1. |
- Acevedo, Nathalie, et al.
(author)
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DNA Methylation Levels in Mononuclear Leukocytes from the Mother and Her Child Are Associated with IgE Sensitization to Allergens in Early Life
- 2021
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22:2
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Journal article (peer-reviewed)abstract
- DNA methylation changes may predispose becoming IgE-sensitized to allergens. We analyzed whether DNA methylation in peripheral blood mononuclear cells (PBMC) is associated with IgE sensitization at 5 years of age (5Y). DNA methylation was measured in 288 PBMC samples from 74 mother/child pairs from the birth cohort ALADDIN (Assessment of Lifestyle and Allergic Disease During INfancy) using the HumanMethylation450BeadChip (Illumina). PBMCs were obtained from the mothers during pregnancy and from their children in cord blood, at 2 years and 5Y. DNA methylation levels at each time point were compared between children with and without IgE sensitization to allergens at 5Y. For replication, CpG sites associated with IgE sensitization in ALADDIN were evaluated in whole blood DNA of 256 children, 4 years old, from the BAMSE (Swedish abbreviation for Children, Allergy, Milieu, Stockholm, Epidemiology) cohort. We found 34 differentially methylated regions (DMRs) associated with IgE sensitization to airborne allergens and 38 DMRs associated with sensitization to food allergens in children at 5Y (Sidak p <= 0.05). Genes associated with airborne sensitization were enriched in the pathway of endocytosis, while genes associated with food sensitization were enriched in focal adhesion, the bacterial invasion of epithelial cells, and leukocyte migration. Furthermore, 25 DMRs in maternal PBMCs were associated with IgE sensitization to airborne allergens in their children at 5Y, which were functionally annotated to the mTOR (mammalian Target of Rapamycin) signaling pathway. This study supports that DNA methylation is associated with IgE sensitization early in life and revealed new candidate genes for atopy. Moreover, our study provides evidence that maternal DNA methylation levels are associated with IgE sensitization in the child supporting early in utero effects on atopy predisposition.
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2. |
- Acevedo, Nathalie, et al.
(author)
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Epigenetic alterations in skin homing CD4(+)CLA(+) T cells of atopic dermatitis patients
- 2020
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Journal article (peer-reviewed)abstract
- T cells expressing the cutaneous lymphocyte antigen (CLA) mediate pathogenic inflammation in atopic dermatitis (AD). The molecular alterations contributing to their dysregulation remain unclear. With the aim to elucidate putative altered pathways in AD we profiled DNA methylation levels and miRNA expression in sorted T cell populations -(CD4(+), -CD4(+)CD45RA(+) naive, -CD4(+)CLA(+), and -CD8(+)) from adult AD patients and healthy controls (HC). Skin homing -CD4(+)CLA(+) T cells from AD patients showed significant differences in DNA methylation in 40 genes compared to HC (p < 0.05). Reduced DNA methylation levels in the upstream region of the interleukin-13 gene (IL13) in -CD4(+)CLA(+) T cells from AD patients correlated with increased IL13 mRNA expression in these cells. Sixteen miRNAs showed differential expression in -CD4(+)CLA(+) T cells from AD patients targeting genes in 202 biological processes (p < 0.05). An integrated network analysis of miRNAs and CpG sites identified two communities of strongly interconnected regulatory elements with strong antagonistic behaviours that recapitulated the differences between AD patients and HC. Functional analysis of the genes linked to these communities revealed their association with key cytokine signaling pathways, MAP kinase signaling and protein ubiquitination. Our findings support that epigenetic mechanisms play a role in the pathogenesis of AD by affecting inflammatory signaling molecules in skin homing -CD4(+)CLA(+) T cells and uncover putative molecules participating in AD pathways.
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3. |
- Alves, A. C., et al.
