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- Assadi, Ghazaleh, et al.
(författare)
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Functional Analyses of the Crohn's Disease Risk Gene LACC1
- 2016
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Ingår i: PLOS ONE. - San Francisco, USA : Public Library of Science. - 1932-6203. ; 11:12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression.Methods: We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function.Results: FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems.Conclusion: FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.
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| 2. |
- Assadi, Ghazaleh
(författare)
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Identification and functional characterization of gastrointestinal disease genes
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The inflammatory bowel diseases (IBD) Crohn’s disease (CD) and ulcerative colitis (UC) are conditions characterized by chronic and relapsing inflammation of the gastrointestinal tract. IBD affects around 2.5 million people of European ancestry and the incidence is increasing worldwide (currently, 1% of the population suffers from IBD in Sweden). IBD patients require life-long medication, hospitalizations, recurring sick-leaves, surgical intervention and may acquire serious complications, such as colorectal cancer. There is as yet no definitive cure, and new treatment modalities are effective, but far from being optimal. A much greater understanding of IBD pathophysiology is therefore needed, in order to delineate improved therapeutic strategies, and to predict disease course and response to treatment. Although the etiology of IBDs is unknown, current consensus is that they occur in genetically predisposed individuals, primarily due to a dysregulated immune response to gut microbiota. IBD genetic research has highlighted the importance of innate immune interactions with the gut microbiota, the regulation of immune functions, the maintenance of gut epithelial barrier, and autophagy in order to maintain gut homeostasis. However, these discoveries have not yet led to the identification of novel pathogenetic pathways that may be amenable to exploitation for renewed therapeutic intervention. Eventually, this may come from the study of risk genes of unknown function. The overall aim of this thesis is the functional characterization of novel gastrointestinal disease genes, and in particular the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene, in order to elucidate the mechanism(s) by which its genetic variation(s) contributes to IBD, and ultimately provide novel opportunities for therapeutic exploitation. In paper I, we tested a series of LACC1 common variants for association with disease in two Swedish cohorts of IBD and non-systemic juvenile idiopathic arthritis (nsJIA). Significant findings were detected for multiple LACC1 markers in the studied cohorts, thereby expanding previous results for CD to both UC and nsJIA. In paper II, we identified FAMIN (the LACC1 encoded protein) as a core metabolic regulator of macrophage function. By forming a complex with fatty acid synthase at peroxisomes, FAMIN promotes carbon flux through de novo lipogenesis (DNL) and drives high levels of fatty-acid oxidation (FAO) alongside high levels of glycolysis. As a consequence, FAMIN deficiency causes defects in DNL, FAO, reactive oxygen species production, inflammasome activation, endotoxin-response and bacterial clearance, thereby providing a plausible explanation to the observed disease phenotype in patients with the variants Ile254Val and Cys284Arg. In paper III, we found higher LACC1 expression in human immune-tissues and cells such as spleen, lymph nodes, monocytes/macrophages, DCs and neutrophils. In addition, FAMIN expression was shown to be regulated by peroxisome proliferator-activated receptor ligands. In paper IV, we identified a number of potential candidate biomarkers that may be followed up in validation experiments in independent IBD case-control cohorts. Of particular interest, FAMIN serum levels were found to differ between IBD patients and healthy controls, with lowest expression in CD patients. This parallels mouse and human data suggesting reduced FAMIN activity predisposes to disease. In summary, this thesis characterizes LACC1/FAMIN as a new major player in IBD pathophysiology, identifying novel biological pathways that may be amenable to modulation for therapeutic purposes, while at the same time providing preliminary data of potential exploitation for biomarkers delineation.
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| 3. |
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| 4. |
- Drobin, Kimi, et al.
(författare)
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Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci
- 2019
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Ingår i: Inflammatory Bowel Diseases. - : Oxford University Press. - 1078-0998 .- 1536-4844. ; 25:2, s. 306-316
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Tidskriftsartikel (refereegranskat)abstract
- Background: Few studies have investigated the blood proteome of inflammatory bowel disease (IBD). We characterized the serum abundance of proteins encoded at 163 known IBD risk loci and tested these proteins for their biomarker discovery potential.Methods: Based on the Human Protein Atlas (HPA) antibody availability, 218 proteins from genes mapping at 163 IBD risk loci were selected. Targeted serum protein profiles from 49 Crohn's disease (CD) patients, 51 ulcerative colitis (UC) patients, and 50 sex- and age-matched healthy individuals were obtained using multiplexed antibody suspension bead array assays. Differences in relative serum abundance levels between disease groups and controls were examined. Replication was attempted for CD-UC comparisons (including disease subtypes) by including 64 additional patients (33 CD and 31 UC). Antibodies targeting a potentially novel risk protein were validated by paired antibodies, Western blot, immuno-capture mass spectrometry, and epitope mapping.Results: By univariate analysis, 13 proteins mostly related to neutrophil, T-cell, and B-cell activation and function were differentially expressed in IBD patients vs healthy controls, 3 in CD patients vs healthy controls and 2 in UC patients vs healthy controls (q < 0.01). Multivariate analyses further differentiated disease groups from healthy controls and CD subtypes from UC (P < 0.05). Extended characterization of an antibody targeting a novel, discriminative serum marker, the laccase (multicopper oxidoreductase) domain containing 1 (LACC1) protein, provided evidence for antibody on-target specificity.Conclusions: Using affinity proteomics, we identified a set of IBD-associated serum proteins encoded at IBD risk loci. These candidate proteins hold the potential to be exploited as diagnostic biomarkers of IBD.
