College Cork, Cork, Ireland.
    Moloney, Gerard
    University College Cork, Cork, Ireland.
    MacSharry, John
    University College Cork, Cork, Ireland; School of Microbiology, University College Cork, Cork, Ireland; School of Medicine, University College Cork, Cork, Ireland.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hurley, Margot
    University College Cork, Cork, Ireland; Department of Medicine, University College Cork, Cork, Ireland.
    Fitzgibbons, Christine
    University College Cork, Cork, Ireland.
    Quigley, Eamonn M M
    University College Cork, Cork, Ireland.
    Shanahan, Fergus
    University College Cork, Cork, Ireland; Department of Medicine, University College Cork, Cork, Ireland.
    Zulquernain, Syed A.
    University College Cork, Cork, Ireland; Department of Medicine, University College Cork, Cork, Ireland.
    McCarthy, Jane
    Department of Gastroenterology, Mercy University Hospital, Cork, Ireland.
    Dodson, G Steven
    Second Genome, CA, Brisbane, United States.
    Dabbagh, Karim
    Second Genome, CA, Brisbane, United States.
    McRae, Bradford L.
    Immunology Discovery, Abbvie Bioresearch Center, MA, Worcester, United States.
    Melgar, Silvia
    University College Cork, Cork, Ireland.
    Nally, Ken
    University College Cork, Cork, Ireland; School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
    DNA sensor-associated type I interferon signaling is increased in ulcerative colitis and induces JAK-dependent inflammatory cell death in colonic organoids2022In: American Journal of Physiology - Gastrointestinal and Liver Physiology, ISSN 0193-1857, E-ISSN 1522-1547, Vol. 323, no 5, p. G439-G460Article in journal (Refereed)
    Abstract [en]

    DNA sensor pathways can initiate inflammasome, cell death, and type I interferon (IFN) signaling in immune-mediated inflammatory diseases (IMIDs), including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC) by analyzing the expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control (n = 31), inactive UC (n = 31), active UC (n = 33), and a UC single-cell RNA-Seq dataset. The effects of type I IFN (IFN-β), IFN-γ, and TNF-α on gene expression, cytokine production, and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLR family pyrin domain-containing 3 (NLRP3), caspase, or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1β/IL-18/CXCL10. The expression of DNA sensor pathway genes-PYHIN family members [absent in melanoma 2 (AIM2), IFI16, myeloid cell nuclear differentiation antigen (MNDA), and pyrin and HIN domain family member 1 (PYHIN1)- as well as Z-DNA-binding protein 1 (ZBP1), cyclic GMP-AMP synthase (cGAS), and DDX41 was increased in active UC and expressed in a cell type-restricted pattern. Inflammasome genes (CASP1, IL1B, and IL18), type I IFN inducers [stimulator of interferon response cGAMP interactor 1 (STING), TBK1, and IRF3), IFNB1, and type I IFN biomarker genes (OAS2, IFIT2, and MX2) were also increased in active UC. Cotreatment of organoids with IFN-β or IFN-γ in combination with TNFα increased expression of IFI16, ZBP1, CASP1, cGAS, and STING induced cell death and IL-1β/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC.NEW & NOTEWORTHY This study found that patients with active UC have significantly increased colonic gene expression of cytosolic DNA sensor, inflammasome, STING, and type I IFN signaling pathways. The type I IFN, IFN-β, in combination with TNF-α induced JAK-dependent but NLRP3 and inflammasome-independent inflammatory cell death of colonic organoids. This novel inflammatory cell death phenotype is relevant to UC immunopathology and may partially explain the efficacy of the JAKinibs tofacitinib and upadacitinib in patients with UC.

  • 19.
    Forsberg, Göte
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hörstedt, Per
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease2004In: American Journal of Gastroenterology, ISSN 0002-9270, E-ISSN 1572-0241, Vol. 99, no 5, p. 894-904Article in journal (Refereed)
    Abstract [en]

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

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

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

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

  • 20.
    Forsberg, Göte
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hernell, O
    Umeå University, Faculty of Medicine, Department of Clinical Sciences.
    Melgar, S
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Israelsson, A
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Paradoxical coexpression of proinflammatory and down-regulatory cytokines in intestinal T cells in childhood celiac disease2002In: Gastroenterology, ISSN 0016-5085, E-ISSN 1528-0012, Vol. 123, no 3, p. 667-678Article in journal (Refereed)
  • 21.
    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.

  • 22. 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.

  • 23.
    Hammarström, Marie-Louise
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lindmark, Gudrun
    Olsson, Lina
    HiloProbe AB.
    Israelsson, Anne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Biomarkör-mRNA analyser som komplement till histopatologisk undersöknig av lymfkörtelstatus vid kolorektal cancer2020In: Cancerläkaren, ISSN 2003-0290, no 2, p. 33-34Article in journal (Other academic)
  • 24.
    Hedberg, Maria E.
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Israelsson, Anne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Moore, Edward R. B.
    Svensson-Stadler, Liselott
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Pietz, Grzegorz
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Sandström, Olof
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    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.
    Hammarstrom, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Prevotella jejuni sp nov., isolated from the small intestine of a child with coeliac disease2013In: International Journal of Systematic and Evolutionary Microbiology, ISSN 1466-5026, E-ISSN 1466-5034, Vol. 63, no 11, p. 4218-4223Article in journal (Refereed)
    Abstract [en]

    Five obligately anaerobic, Gram-stain-negative, saccharolytic and proteolytic, non-spore-forming bacilli (strains CD3 :27, CD3 :28(T), CD3 :33, CD3 :32 and CD3 :34) are described. All five strains were isolated from the small intestine of a female child with coeliac disease. Cells of the five strains were short rods or coccoid cells with longer filamentous forms seen sporadically. The organisms produced acetic acid and succinic acid as major metabolic end products. Phylogenetic analysis based on comparative 16S rRNA gene sequence analysis revealed close relationships between CD3 : 27, CD3 :28(T) and CD3 :33, between CD3 :32 and Prevotella histicola CCUG 55407(T), and between CD3 :34 and Prevotella melaninogenica CCUG 4944B(T). Strains CD3 : 27, CD3 :28(T) and CD3 :33 were clearly different from all recognized species within the genus Prevotella and related most closely to but distinct from P. melaninogenica. Based on 16S rRNA, RNA polymerase) beta-subunit (rpoB) and 60 kDa chaperonin protein subunit (cpn60) gene sequencing, and phenotypic, chemical and biochemical properties, strains CD3 :27, CD3 :28(T) and CD3 :33 are considered to represent a novel species within the genus Prevotella, for which the name Prevotella jejuni sp. nov. is proposed. Strain CD3 : 28(T) (=CCUG 60371(T)=DSM 26989(T)) is the type strain of the proposed novel species. All five strains were able to form homologous aggregates, in which tube-like structures were connecting individual bacteria cells. The five strains were able to bind to human intestinal carcinoma cell lines at 37 degrees C.