(author)
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Dysregulation of Complement System and CD4+T Cell Activation Pathways Implicated in Allergic Response
- 2013
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In: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 8:10
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Journal article (peer-reviewed)abstract
- Allergy is a complex disease that is likely to involve dysregulated CD4+ T cell activation. Here we propose a novel methodology to gain insight into how coordinated behaviour emerges between disease-dysregulated pathways in response to pathophysiological stimuli. Using peripheral blood mononuclear cells of allergic rhinitis patients and controls cultured with and without pollen allergens, we integrate CD4+ T cell gene expression from microarray data and genetic markers of allergic sensitisation from GWAS data at the pathway level using enrichment analysis; implicating the complement system in both cellular and systemic response to pollen allergens. We delineate a novel disease network linking T cell activation to the complement system that is significantly enriched for genes exhibiting correlated gene expression and protein-protein interactions, suggesting a tight biological coordination that is dysregulated in the disease state in response to pollen allergen but not to diluent. This novel disease network has high predictive power for the gene and protein expression of the Th2 cytokine profile (IL-4, IL-5, IL-10, IL-13) and of the Th2 master regulator (GATA3), suggesting its involvement in the early stages of CD4+ T cell differentiation. Dissection of the complement system gene expression identifies 7 genes specifically associated with atopic response to pollen, including C1QR1, CFD, CFP, ITGB2, ITGAX and confirms the role of C3AR1 and C5AR1. Two of these genes (ITGB2 and C3AR1) are also implicated in the network linking complement system to T cell activation, which comprises 6 differentially expressed genes. C3AR1 is also significantly associated with allergic sensitisation in GWAS data.
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4. |
- Barrenäs, Fredrik, et al.
(author)
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Disease-Associated MRNA Expression Differences in Genes with Low DNA Methylation
- 2012
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Other publication (other academic/artistic)abstract
- Although the importance of DNA methylation for mRNA expression has been shown for individualgenes in several complex diseases, such a relation has been difficult to show on a genome-wide scale.Here, we used microarrays to examine the relationship between DNA methylation and mRNAexpression in CD4+ T cells from patients with seasonal allergic rhinitis (SAR) and healthy controls.SAR is an optimal disease model because the disease process can be studied by comparing allergenchallengedCD4+ T cells obtained from patients and controls, and mimicked in Th2 polarised T cellsfrom healthy controls. The cells from patients can be analyzed to study relations between methylationand mRNA expression, while the Th2 cells can be used for functional studies. We found that DNAmethylation, but not mRNA expression clearly separated patients from controls. Similar to studies ofother complex diseases, we found no general relation between DNA methylation and mRNAexpression. However, when we took into account the absence or presence of CpG islands in thepromoters of disease associated genes an association was found: low methylation genes without CpGislands had significantly higher expression levels of disease-associated genes. This association wasconfirmed for genes whose expression levels were regulated by a transcription factor of knownrelevance for allergy, IRF4, using combined ChIP-chip and siRNA mediated silencing of IRF4expression. In summary, disease-associated increases of mRNA expression were found in lowmethylation genes without CpG islands in CD4+ T cells from patients with SAR. Further studies arewarranted to examine if a similar association is found in other complex diseases.
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5. |
- Bruhn, Sören, et al.
(author)
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A Generally Applicable Translational Strategy Identifies S100A4 as a Candidate Gene in Allergy
- 2014
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In: Science Translational Medicine. - : American Association for the Advancement of Science (AAAS). - 1946-6234 .- 1946-6242. ; 6:218
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Journal article (peer-reviewed)abstract
- The identification of diagnostic markers and therapeutic candidate genes in common diseases is complicated by the involvement of thousands of genes. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to identify candidate genes. We identified a T helper 2 (T(H)2) cell module by small interfering RNA-mediated knockdown of 25 putative IL13-regulating transcription factors followed by expression profiling. The module contained candidate genes whose diagnostic potential was supported by clinical studies. Functional studies of human TH2 cells as well as mouse models of allergy showed that deletion of one of the genes, S100A4, resulted in decreased signs of allergy including TH2 cell activation, humoral immunity, and infiltration of effector cells. Specifically, dendritic cells required S100A4 for activating T cells. Treatment with an anti-S100A4 antibody resulted in decreased signs of allergy in the mouse model as well as in allergen-challenged T cells from allergic patients. This strategy, which may be generally applicable to complex diseases, identified and validated an important diagnostic and therapeutic candidate gene in allergy.
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6. |
- Bruhn, Sören, 1976-, et al.
