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1.
J Immunol Methods ; 494: 113058, 2021 07.
Article in English | MEDLINE | ID: mdl-33891922

ABSTRACT

Inflammatory bowel disease (IBD) is thought to be caused by an aberrant host response to the commensal enteric flora in genetically susceptible individuals. Dendritic cells (DCs) play a key role in the regulation of this response as they sample gut commensals. In healthy individuals DCs actively contribute to tolerance upon recognition of these resident bacteria, whereas in individuals with IBD, DCs will initiate an inflammatory response. To mimic the disease response in vitro, human monocyte-derived DCs were matured with E. coli causing the cells to produce high levels of the pro-inflammatory cytokine IL-12/IL-23p40 (p40) and low levels of the anti-inflammatory cytokine IL-10. A siRNA-based screening assay was developed and screened to identify potential therapeutic targets that shift this balance towards an immunosuppressive state with lower levels of p40 and higher levels of IL-10. The screening assay was optimized and quality controlled using non-targeting controls and positive control siRNAs targeting IL12B and TLR4 transcripts. In the primary screen, smartpool siRNAs were screened for reduction in p40 expression, induction of IL-10 levels, or increase in IL-10:p40 ratios without affecting cell viability. All potential targets were taken forward into a confirmation screen in a different DC donor in which four individual siRNAs per target were screened. At least two siRNAs per target should have an effect to be considered a valid target. This screen resulted in a concise list of ten genes, of which their role in DC maturation is currently being investigated.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Dendritic Cells/immunology , Inflammatory Bowel Diseases/immunology , Intestinal Mucosa/immunology , RNA, Small Interfering/genetics , Antigen Presentation , Antigens, Bacterial/immunology , Cell Differentiation , Cells, Cultured , Dendritic Cells/drug effects , Humans , Immune Tolerance , Inflammatory Bowel Diseases/drug therapy , Interleukin-10/metabolism , Interleukin-12/metabolism , Mass Screening , Symbiosis
2.
Eur J Hum Genet ; 17(3): 319-28, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18830229

ABSTRACT

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34 or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). Here we investigate the effects of putative TSC1 missense mutations identified in individuals with signs and/or symptoms of TSC on TSC1-TSC2 complex formation and mTOR signalling. We show that specific amino-acid substitutions close to the N-terminal of TSC1 reduce steady-state levels of TSC1, resulting in the activation of mTOR signalling and leading to the symptoms of TSC.


Subject(s)
Mutation, Missense , Tuberous Sclerosis/genetics , Tumor Suppressor Proteins/genetics , Amino Acid Substitution , Humans , Pedigree , Protein Kinases/genetics , Protein Kinases/metabolism , Signal Transduction , TOR Serine-Threonine Kinases , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/metabolism
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