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  • 25.
    Hedberg, Maria E
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Moore, Edward RB
    Svensson-Stadler, Liselott
    Hörstedt, Per
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    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.
    Lachnoanaerobaculum a new genus in Lachnospiraceae; characterization of Lachnoanaerobaculum umeaense gen. nov., sp. nov., isolated from human small intestine, Lachnoanaerobaculum orale gen. nov., sp. nov., isolated from saliva and reclassification of Eubacterium saburreum (Prevot) Holdeman and Moore 1970 as Lachnoanaerobaculum saburreum comb. nov.2012In: International Journal of Systematic and Evolutionary Microbiology, ISSN 1466-5026, E-ISSN 1466-5034, Vol. 62, no 11, p. 2685-2690Article in journal (Refereed)
    Abstract [en]

    Two new obligately anaerobic Gram-positive, saccharolytic and non-proteolytic spore-forming bacilli (strain CD3:22 and N1) are described. Strain CD3:22 was isolated from a biopsy of the small intestine of a child with celiac disease and strain N1 from the saliva of a healthy young man. The cells of both strains were observed to be filamentous with lengths of approximately 5 to >20 µm, some of them curving and with swellings. The novel organisms produced H2S, NH3, butyric acid and acetic acid as major metabolic end products. Phylogenetic analyses, based on comparative 16S rRNA gene sequencing, revealed close relationships (98 % sequence similarity) between the two isolates, as well as the type strain of Eubacterium saburreum CCUG 28089T and four other Lachnospiraceae bacterium/E. saburreum-like organisms. This group of bacteria were clearly different from any of the 19 known genera in the family Lachnospiraceae. While Eubacterium spp. are reported to be non-spore-forming, reanalysis of E. saburreum CCUG 28089T confirmed that the bacterium, indeed, is able to form spores. Based on 16S rRNA gene sequencing, phenotypic and biochemical properties, CD3:22 (CCUG 58757T) and N1 (CCUG 60305T) represent new species of a new and distinct genus, named Lachnoanaerobaculum, in the family Lachnospiraceae [within the order Clostridiales, class Clostridia, phylum Firmicutes]. Strain CD3:22 is the type strain of the type species, Lachnoanaerobaculum umeaense gen. nov., sp. nov., of the proposed new genus. Strain N1 is the type strain of the species, Lachnoanaerobaculum orale gen. nov., sp. nov. Moreover, E. saburreum CCUG 28089T is reclassified as Lachnoanaerobaculum saburreum comb. nov.

  • 26.
    Hedberg, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Moore, Edward
    Sahlgrenska Universitetssjukhuset, Göteborgs Universitet.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Clostridiales bacterium CD3:22-an anaerobic spore-forming bacterium isolated from small intestine in a celiac disease patient2010Report (Other academic)
  • 27.
    Ildgruben, A.
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Sjöberg, I.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Influence of Hormonal Contraceptives on the Immune Cells and Thickness of Human Vaginal Epithelium2003In: Obstetrics and Gynecology, ISSN 0029-7844, E-ISSN 1873-233X, Vol. 102, no 3, p. 571-582Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: To determine whether use of steroid hormone contraceptives modifies immune defense parameters of the vaginal epithelium in humans. METHODS: Vaginal biopsies were collected during the follicular and luteal phases in regularly menstruating women (controls) and in women using combined oral contraceptives, depot-medroxyprogesterone acetate injections, or levonorgestrel implants. Fifteen healthy women (aged 20-34 years) were enrolled in each group. Biopsies were analyzed in a blinded manner. Epithelial thickness was estimated by morphometry. Immune cells were analyzed by immunomorphometry with cell-type-specific monoclonal antibodies. RESULTS: The epithelium of controls harbored 241+/-35 leukocytes (CD45+ cells) per mm2 (mean+/-1 standard error of the mean), and the thickness was 261+/-16 microm. T lymphocytes (CD3+) dominated, and cytotoxic or suppressor T cells (CD8+) were more frequent than T helper cells (CD4:CD8 ratio: 0.7+/- 0.1). Macrophages (CD68+) constituted the second-largest population, followed by Langerhans cells (CD1a+). B cells, natural killer cells, monocytes, and granulocytes were generally absent. There were no significant differences between the follicular and luteal phases. The epithelium was significantly thicker in all three groups that used hormonal contraceptive (333+/-9 microm) compared with controls, and it exhibited superficial hyperplasia. The frequency of intraepithelial leukocytes (CD45+) was increased in depot-medroxyprogesterone acetate (P<.001) and levonorgestrel implant users (P<.04). In depot-medroxyprogesterone acetate users, this was explained by an increased frequency of the CD8+ T lymphocyte subset. CONCLUSION: Hormonal contraceptives induce hyperplasia of the vaginal epithelium. The significant changes in the intraepithelial leukocyte population in depot-medroxyprogesterone acetate and levonorgestrel implant users most probably reflect altered local immune capacity.

  • 28.
    Ismail, Hager
    et al.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    AbdelMageed, Manar
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
    Lindmark, Gudrun
    Institution of Clinical Sciences, Lund University, Helsingborg, Sweden.
    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.
    Sitohy, Basel
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Prognostic Significance of GPR55 mRNA Expression in Colon Cancer2022In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 9, article id 4556Article in journal (Refereed)
    Abstract [en]

    G protein-coupled receptor 55 (GPR55) probably plays a role in innate immunity and tumor immunosurveillance through its effect on immune cells, such as T cells and NK cells. In this study, the prognostic value of GPR55 in colon cancer (CC) was investigated. mRNA expression levels of GPR55 were determined in 382 regional lymph nodes of 121 CC patients with 12 years observation time after curative surgery. The same clinical material had previously been analyzed for expression levels of CEA, CXCL16, CXCL17, GPR35 V2/3 and LGR5 mRNAs. Clinical cutoffs of 0.1365 copies/18S rRNA unit for GPR55 and 0.1481 for the GPR55/CEA ratio were applied to differentiate between the high-and low-GPR55 expression groups. Kaplan–Meier survival analysis and Cox regression risk analysis were used to determine prognostic value. Improved discrimination between the two groups was achieved by combining GPR55 with CEA, CXCL16 or CXCL17 compared with GPR55 alone. The best result was obtained using the GPR55/CEA ratio, with an increased mean survival time of 14 and 33 months at 5 and 12 years observation time, respectively (p = 0.0003 and p = 0.003) for the high-GPR55/CEA group. The explanation for the observed improvement is most likely that GPR55 is a marker for T cells and B cells in lymph nodes, whereas CEA, CXCL16 and CXCL17, are markers for tumor cells of epithelial origin.