(author)
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Combining gene expression microarray- and cluster analysis with sequence-based predictions to identify regulators of IL-13 in allergy
- 2012
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In: Cytokine. - : Elsevier. - 1043-4666 .- 1096-0023. ; 60:3, s. 736-740
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Journal article (peer-reviewed)abstract
- The Th2 cytokine IL-13 plays a key role in allergy, by regulating IgE, airway hyper secretion, eosinophils and mast cells. In this study, we aimed to identify novel transcription factors (TFs) that potentially regulated IL-13. We analyzed Th2 polarized naïve T cells from four different blood donors with gene expression microarrays to find clusters of genes that were correlated or anti-correlated with IL13. These clusters were further filtered, by selecting genes that were functionally related. In these clusters, we identified three transcription factors (TFs) that were predicted to regulate the expression of IL13, namely CEBPB, E2F6 and AHR. siRNA mediated knockdowns of these TFs in naïve polarized T cells showed significant increases of IL13, following knockdown of CEBPB and E2F6, but not AHR. This suggested an inhibitory role of CEBPB and E2F6 in the regulation of IL13 and allergy. This was supported by analysis of E2F6, but not CEBPB, in allergen-challenged CD4+ T cells from six allergic patients and six healthy controls, which showed decreased expression of E2F6 in patients. In summary, our findings indicate an inhibitory role of E2F6 in the regulation of IL-13 and allergy. The analytical approach may be generally applicable to elucidate the complex regulatory patterns in Th2 cell polarization and allergy.
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7. |
- Bruhn, Sören, et al.
(author)
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Increased expression of IRF4 and ETS1 in CD4+ cells from patients with intermittent allergic rhinitis
- 2012
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In: Allergy. European Journal of Allergy and Clinical Immunology. - : John Wiley and Sons. - 0105-4538 .- 1398-9995. ; 67:1, s. 33-40
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Journal article (peer-reviewed)abstract
- Background: The transcription factor (TF) IRF4 is involved in the regulation of Th1, Th2, Th9, and Th17 cells, and animal studies have indicated an important role in allergy. However, IRF4 and its target genes have not been examined in human allergy. Methods: IRF4 and its target genes were examined in allergen-challenged CD4+ cells from patients with IAR, using combined gene expression microarrays and chromatin immunoprecipitation chips (ChIP-chips), computational target prediction, and RNAi knockdowns. Results: IRF4 increased in allergen-challenged CD4+ cells from patients with IAR, and functional studies supported its role in Th2 cell activation. IRF4 ChIP-chip showed that IRF4 regulated a large number of genes relevant to Th cell differentiation. However, neither Th1 nor Th2 cytokines were the direct targets of IRF4. To examine whether IRF4 induced Th2 cytokines via one or more downstream TFs, we combined gene expression microarrays, ChIP-chips, and computational target prediction and found a putative intermediary TF, namely ETS1 in allergen-challenged CD4+ cells from allergic patients. ETS1 increased significantly in allergen-challenged CD4+ cells from patients compared to controls. Gene expression microarrays before and after ETS1 RNAi knockdown showed that ETS1 induced Th2 cytokines as well as disease-related pathways. Conclusions: Increased expression of IRF4 in allergen-challenged CD4+ cells from patients with intermittent allergic rhinitis leads to activation of a complex transcriptional program, including Th2 cytokines.
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8. |
- Chavali, Sreenivas, et al.
(author)
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MicroRNAs act complementarily to regulate disease-related mRNA modules in human diseases
- 2013
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In: Rna-a Publication of the Rna Society. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 19:11, s. 1552-1562
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Journal article (peer-reviewed)abstract
- MicroRNAs (miRNAs) play a key role in regulating mRNA expression, and individual miRNAs have been proposed as diagnostic and therapeutic candidates. The identification of such candidates is complicated by the involvement of multiple miRNAs and mRNAs as well as unknown disease topology of the miRNAs. Here, we investigated if disease-associated miRNAs regulate modules of disease-associated mRNAs, if those miRNAs act complementarily or synergistically, and if single or combinations of miRNAs can be targeted to alter module functions. We first analyzed publicly available miRNA and mRNA expression data for five different diseases. Integrated target prediction and network-based analysis showed that the miRNAs regulated modules of disease-relevant genes. Most of the miRNAs acted complementarily to regulate multiple mRNAs. To functionally test these findings, we repeated the analysis using our own miRNA and mRNA expression data from CD4+ T cells from patients with seasonal allergic rhinitis. This is a good model of complex diseases because of its well-defined phenotype and pathogenesis. Combined computational and functional studies confirmed that miRNAs mainly acted complementarily and that a combination of two complementary miRNAs, miR-223 and miR-139-3p, could be targeted to alter disease-relevant module functions, namely, the release of type 2 helper T-cell (Th2) cytokines. Taken together, our findings indicate that miRNAs act complementarily to regulate modules of disease-related mRNAs and can be targeted to alter disease-relevant functions.
