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- Fredin, Maria Fritsch, 1970, et al.
(author)
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The application and relevance of ex vivo culture systems for assessment of IBD treatment in murine models of colitis
- 2008
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In: Pharmacological Research. - : Elsevier BV. - 1043-6618 .- 1096-1186. ; 58:3-4, s. 222-231
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Journal article (peer-reviewed)abstract
- The aim of this study was to investigate the relevance of mouse ex vivo cultures as a first screening model for new therapeutic agents of Inflammatory Bowel Disease (IBD). Two murine models (dextran sodium sulphate (DSS)-induced colitis and Gαi2-deficient mice) and two anti-inflammatory agents (methyl-prednisolone and the proteasome inhibitor MG132) were evaluated. The in vivo effects of methyl-prednisolone were assessed in both models. Ex vivo colonic tissue from both mouse models were cultured in the presence or absence of the drugs and TaqMan Low-Density arrays were used to assess the regulation of inflammatory genes before and after drug treatment. Colitis induced a similar inflammatory gene profile in both mouse models in in vivo studies and in ex vivo cultures. The differences encountered reflected the different phases of colitis in the models, e.g. innate cytokine/chemokine profile in the DSS model and T cell related markers in Gαi2-deficient mice. After steroid treatment, a similar pattern of genes was suppressed in the two mouse models. We confirmed the suppression of inflammatory gene expression for IL-1β, IL-6 and iNOS in ex vivo and in vivo colons from both mouse models by quantitative RT-PCR. Importantly, the inflammatory responses in the murine ex vivo culture system reflected the in vivo response in the inflamed colonic tissue as assessed by changes in inflammatory gene expression, suggesting that the murine culture system can be used for validation of future IBD therapies.
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| 3. |
- Götlind, Yu-Yuan Chiu, 1964
(author)
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Interplay between Pathogenic and Immune Regulatory Mechanisms in G-alpha-i2 deficient colitis
- 2011
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Doctoral thesis (other academic/artistic)abstract
- The two major forms of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC) are gastrointestinal disorders characterized by chronic and relapsing inflammation. Mice deficient in the inhibitory G protein subunit Gαi2 spontaneously develop chronic colitis and have been used as a model for IBD, with particular similarities to UC. They have been used in this thesis to study the changes in immune pathology during disease progression; to investigate the role of regulatory T cells (Tregs) in chronic intestinal inflammation; and as a model for testing anti-inflammatory agents. During the progression of the colitis, continuing increases in colon weight/cm and spleen weight, and a gradual decrease in thymus weight were observed. The proteins levels of proinflammatory cytokines/chemokines IL-1β, IL-6, IL-12p40, IL-17, TNF-α, CCL2 and CXCL1 increased during colitis progression and were all significantly increased in mice with moderate and severe colitis in colonic, but not small intestinal tissues. In colon, IFN-γ mRNA and IL-27 mRNA were gradually elevated during the course of the colitis, whereas IL-21 mRNA expression was significantly enhanced in mice with severe colitis. Thus, the lack of Gαi2 elicits an expansion of gut Th17 responses, possibly as a result of a Gαi2–/–-driven epithelial barrier defect; this drives the production of neutrophil-attractant chemokines, resulting in the influx of neutrophils; in turn promoting an adaptive Th1 response. Numbers of lamina propria CD4+FoxP3+ cells were significantly increased in colitis and were dispersed in the tissue, in contrast to non-inflamed colon in which they concentrated within organized lymphoid structures. In vitro data showed that both Gαi2–/– and wild-type (WT) splenic Tregs were able to suppress wild type (WT) effector T cells (Teffs) , but that pathogenetic Gαi2–/– Teffs could not be suppressed by either type of Tregs. Adoptive transfer experiments in the colitis model showed that neither type of Tregs could prevent disease induced by co-transfer of Gαi2–/– Teffs. It is possible that Treg regulatory function is suppressed in the inflamed colonic milieu, and/or that they