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  • 29.
    Lindmark, Gudrun
    et al.
    Department of Clinical Sciences, Lund University, Helsingborg, Sweden; Specialistläkarna, Malmö, Sweden.
    Olsson, Lina
    HiloProbe AB, Umeå, Sweden.
    Sitohy, Basel
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Israelsson, Anne
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Blomqvist, Joel
    HiloProbe AB, Umeå, Sweden.
    Kero, Sara
    HiloProbe AB, Umeå, Sweden.
    Roshdy, Tamer
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Department of Molecular Biology, Genetic Engineering, and Biotechnology Research Institute, University of Sadat City, Sadat City, Menoufia, Egypt.
    Söderholm, Mattias
    Department of Surgery, Blekinge Hospital, Karlskrona, Sweden.
    Turi, Annamaria
    Department of Clinical Pathology and Cytology, Blekinge Hospital, Karlskrona, Sweden.
    Isaksson, Jessica
    Department of Clinical Pathology and Cytology, Blekinge Hospital, Karlskrona, Sweden.
    Sakari, Thorbjörn
    Department of Surgical Sciences, Uppsala University Hospital, Uppsala, Sweden; Department of Surgery, Gävle Hospital, Gävle, Sweden.
    Dooper, Michiel
    Department of Clinical Pathology and Cytology, Gävle Hospital, Gävle, Sweden.
    Dafnis, George
    Colorectal Unit, Department of Surgery and Urology, Mälarsjukhuset, Eskilstuna, Sweden.
    Forsberg, Pehr
    Unilabs, Clinical Pathology and Cytology, Mälarsjukhuset, Eskilstuna, Sweden.
    Skovsted, Susanne
    Unit for Surgery, Örnsköldsvik Hospital, Örnsköldsvik, Sweden.
    Walldén, Maria
    Centrum for Surgery, Sundsvall Hospital, Sundsvall, Sweden.
    Kung, Chih-Han
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery. Department of Surgery, Skellefteå Hospital, Skellefteå, Sweden.
    Rutegård, Martin
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Nordmyr, Johanna
    Department of Clinical Pathology, Linköping University Hospital, Linköping, Sweden.
    Muhrbeck, Måns
    Department of Surgery in Norrköping, Linköping University, Norrköping, Sweden; Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    qRT-PCR analysis of CEACAM5, KLK6, SLC35D3, MUC2 and POSTN in colon cancer lymph nodes: An improved method for assessment of tumor stage and prognosis2024In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 154, no 3, p. 573-584Article in journal (Refereed)
    Abstract [en]

    One fourth of colorectal cancer patients having curative surgery will relapse of which the majority will die. Lymph node (LN) metastasis is the single most important prognostic factor and a key factor when deciding on postoperative treatment. Presently, LN metastases are identified by histopathological examination, a subjective method analyzing only a small LN volume and giving no information on tumor aggressiveness. To better identify patients at risk of relapse we constructed a qRT-PCR test, ColoNode, that determines levels of CEACAM5, KLK6, SLC35D3, MUC2 and POSTN mRNAs. Combined these biomarkers estimate the tumor cell load and aggressiveness allocating patients to risk categories with low (0, −1), medium (1), high (2) and very high (3) risk of recurrence. Here we present result of a prospective, national multicenter study including 196 colon cancer patients from 8 hospitals. On average, 21 LNs/patient, totally 4698 LNs, were examined by both histopathology and ColoNode. At 3-year follow-up, 36 patients had died from colon cancer or lived with recurrence. ColoNode identified all patients that were identified by histopathology and in addition 9 patients who were undetected by histopathology. Thus, 25% of the patients who recurred were identified by ColoNode only. Multivariate Cox regression analysis proved ColoNode (1, 2, 3 vs 0, −1) as a highly significant risk factor with HR 4.24 [95% confidence interval, 1.42-12.69, P =.01], while pTN-stage (III vs I/II) lost its univariate significance. In conclusion, ColoNode surpassed histopathology by identifying a significantly larger number of patients with future relapse and will be a valuable tool for decisions on postoperative treatment.

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  • 30. Lundqvist, C
    et al.
    Melgar, S
    Yeung, M M
    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.
    Intraepithelial lymphocytes in human gut have lytic potential and a cytokine profile that suggest T helper 1 and cytotoxic functions.1996In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 157, no 5, p. 1926-34Article in journal (Refereed)
    Abstract [en]

    The functional properties of intraepithelial lymphocytes (IEL) in normal human jejunum, ileum, and colon were investigated. Cytokine mRNA expression in IEL and enterocytes was determined by reverse transcriptase-PCR and IFN-gamma+ IEL by immunohistochemistry. Polyclonal activators were used to study proliferation and IFN-gamma secretion of IEL, and an anti-CD3-mediated redirected cytotoxicity assay was used to determine the lytic potential of IEL. Freshly isolated IEL at all three gut levels expressed mRNA for IL-1 beta, IL-2, IL-8, IFN-gamma, and TNF-alpha. Approximately 10% of IEL produced IFN-gamma, suggesting that IEL are immunologically active in vivo, performing similar functions along the intestine. IEL could be stimulated further in vitro to express IL-10, TNF-beta, and TGF-beta 1, while no Th2-type cytokines were induced, suggesting suppressive and cytolytic functions for IEL. All three jejunal IEL subpopulations (CD4-CD8-TCR-gamma delta+, CD4+TCR-alpha beta+, CD8+TCR-alpha beta+) expressed the same four cytokines, IL-2, IL-8, IFN-gamma, and TNF-alpha, indicating that CD4+TCR-alpha beta+ IEL are Th1 cells and that TCR-gamma delta+ IEL and CD8+TCR-alpha beta+ IEL include cytotoxic effector cells. Indeed, freshly isolated jejunal IEL displayed cytolytic activity. IEL were induced to proliferation by anti-CD3/TCR complex mAbs and leukoagglutinin, but not by Con A. There was no correlation between the magnitude of the proliferative response and the amounts of secreted IFN-gamma. Enterocytes expressed IL-1 beta and IL-8, and sometimes TNF-alpha. Although jejunal enterocytes express HLA-DR and hsp60, Ag presentation by these cells may induce anergy since their cytokine profile is different from that of classical APCs.