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9. |
- Gasser, Andreas, et al.
(author)
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Second Messenger Function of Nicotinic Acid Adenine Dinucleotide Phosphate Revealed by an Improved Enzymatic Cycling Assay
- 2006
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In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 281:25, s. 16906-16913
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Journal article (peer-reviewed)abstract
- Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 +/- 1.6 nm (0.055 +/- 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+-mobilizing second messenger during T cell activation.
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10. |
- Juul, Louise, et al.
(author)
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Ulva fenestrata protein – comparison of three extraction methods with respect to protein yield and protein quality
- 2021
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In: Algal Research. - : Elsevier BV. - 2211-9264. ; 60
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Journal article (peer-reviewed)abstract
- Seaweed is gaining attention as a possible alternative and sustainable source of proteins. This study investigates three protein extraction methods and their effect on protein yield and quality when applied to Ulva fenestrata . Two of the methods included alkaline extractions (pH-shifts); one version solubilizing the proteins at pH 8.5 and one solubilizing them at pH 8.5 followed by pH 12 (pH 8.5+12). The third method was a mechanical pressing, using a double screw press. All extraction methods were followed by isoelectric precipitation to concentrate the proteins. Extraction at pH 8.5 gave the significantly highest total protein yield after the isoelectric precipitation, followed by extraction at pH 8.5+12 and lastly mechanical extraction gave the lowest yield. Proteins extracted with both alkaline methods had a significantly higher solubility at pH 7 and pH 9, compared to proteins from the mechanical pressing. There were no significant differences between the three methods in total D/L-amino acid ratio. Amino acid cross-links measured as lysinoalanine (LAL) and lanthionine (LAN) where found in significantly higher amounts in alkali-extracted proteins compared to mechanically extracted, however not to a degree that expect to compromise functional or nutritional quality. Further, no significant difference in protein in vitro digestibility was found between extraction methods. In conclusion, results indicated that protein extraction at pH 8.5 can be recommended, especially regarding total protein yield and solubility of the final protein extract.
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11. |
- Kirchberger, Tanja, et al.
(author)
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Cellular effects and metabolic stability of N1-cyclicinosinediphosphoribose and its derivatives
- 2006
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In: British Journal of Pharmacology. - : John Wiley & Sons. - 0007-1188 .- 1476-5381. ; 149:4
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Journal article (peer-reviewed)abstract
- Background and purpose:Recently, a number of mimics of the second messenger cyclic ADP-ribose (cADPR) with replacement of adenosine by inosine were introduced. In addition, various alterations in the molecule ranging from substitutions at C8 of the base up to full replacement of the ribose moieties still retained biological activity. However, nothing is known about the metabolic stability and cellular effects of these novel analogues.Experimental approach:cADPR and the inosine-based analogues were incubated with CD38, ADP-ribosyl cyclase and NAD-glycohydrolase and metabolism was analysed by RP-HPLC. Furthermore, the effect of the analogues on cytokine expression and proliferation was investigated in primary T-lymphocytes and T-lymphoma cells.Key results:Incubation of cADPR with CD38 resulted in degradation to adenosine diphosphoribose. ADP-ribosyl cyclase weakly catabolised cADPR whereas NAD-glycohydrolase showed no such activity. In contrast, N1-cyclic inosine 5′-diphosphoribose (N1-cIDPR) was not hydrolyzed by CD38. Three additional N1-cIDPR analogues showed a similar stability. Proliferation of Jurkat T-lymphoma cells was inhibited by N1-cIDPR, N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (N1-cIDP-DE) and N1-ethoxymethyl-cIDPR (N1-cIDPRE). In contrast, in primary T cells neither proliferation nor cytokine expression was affected by these compounds.Conclusions and Implications:The metabolic stability of N1-cIDPR and its analogues provides an advantage for the development of novel pharmaceutical compounds interfering with cADPR mediated Ca2+ signalling pathways. The differential effects of N1-cIDPR and N1-cIDPRE on proliferation and cytokine expression in primary T cells versus T-lymphoma cells may constitute a starting point for novel anti-tumor drugs.