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| 7. |
- Westerlind, Helga, et al.
(författare)
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Dense genotyping of immune-related loci identifies HLA variants associated with increased risk of collagenous colitis.
- 2017
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Ingår i: Gut. - : BMJ Publishing Group Ltd. - 0017-5749 .- 1468-3288. ; 66:3, s. 421-428
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Collagenous colitis (CC) is a major cause of chronic non-bloody diarrhoea, particularly in the elderly female population. The aetiology of CC is unknown, and still poor is the understanding of its pathogenesis. This possibly involves dysregulated inflammation and immune-mediated reactions in genetically predisposed individuals, but the contribution of genetic factors to CC is underinvestigated. We systematically tested immune-related genes known to impact the risk of several autoimmune diseases for their potential CC-predisposing role.DESIGN: Three independent cohorts of histologically confirmed CC cases (N=314) and controls (N=4299) from Sweden and Germany were included in a 2-step association analysis. Immunochip and targeted single nucleotide polymorphism (SNP) genotype data were produced, respectively, for discovery and replication purposes. Classical human leucocyte antigen (HLA) variants at 2-digit and 4-digit resolution were obtained via imputation from single marker genotypes. SNPs and HLA variants passing quality control filters were tested for association with CC with logistic regression adjusting for age, sex and country of origin.RESULTS: Forty-two markers gave rise to genome-wide significant association signals, all contained within the HLA region on chromosome 6 (best p=4.2×10(-10) for SNP rs4143332). Among the HLA variants, most pronounced risk effects were observed for 8.1 haplotype alleles including DQ2.5, which was targeted and confirmed in the replication data set (p=2.3×10(-11); OR=2.06; 95% CI (1.67 to 2.55) in the combined analysis).CONCLUSIONS: HLA genotype associates with CC, thus implicating HLA-related immune mechanisms in its pathogenesis.
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| 8. |
- Zucchelli, Marco, et al.
(författare)
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PepT1 oligopeptide transporter (SLC15A1) gene polymorphism in inflammatory bowel disease
- 2009
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Ingår i: Inflammatory Bowel Diseases. - : Oxford University Press (OUP). - 1078-0998 .- 1536-4844. ; 15:10, s. 1562-1569
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Human polymorphisms affecting gut epithelial barrier and interactions with bacteria predispose to the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC). The intestinal transporter PepT1, encoded by the SLC15A1 gene, mediates intracellular uptake of bacterial products that can induce inflammation and NF-kappaB activation upon binding to NOD2, a protein often mutated in CD. Hence, we tested SLC15A1 polymorphisms for association with IBD.METHODS: Twelve SLC15A1 single nucleotide polymorphisms (SNPs) were genotyped in 1783 individuals from 2 cohorts of Swedish and Finnish IBD patients and controls. An in vitro system was set up to evaluate the potential impact of SLC15A1 polymorphism on PepT1 transporter function by quantification of NOD2-mediated activation of NF-kappaB.RESULTS: The common allele (C) of a coding polymorphism (rs2297322, Ser117Asn) was associated with CD susceptibility both in Sweden and in Finland, but with genetic effects in opposite directions (risk and protection, respectively). The best evidence of association was found in both populations when the analysis was performed on individuals not carrying NOD2 common risk alleles (Sweden allelic P = 0.0007, OR 1.97, 95% confidence interval [CI] 1.34-2.92; Finland genotype P = 0.0013, OR 0.63, 95% CI 0.44-0.90). The PepT1 variant encoded by the C allele (PepT1-Ser117) was associated with reduced signaling downstream of NOD2 (P < 0.0001 compared to Pept1-Asn117).CONCLUSIONS: A functional polymorphism in the SLC15A1 gene might be of relevance to inflammation and antibacterial responses in IBD. Whether this polymorphism truly contributes to disease susceptibility needs to be further addressed, and should stimulate additional studies in other populations.
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