  • 31.
    Lundqvist, Carina
    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.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Athlin, Leif
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Cytokine profile and ultrastructure of intraepithelial lymphocytes in human gut suggest that they are activated cells with T helper 1 and cytotoxic functionsManuscript (preprint) (Other academic)
  • 32.
    Lundqvist, Carina
    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.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Athlin, Leif
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Intra-epithelial lymphocytes. Evidence for regional specialization and extrathymic T cell maturation in the human gut epithelium1995In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 7, no 9, p. 1473-1487Article in journal (Refereed)
    Abstract [en]

    The human gut epithelium is a unique immunological compartment, containing substantial amounts of intra-epithelial lymphocytes (IEL) with unknown functions. In this study we show that distinct and unusual subpopulations of IEL are present at different levels of human intestine. IEL phenotypes in normal jejunum, ileum and colon were compared using immunoflow cytometry and immunohistochemistry. The expression of mRNA for recombination-activating gene-1 (RAG-1) in IEL from all three levels was compared using reverse-transcription polymerase chain reaction, and the morphology of IEL in situ was determined using immunoelectron microscopy. Surface marker profiles of isolated intestinal epithelial cells at all three levels were also investigated. On average the proportion of TCR gamma delta IEL was comparable in jejunum than ileum and colon and varied in phenotype with gut level. CD4-CD8-TCR alpha beta IEL dominated in colon but were absent in jejunum. CD8+ TCR alpha beta IEL were present at all levels but only in jejunum did they constitute the majority of all IEL. CD4+ TCR alpha beta IEL were present in similar frequencies at all levels of the gut. In general, the majority of IEL had an activated phenotype (CD45RO+, alpha E beta 7+). Furthermore, IEL exhibited phenotypes which are rare in peripheral blood. The thymocyte markers CD1a and CD1c as well as the NK cell marker CD56 were expressed on a fraction of TCR alpha beta and TCR gamma delta IEL. A small population of 'null' cells (CD45+ TCR/CD#-CD20-CD14-CD15- cells) was also present at equal proportions along the gut. Jejunal but not colonic IEL expressed RAG-1 mRNA suggesting that extrathymic T cell maturation occurs in the epithelium of small intestine. RAG-1 was expressed in CD2+TCR/CD3- and CD3+/TCR-IEL. Ultrastructurally, IEL often formed small clusters and intimate contacts with epithelial cells, suggesting cell cooperation within the epithelium. Some IEL had pseudopodium-like extensions penetrating the epithelial basement membrane suggesting transmigration. Epithelial cells in small intestine but not colon expressed heat shock protein 60 and HLA-DR. CD1a, CD1b and CD1c were not expressed on intestinal epithelial cells at any level. The distinct surface marker profiles of IEL and epithelial cells along small and large intestine suggest functional regional specialization and are compatible with the hypothesis that TCR alpha beta IEL participate in immune reactions to lumenal antigens while TCR gamma delta IEL perform surveillance of the epithelium.

  • 33.
    Lundqvist, Carina
    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.
    Teglund, S
    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.
    Cytokine profile and ultrastructure of intraepithelial gamma delta T cells in chronically inflamed human gingiva suggest a cytotoxic effector function1994In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 153, no 5, p. 2302-2312Article in journal (Refereed)
    Abstract [en]

    We have shown that gamma delta T cells in human gingiva have an intraepithelial location and, that in the chronic inflammatory disease periodontitis, the expression of CD45RO and CD8 or CD4 is induced on gamma delta T cells. To study the role of gamma delta T cells in local antibacterial responses, we determined the cytokine profiles of isolated human gingival cells. Different T cell subpopulations, isolated by positive selection with mAb-coated magnetic beads and macrophages, as well as epithelial cells, were analyzed for expression of mRNA for 15 cytokines by reverse transcriptase-PCR. The ultrastructure of gingival gamma delta T cells was also studied. The gamma delta T cells expressed mRNA for IFN-gamma, TNF-alpha, TGF-beta 1, and IL-6. Expression of IFN-gamma was a consequence of inflammation. CD4+ gamma delta T cells expressed IFN-gamma only, whereas CD8+ gamma delta T cells expressed all four cytokines. CD8+ cells expressing IFN-gamma, TNF-alpha, and IL-6 in combination suggest a cytotoxic effector function. Gingival gamma delta T cells contained cytoplasmic electron-dense membrane-bound granules and multivesicular bodies that are ultrastructural characteristics of cytotoxic cells. Epithelial cells from inflamed gingiva expressed HLA-DR, CD1a, CD1c, and heat shock protein 60 on the cell surface. They also expressed mRNA for IL-1 beta, IL-6, IL-8, TNF-alpha, and TGF-beta 1. Thus, epithelial cells may function as accessory cells in immune activation and, at the same time, be target cells for CD8+ gamma delta T cells reactive with CD1 Ag or heat shock protein. These results suggest that gamma delta T cells constitute a first line of defense in gingiva, preventing entrance of pathogens by cytotoxicity against infected and stressed epithelial cells, and by control of epithelial cell growth through secretion of regulatory cytokines.

  • 34.
    Lundqvist, Carina
    et al.
    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.
    T-cell receptor gamma delta-expressing intraepithelial lymphocytes are present in normal and chronically inflamed human gingiva1993In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 79, no 1, p. 38-45Article in journal (Refereed)
    Abstract [en]

    The phenotypic profile of leucocytes in diseased and normal gingival tissue was studied in situ and in isolated gingival mononuclear cell (GMC) preparations. T-cell receptor (TcR)gamma delta + cells showed preferential localization to epithelium, both in normal and inflamed gingiva, and were present in crevicular as well as oral epithelium. In normal gingiva > or = 30% of the isolated leucocytes expressed TcR gamma delta, of which the majority were CD4- CD8-, and expressed CD45RA. The proportion of TcR gamma delta + cells in GMC from periodontitis tissue varied between 2 and 32%. In contrast to normal gingiva the majority of TcR gamma delta + cells in diseased tissue were CD8+ and expressed CD45RO. Thus expression of the CD8 antigen on gingival TcR gamma delta + cells is probably a consequence of immune activation. Numerous Langerhans' cells and keratinocytes expressing the major histocompatibility complex (MHC) class I-like antigen, CD1, were present within normal and inflamed gingival epithelium in close proximity to the TcR gamma delta + cells. Most CD1a+ cells were scattered within oral epithelium. CD1c+ cells were localized close to the basal layer of crevicular epithelium. No CD1b+ cells were found. TcR alpha beta + cells, CD4+ and B cells were restricted to lamina propria of periodontitis lesions. The presence of intraepithelial TcR gamma delta + cells in normal gingiva suggests that they constitute the 'first line of defence' against the potentially harmful microflora in the oral cavity. Induction of CD8 and CD45RO antigens on TcR gamma delta + cells in periodontitis tissue indicate that they play a significant role in the disease. CD1 molecules on Langerhans' cells and keratinocytes may be the restriction elements for the CD8+ TcR gamma delta + cells.