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12. |
- Nestor, Colm, et al.
(author)
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DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4⁺ T-Cell Population Structure
- 2014
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In: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 10:1
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Journal article (peer-reviewed)abstract
- Altered DNA methylation patterns in CD4+ T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (Npatients = 8, Ncontrols = 8) and gene expression (Npatients = 9, Ncontrols = 10) profiles of CD4+ T-cells from SAR patients and healthy controls using Illumina's HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (Npatients = 12, Ncontrols = 12), but not by gene expression (Npatients = 21, Ncontrols = 21) was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (Npatients = 35) and controls (Ncontrols = 12), which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4+ T cells.
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13. |
- Schmid, Frederike, et al.
(author)
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CD38 : a NAADP degrading enzyme
- 2011
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In: FEBS Letters. - : Elsevier. - 0014-5793 .- 1873-3468. ; 585:22, s. 3544-3548
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Journal article (peer-reviewed)abstract
- The role of the multifunctional enzyme CD38 in formation of the Ca2+-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) was investigated. Gene silencing of CD38 did neither inhibit NAADP synthesis in intact Jurkat T cells nor in thymus or spleen obtained from CD38 knock out mice. In vitro, both NAADP formation by base-exchange and degradation to 2-phospho adenosine diphosphoribose were efficiently decreased. Thus in vivo CD38 appears to be a NAADPdegrading rather than a NAADP forming enzyme, perhaps avoiding desensitizing NAADP levels in intact cells.
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14. |
- Skogberg, Gabriel, et al.
(author)
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Characterization of Human Thymic Exosomes
- 2013
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In: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
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Journal article (peer-reviewed)abstract
- Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.
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15. |
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16. |
- Zhao, Yelin, et al.
(author)
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Combined Multivariate and Pathway Analyses Show That Allergen-Induced Gene Expression Changes in CD4(+) T Cells Are Reversed by Glucocorticoids
- 2012
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In: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 7:6
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Journal article (peer-reviewed)abstract
- Background: Glucocorticoids (GCs) play a key role in the treatment of allergy. However, the genome-wide effects of GCs on gene expression in allergen-challenged CD4(+) T cells have not been described. The aim of this study was to perform a genome-wide analysis to investigate whether allergen-induced gene expression changes in CD4(+) T cells could be reversed by GCs. Methodology/Principal Findings: Gene expression microarray analysis was performed to profile gene expression in diluent( D), allergen- (A), and allergen + hydrocortisone- (T) challenged CD4(+) T cells from patients with seasonal allergic rhinitis. Principal component analysis (PCA) showed good separation of the three groups. To identify the correlation between changes in gene expression in allergen-challenged CD4(+) T cells before and after GC treatment, we performed orthogonal partial least squares discriminant analysis (OPLS-DA) followed by Pearson correlation analysis. This revealed that allergen-induced genes were widely reversed by GC treatment (r = -0.77, Pless than0.0001). We extracted 547 genes reversed by GC treatment from OPLS-DA models based on their high contribution to the discrimination and found that those genes belonged to several different inflammatory pathways including TNFR2 Signalling, Interferon Signalling, Glucocorticoid Receptor Signalling and T Helper Cell Differentiation. The results were supported by gene expression microarray analyses of two independent materials. Conclusions/Significance: Allergen-induced gene expression changes in CD4(+) T cells were reversed by treatment with glucocorticoids. The top allergen-induced genes that reversed by GC treatment belonged to several inflammatory pathways and genes of known or potential relevance for allergy.
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