are unable to overcome the heightened activity of Gαi2–/– Teffs. Acute colitis induced by dextran sodium sulphate (DSS) and spontaneous Gαi2−/− chronic colitis were used to assess the efficacy of ex vivo anti-inflammatory treatment. The gene profiles reflected the different mechanisms underpinning these two types of colitis. Thus, genes related to pro-inflammatory innate cytokines, chemokines and chemokine receptors were up-regulated in DSS-induced colitis, whereas genes related to T cell markers were preferentially elevated in Gαi2−/− colitic tissues. In general, the same panel of genes displayed increased transcription in the in vivo and ex vivo cultured tissues in DSS model. The well-characterised corticosteroid methyl-prednisolone and the proteasome inhibitor MG132, were used to compare the efficacy between in vivo treatment and ex vivo cultures of colon obtained from DSS-induced and Gαi2–/– colitic mice. After steroid treatment, IL-1β, IL-6 and iNOS were suppressed in both models, both in in vivo and ex vivo. The anti-inflammatory function of methyl-prednisolone was mostly at the innate level, as shown in DSS-colitis, but MG132 acted effectively on the chronically activated adaptive immune response in the Gαi2−/− colitis model. Thus the changes in inflammatory gene expression in the murine ex vivo culture system reflected the in vivo response in the inflamed colonic tissue, suggesting that the murine culture system can be used for validation of future IBD therapies. We conclude that the genetic Gαi2 deficiency leads to amplification of gut Th17 responses, possibly mediated by a gut barrier defect, and this leads to a mixed Th17/Th1 effector phenotype in late-stage colitis. The hyper-reactive Gαi2−/− effector T cells further amplify the inflammation and increased numbers of memory effector T cells are generated as disease progresses. The lack of appropriate regulation by Tregs further exacerbes the Gαi2−/− colitis. Key words: IBD, Gαi2−/− colitis, Tregs, Teffs, Th17/Th1, pathogenesis/immunoregulation
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| 4. |
- Götlind, Yu-Yuan Chiu, 1964, et al.
(author)
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Interplay between Th1 and Th17 effector T cell pathways in the pathogenesis of spontaneous colitis and colon cancer in the Gai2-deficient mouse
- 2013
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In: International Immunology. - Oxford, United Kingdom : Oxford University Press. - 0953-8178 .- 1460-2377. ; 25:1, s. 35-44
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Journal article (peer-reviewed)abstract
- Gαi2-deficient mice spontaneously develop colitis. Using xMAP technology and RT-PCR, we investigated cytokine/chemokine profiles during histologically defined phases of disease: (i) no/mild, (ii) moderate, (iii) severe colitis without dysplasia/cancer and (iv) severe colitis with dysplasia/cancer, compared with age-matched wild-type (WT) littermates. Colonic dysplasia was observed in 4/11 mice and cancer in 1/11 mice with severe colitis. The histology correlated with progressive increases in colon weight/cm and spleen weight, and decreased thymus weight, all more advanced in mice with dysplasia/cancer. IL-1β, IL-6, IL-12p40, IL-17, TNF-α, CCL2 and CXCL1 protein levels in colons, but not small intestines increased with colitis progression and were significantly increased in mice with moderate and severe colitis compared with WT mice, irrespective of the absence/presence of dysplasia/cancer. CCL5 did not change during colitis progression. Colonic IL-17 transcription increased 40- to 70-fold in all stages of colitis, whereas IFN-γ mRNA was gradually up-regulated 12- to 55-fold with colitis progression, and further to 62-fold in mice with dysplasia/cancer. IL-27 mRNA increased 4- to 15-fold during the course of colitis, and colonic IL-21 transcription increased 3-fold in mice with severe colitis, both irrespective of the absence/presence of dysplasia/cancer. FoxP3 transcription was significantly enhanced (3.5-fold) in mice with moderate and severe colitis, but not in mice with dysplasia/cancer, compared with WT mice. Constrained correspondence analysis demonstrated an association between increased protein levels of TNF-α, CCL2, IL-1β, IL-6 and CXCL1 and dysplasia/cancer. In conclusion, colonic responses are dominated by a mixed T(h)1/T(h)17 phenotype, with increasing T(h)1 cytokine transcription with progression of colitis in Gαi2(-/-) mice.
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