  • 35.
    Lundqvist, Carina
    et al.
    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.
    Athlin, Leif
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine1992In: JIM - Journal of Immunological Methods, ISSN 0022-1759, E-ISSN 1872-7905, Vol. 152, no 2, p. 253-263Article in journal (Refereed)
    Abstract [en]

    A mild purification method has been developed for the isolation of human intraepithelial lymphocytes (IEL) and enterocytes from the same individual. The isolation procedure includes mechanical disruption of the mucosal layer, treatment with reducing agent and sedimentation followed by Percoll gradient centrifugation. Finally, epithelial cells are removed from the IEL fraction using magnetic beads coated with the anti-epithelial antigen monoclonal antibody (mAb) BerEP4. Leucocytes are removed from the enterocyte fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Using this procedure IEL and enterocytes have been isolated from apparently normal jejunal, ileal and colonic tissue specimens. Recoveries of IEL were 7 x 10(5), 4 x 10(5) and 1 x 10(5)/cm2 mucosa from jejunum, ileum and colon respectively. 1-2 x 10(6) enterocytes/cm2 mucosa were recovered from small intestine while the corresponding value for colonic biopsies was approximately 2 x 10(5) enterocytes/cm2. The IEL fraction was pure as judged by the low percentages of B cells, macrophages and BerEP4 positive cells (less than 4%) present in the purified fraction. The enterocyte fraction contained less than 2% CD45+ cells. The two cell fractions were viable and expanded in vitro. Enterocytes expanded spontaneously while IEL required initial stimulation with mitogens. The isolation procedure described here will make it possible to study the function of human IEL, interactions between IEL and enterocytes and the role of both cell types in local immunity.

  • 36.
    Melgar, S
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Öberg, Åke
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.
    Danielsson, Åke
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Cytolytic capabilities of lamina propria and intraepithelial lymphocytes in normal and chronically inflamed human intestine2004In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 60, no 1-2, p. 167-177Article in journal (Refereed)
    Abstract [en]

    Cell-mediated lymphocyte cytotoxicity in ileum and colon of patients with ulcerative colitis (UC), Crohn's disease (CD) and controls was investigated. Frequencies of cells expressing perforin and Fas-ligand (FasL) were determined by immunomorphometry. mRNA expression of perforin, granzyme B and FasL in T cells and subsets was assayed by reverse transcriptase-polymerase chain reaction. Cytotoxicity of intraepithelial and lamina propria lymphocytes was analysed without ex vivo activation in three functional assays: (1) anti-CD3-dependent T-cell receptor (TCR)-/CD3-mediated redirected cytotoxicity, (2) Fas-/FasL-mediated TCR-/CD3-independent cytotoxicity and (3) natural killer (NK) cell cytotoxicity. Inflammation in ileum of CD patients caused increased frequency of perforin-expressing cells and enhanced perforin-dependent TCR-/CD3-mediated cytotoxicity. In contrast, lymphocytes in the inflamed colon of UC or Crohn's colitis patients did not display this cytotoxicity nor did lymphocytes of normal colon. Normal colon lymphocytes showed spontaneous Fas-/FasL-mediated cytotoxicity. This activity was retained but not enhanced in inflamed UC colon. In contrast, a significant increase of FasL-expressing cells was seen in situ. Inflammation did not induce NK cell activity in colonic lymphocytes. Intestinal lymphocytes comprise effectors active in two different cytolytic processes. 'Classical' cytotoxic T lymphocytes in small intestine and lymphocytes executing TCR-/CD3-independent FasL-/Fas-mediated killing of unknown biological role present throughout the intestinal mucosa. Ongoing normal cytolytic processes seem to be enhanced by chronic inflammation.

  • 37. Melgar, S
    et al.
    Yeung, M M-W
    Bas, A
    Forsberg, G
    Suhr, O
    Oberg, A
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Danielsson, A
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Over-expression of interleukin 10 in mucosal T cells of patients with active ulcerative colitis.2003In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 134, no 1, p. 127-37Article in journal (Refereed)
    Abstract [en]

    Ulcerative colitis (UC), a chronic inflammatory bowel disease, exhibits pronounced increase of T lymphocytes in the inflamed mucosa. To understand the role of intestinal T lymphocytes in the pathogenesis of UC their cytokine production in the mucosa was analysed. Intestinal T lymphocytes of UC, Crohn's disease and control patients were analysed for cytokine mRNA levels by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) directly after isolation without in vitro stimulation. Frequencies of cytokine positive cells were determined in UC and control colon by immunomorphometry. T lymphocytes in normal colon expressed interleukin (IL)-2, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1, but not IL-4, IL-5 or IL-10. In UC, a highly significant increase in IL-10 mRNA levels in T lymphocytes and an increased frequency of IL-10 positive cells was seen in colon. IL-10 mRNA levels were also elevated in T lymphocytes of the non-inflamed ileum and correlated with disease activity at both locations. CD4+ T lymphocytes were the major source of IL-10 mRNA. IL-2, IFN-gamma and TNF-alpha mRNA levels were decreased in colonic T lymphocytes, and virtually no IL-2, IFN-gamma, TNF-alpha or TGF-beta positive cells were detected in basal lymphoid aggregates. However, scattered IL-10 positive cells were found here. Lamina propria outside the aggregates contained IL-10-, IFN-gamma, TNF-alpha and TGF-beta but not IL-2 positive cells. T cells of UC patients did not express IL-4 or IL-5. Taken, together the data suggest a generalized activation of IL-10 producing CD4+ T cells along the intestine of UC patients. The local environment seems to determine the biological consequences of elevated IL-10.

  • 38. Melgar, Silvia
    et al.
    Bas, Anna
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Human small intestinal mucosa harbours a small population of cytolytically active CD8+ alphabeta T lymphocytes.2002In: Immunology, Vol. 106, no 4, p. 476-485Article in journal (Refereed)
  • 39.
    Melgar, Silvia
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Yeung, M.M.
    Forsberg, G.
    Omholt, K.
    Suhr, Ole
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Öberg, Å.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Danielsson, Åke
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Mucosal T cells of patients with active ulcerative colitis express increased levels of the immune-regulatory cytokine interleukin-10Manuscript (preprint) (Other academic)
  • 40.
    Mincheva-Nilsson, Lucia
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Yeung, M M
    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.
    Immunomorphologic studies of human decidua-associated lymphoid cells in normal early pregnancy.1994In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 152, no 4, p. 2020-32Article in journal (Refereed)
    Abstract [en]

    Human decidual lymphocytes from early, normal pregnancy were characterized in situ with respect to ultrastructure and distribution of subsets. The ultrastructure of isolated decidual gamma delta T cells was also studied. CD45+ cells comprised 11 +/- 2% of all decidual cells. The majority were localized in large lymphoid cell clusters (LCC), near endometrial glands, or as intraepithelial lymphocytes (IEL) in glandular epithelium. The major cell populations in LCC were CD56+TCR-gamma delta+ cells, CD56+ cells, TCR-alpha beta+CD4+ cells, and TCR-alpha beta+CD8+ cells. All expressed activation markers (CD45RO, Kp43, and/or HML-1) and MHC class II Ag (HLA-DR, HLA-DP, and/or HLA-DQ). No B cells were found. Almost all IEL were activated TCR-gamma delta+ cells (CD56+ and CD56-). The glandular epithelial cells expressed heat shock protein 60 at the basolateral side facing the TCR-gamma delta+ IEL. Decidual lymphocytes displayed cytoplasmic processes, microvilli, characteristic cytoplasmic granules, and had intimate contact with neighboring cells. Lymphocytes in the outer rim of LCC and the stroma showed signs of cellular movement. Two main morphotypes of gamma delta T cells could be distinguished. One had single microvilli, membrane-bound granules, and nuclear inclusions. The other had many microvilli, nonmembrane-bound granules and cytoplasmic multivesicular bodies. Our data suggest that LCC are centers of immune reactivity where T and NK cells become activated. The activated cells may guard against infections and undue trophoblast invasion and/or be involved in modulating the local maternal immune system toward unresponsiveness against the semiallogeneic fetus.

  • 41.
    Mincheva-Nilsson, Lucia
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Juto, Per
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Human milk contains proteins that stimulate and suppress T lymphocyte proliferation1990In: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 79, no 3, p. 463-469Article in journal (Refereed)
    Abstract [en]

    The modulatory effect of human milk proteins from colostrum and late milk on the proliferative response of human T lymphocytes activated by mitogens (OKT3 and leucoagglutinin from Phaseolus vulgaris) and alloantigens was studied. High concentrations (10-100 micrograms/ml) of crude colostral milk proteins had an inhibitory effect on T cell growth while low concentrations (0.1-1 microgram/ml) enhanced T cells growth. In contrast, proteins from late milk did not inhibit T lymphocyte proliferation while the enhancing effect was retained. Colostrum was fractionated by ammonium sulphate precipitation and gel filtration on sepharose 6B. The inhibitory activity was recovered in a protein fraction containing lactoferrin as its major component. Lactoferrin was, however, not responsible for the observed inhibition. On the contrary, lactoferrin in most cases augmented the proliferative response induced by polyclonal activators. The inhibitory activity was found to bind concanavalin A-sepharose suggesting an association with glycoprotein. Inhibitory fractions contained glycoproteins of the following molecular sizes 26, 74/76 (doublet), 84, 145 and 160 kD under reducing conditions. The inhibitory effect appeared to be lymphocyte specific since the active fraction did not inhibit the growth of tissue culture cells (HeLa cells and human fibroblasts) or bacteria. Furthermore, the fraction was not toxic for lymphocytes. The inhibitory colostrum factor may prevent the newborn from overreacting immunologically against the environmental antigens encountered at birth.

  • 42.
    Mincheva-Nilsson, Lucia
    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.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Activated human gamma delta T lymphocytes express functional lactoferrin receptors.1997In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 46, no 6, p. 609-18Article in journal (Refereed)
    Abstract [en]

    Lactoferrin (Lf), an iron-binding protein in milk, mucosal secretions and neutrophil granules has bactericidal properties and is a source of iron for breast-fed infants. In this paper the authors show that most in vivo activated lymphocytes, i.e. freshly isolated lymphocytes from first trimester human decidua, and most in vitro activated human blood lymphocytes, express lactoferrin receptors (Lf-R), while unstimulated blood lymphocytes do not. All major lymphocyte subsets, i.e. alpha beta T cells, gamma delta T cells, CD8+ T cells, CD4+ T cells, B cells and NK cells, express Lf-R after activation. The proportion of Lf-R expressing activated gamma delta T cells is significantly larger than that of activated alpha beta T cells. Lf-R and transferrin receptors (Tr-R/CD71) show the same kinetics of appearance on activated blood lymphocytes and are, to a large extent, expressed on the same cells. However, 35% of decidual lymphocytes and 15% of activated blood lymphocytes express Lf-R only. Addition of Lf to cultures containing an optimal concentration of Tr augments the proliferative response to polyclonal T cell activators and alloantigens, suggesting that presently used standard culture conditions for in vitro activation are suboptimal in particular for gamma delta T cells. Lf-R on decidual lymphocytes contain bound Lf, which probably is produced locally. The results suggest that Lf is a growth-supporting factor, especially important in local immune responses in the mucosa.

  • 43.
    Mincheva-Nilsson, Lucia
    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.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Human decidual leukocytes from early pregnancy contain high numbers of gamma delta+ cells and show selective down-regulation of alloreactivity.1992In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 149, no 6, p. 2203-11Article in journal (Refereed)
    Abstract [en]

    The mononuclear lymphoid cell population in human pregnant uterus mucosa, decidua, from early normal pregnancies was studied phenotypically and functionally. The phenotype was determined in situ by immunohistochemistry, and in isolated decidual mononuclear cell preparations by immunofluorescence and flow cytometry. A mild isolation procedure of gentle mechanical disruption followed by density gradient centrifugation was used. Leukocytes comprised a large part of the decidual tissue. They were present in aggregates mainly situated adjacent to the glandular epithelium. In addition, individual leukocytes were present intraepithelially, as well as scattered between the stromal cells and around vessels and lacunes. Four lymphocyte populations of approximately the same size were identified: TCR gamma delta+/CD56+ cells, TCR gamma delta+/CD56- cells, TCR gamma delta-/CD56+ cells, and TCR alpha beta+/CD8+ cells. TCR gamma delta- expressing cells comprised about 60% of the T cells. They were CD4-/CD8-, and about half of the TCR gamma delta+ cells expressed the memory/activation marker CD45RO. The Kp 43 Ag, earlier described on activated CD56+ and TCR gamma delta+ cells in peripheral blood, was essentially only expressed on the TCR gamma delta-/CD56+ cell population in decidua. At least 50% of the TCR alpha beta+ cells were CD8+. The function(s) of either one of these populations might be to prevent immunologic reactions against the fetus, to protect the uterus from unwanted extensive invasion of trophoblasts, or to protect the uteroplacental unit from infection. Decidual T cells did not respond to stimulation by alloantigens or mitogenic anti-CD3 mAb but responded to the same extent as PBMC to mitogenic lectins. The surface density of the TCR/CD3 complex was low on freshly isolated decidual lymphocytes, but could be up-regulated upon stimulation with PMA/Ionomycin. Local selective down-regulation of surface expression of the TCR/CD3 complex and of activation involving this complex might be one of the mechanisms by which a maternal immunologic reaction against the semiallogeneic fetus is prevented.

  • 44.
    Mincheva-Nilsson, Lucia
    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.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Immunomodulatory role of decidual epithelial cells in early human pregnancyManuscript (preprint) (Other academic)
  • 45.
    Mincheva-Nilsson, Lucia
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Kling, M
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Nagaeva, O
    Sundqvist, K G
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Gamma delta T cells of human early pregnancy decidua: evidence for local proliferation, phenotypic heterogeneity, and extrathymic differentiation.1997In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 159, no 7, p. 3266-77Article in journal (Refereed)
    Abstract [en]

    The uterine mucosa in pregnancy, the decidua, allows placenta formation and survival of the fetus despite the fact that it is semiallogeneic. Decidua contains large numbers of lymphocytes, of which CD56+ cells dominate, followed by T cells expressing either alpha beta or gamma delta TCR. We have investigated the developmental relationship between the CD56- and TCR gamma delta-expressing cells in early pregnancy decidua using dual labeling immunoelectron microscopy, immunoflow cytometry, and cell fractionation. Lymphocyte subpopulations were, in addition, analyzed for expression of the cytokine receptor for IL-7 and c-kit and for mRNA expression of recombinase-activating genes 1 and 2. Four different cell populations could be distinguished: CD56+bright, CD56+dim/TCR gamma delta+low, CD56+dim/TCR gamma delta+high, and TCR gamma delta+low. Recombinase-activating genes 1 and 2 were expressed in the CD56+bright cells and to a limited degree in CD56+dim/TCR gamma delta+low cells. c-kit was preferentially expressed on the CD56+bright cells, while IL-7R was preferentially expressed on CD56+dim/TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells. The CD56+dim TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells displayed the characteristic morphology of large granular lymphocytes, while single positive TCR gamma delta+low cells were usually smaller and did not contain cytoplasmic granules. The gamma delta 1 gene segment was almost exclusively used in the TCR. Gamma delta T cells in mitosis were seen. We suggest that human early pregnancy decidua is a transient site for extrathymic maturation and that the progenitors of TCR gamma delta+ cells are bone marrow-derived immature cells expressing the CD56 (neural cell adhesion molecule) homing receptor.

  • 46.
    Mincheva-Nilsson, Lucia
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Nagaeva, Olga
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Sundqvist, Karl-Gösta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    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.
    Baranov, Vladimir
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    gammadelta T cells of human early pregnancy decidua: evidence for cytotoxic potency.2000In: International Immunology, ISSN 0953-8178, E-ISSN 1460-2377, Vol. 12, no 5, p. 585-96Article in journal (Refereed)
    Abstract [en]

    The immune compromise in decidua allows a semiallogeneic fetus to survive without impairing the ability of the maternal immune system to fight infections. Cytotoxic mechanisms are likely to be important in this compromise. Using RT-PCR, immunoflow cytometry and immunoelectron microscopy, the cytotoxic potential of isolated human decidual gammadelta T cells was studied. mRNA for perforin (Pf), granzymes A and B, granulysin and Fas ligand (FasL) was simultaneously expressed in decidual gammadelta T cells. Pf and FasL were not expressed on the cell surface. However, the cells constitutively synthesized Pf and stored it in cytolytic granules. Within the granules Pf mainly resided in the granule core formed by Pf-containing microvesicles. Ultrastructurally, three groups of Pf-containing granules were distinguished. They probably represent different stages of granule maturation in a process where Pf-containing microvesicles first attach to the core cortex and then are translocated across the cortex into the core. Presynthesized FasL was also stored in the core and microvesicles of the cytolytic granules. Upon degranulation by ionomycin/Ca(2+) treatment, FasL was rapidly translocated to the cell surface, demonstrating that its surface expression was not controlled by de novo biosynthesis. Thus decidual gammadelta T cells appear to perform Pf- and FasL-mediated cytotoxicity utilizing a common secretory mechanism based on cytolytic granule exocytosis. The first cytochemical visualization of lipids in the cytolytic granules is provided. These intragranular lipids probably wrap up the core and participate in packaging of the cytotoxic proteins as well as in the killing process. An ultrastructural model of a cytolytic granule is presented.

  • 47. Moore, Edward R.B.
    et al.
    Salvà‐Serra, Francisco
    Jaén‐Luchoro, Daniel
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hedberg, Maria E.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lachnoanaerobaculum2021In: Bergey's Manual of Systematics of Archaea and Bacteria (BMSAB), John Wiley & Sons, 2021Chapter in book (Refereed)
    Abstract [en]

    The genus Lachnoanaerobaculum comprises obligately anaerobic, Gram-stain-positive chemoorganotrophic, saccharolytic, and nonproteolytic bacilli. Cells are spore forming, rod shaped, and filamentous, 5 to greater than 20 μm in length, some cells with curving and swelling. All species of the genus grow with glucose as the sole carbon source. All species produce H2S, NH3, butyric acid, acetic acid, and lactic acid as metabolic end products. The predominant cellular fatty acids are C14:0, C16:0, and C18:1 ω7c DMA. The G + C contents of genomic DNA of the species are 35.0–37.8 mol%. Phylogenetic relationships of the species of Lachnoanaerobaculum, based on comparative 16S rRNA gene sequence analyses, indicate that they cluster within the phylum Firmicutes, within the family Lachnospiraceae, and exhibit a clear delineation to the other genera of the family, with a relatively close relationship to Johnsonella species. The first described strain of Lachnoanaerobaculum umeaense, the type species of the genus, was isolated from the jejunal mucosa of a child with coeliac disease. Strains of the species of Lachnoanaerobaculum are typically found in intestinal microbiota and oral microbiota of the human microbiome, isolated from the human gut, saliva, blood, amniotic fluid, and, predominantly, from the oral cavity.

  • 48.
    Myleus, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Epidemiology and Global Health.
    Stenlund, Hans
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Epidemiology and Global Health.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Gothefors, Leif
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Persson, Lars-åke
    Women's and Children's Health, International Maternal and Child Health, Uppsala University, Uppsala, Sweden.
    Ivarsson, Anneli
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Epidemiology and Global Health.
    Early vaccinations are not risk factors for Celiac Disease2012In: Pediatrics, ISSN 0031-4005, E-ISSN 1098-4275, Vol. 130, no 1, p. E63-E70Article in journal (Refereed)
    Abstract [en]

    OBJECTIVES: To investigate if changes in the national Swedish vaccination program coincided with changes in the celiac disease (CD) incidence rate in infants (ie, the Swedish CD Epidemic), and to assess the potential association between these vaccinations and CD risk.

    METHODS: All studies were based on the National Swedish Childhood Celiac Disease Register. Using an ecological approach, we plotted changes over time in the national vaccination program in the graph displaying CD incidence rate. A population-based incident case-referent study of invited infants was performed. Exposure information was received through a questionnaire and child health clinic records. Vaccines explored were diphtheria/tetanus, pertussis (acellular), polio (inactivated), Haemophilus influenzae type b (conjugated), measles/mumps/rubella, and live attenuated bacillus Calmette-Guerin (BCG) in children with increased tuberculosis risk. Findings were subjected to a birth cohort analysis.

    RESULTS: Introduction of pertussis vaccine coincided in time with decreasing CD incidence rates. In the infant case-referent study, however, neither vaccination against pertussis (odds ratio 0.91; 95% confidence interval 0.60-1.4), nor against Haemophilus influenzae type b or measles/mumps/rubella was associated with CD. Coverage for the diphtheria/tetanus and polio vaccines was 99%. BCG was associated with reduced risk for CD (adjusted odds ratio 0.54; 95% confidence interval 0.31-0.94). Discontinuation of general BCG vaccination did not affect the cumulative incidence of CD at age 15 years.

    CONCLUSIONS: Early vaccinations within the national Swedish program were not associated with CD risk, nor could changes in the program explain the Swedish epidemic. A protective effect by BCG was suggested, which could be subject to further studies. Pediatrics 2012;130:e63-e70

  • 49.
    Myléus, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Epidemiology and Global Health.
    Hernell, Olle
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Gothefors, Leif
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Persson, Lars-Åke
    International Maternal and Child Health, Department of Women's and Children's Health, Uppsala University, Uppsala, Sweden.
    Stenlund, Hans
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Epidemiology and Global Health.
    Ivarsson, Anneli
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Epidemiology and Global Health.
    Early infections are associated with increased risk for celiac disease: an incident case-referent study2012In: BMC Pediatrics, E-ISSN 1471-2431, Vol. 12, no 1, p. 194-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Celiac disease is defined as a 'chronic small intestinal immune-mediated enteropathy precipitated by exposure to dietary gluten in genetically predisposed individuals'. Sweden has experienced an "epidemic" of celiac disease in children below two years of age. Celiac disease etiology is considered multifactorial; however, little is known regarding potential risk- or protecting factors. We present data on the possible association between early infectious episodes and celiac disease, including their possible contribution to the Swedish celiac disease epidemic.

    METHODS: A population-based incident case-referent study (475 cases, 950 referents) with exposure information obtained via a questionnaire (including family characteristics, infant feeding, and the child's general health) was performed. Celiac disease cases were diagnosed before two years of age, fulfilling the diagnostic criteria of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition. Referents were randomly selected from the national population register after fulfilling matching criteria. The final analyses included 954 children, 373 (79%) cases and 581 (61%) referents, with complete information on main variables of interest in a matched set of one case with one or two referents.

    RESULTS: Having three or more parental-reported infectious episodes, regardless of type of infection, during the first six months of life was associated with a significantly increased risk for later celiac disease, and this remained after adjusting for infant feeding and socioeconomic status (odds ratio [OR] 1.5; 95% confidence interval [CI], 1.1-2.0; P=0.014). The celiac disease risk increased synergistically if, in addition to having several infectious episodes, infants were introduced to dietary gluten in large amounts, compared to small or medium amounts, after breastfeeding was discontinued (OR 5.6; 95% CI, 3.1-10; P<0.001).

    CONCLUSION: This study suggests that having repeated infectious episodes early in life increases the risk for later celiac disease. In addition, we found a synergistic effect between early infections and daily amount of gluten intake, more pronounced among infants for whom breastfeeding had been discontinued prior to gluten introduction. Regarding contribution to the Swedish celiac disease epidemic, which partly was attributed to concurrent changes in infant feeding, early infections probably made a minor contribution via the synergistic effect with gluten amount.

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  • 50. Nap, M
    et al.
    Hammarström, Marie-Louise
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Börmer, O
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Wagener, C
    Handt, S
    Schreyer, M
    Mach, J P
    Buchegger, F
    von Kleist, S
    Specificity and affinity of monoclonal antibodies against carcinoembryonic antigen.1992In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 52, no 8, p. 2329-39Article in journal (Refereed)
    Abstract [en]

    The binding specificities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) from 12 different research groups were studied by immunohistochemistry and immuno flow cytometry. In addition, the binding constant for the interaction between Mab and CEA was determined by a solution-phase assay. Cryostat sections of colon carcinoma and normal colon, stomach, liver, pancreas, and spleen were studied by immunohistochemistry. Peripheral blood granulocytes, monocytes, and lymphocytes were assayed by immuno flow cytometry. The Mabs used here have previously been classified into five essentially nonoverlapping epitope groups (GOLD 1-5) (Cancer Res., 49: 4852-4858, 1989). Most Mabs cross-reacted with different normal tissues, ranging from highly cross-reactive Mabs (positive reaction with 8 of 9 discriminating tissues) to relatively specific Mabs (positive reaction with 1 of 9 discriminating tissues). Five Mabs (10%) were specific, reacting only with colon carcinoma, normal colon mucosa, and normal gastric foveola. There was a correlation between epitope group and binding specificity. Mabs with a high degree of CEA specificity almost exclusively belonged to epitope groups 1, 2, and 3, while highly cross-reactive Mabs belonged to epitope groups 4 and 5. There was no correlation between antibody specificity and affinity for CEA. Specific Mabs with high as well as low affinity were found.

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