subcellular-fractionation-from-fresh-frozen-gastrointestinal

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the ...

University of the Basque Country (UPV-EHU), Biocruces Health Research Institute

It is known that the NFκB route is constitutively upregulated in celiac disease (CD), an immune-mediated disorder of the gut caused by intolerance to ingested gluten. Our aim was to scrutinize the expression patterns of several of the most biologically relevant components of the NFκB route in intestinal biopsies from active and treated patients and after in vitro gliadin challenge, and to assess normalization of the expression using an inhibitor of the MALT1 paracaspase. The expression of 93 NFκB genes was measured by RT-PCR in a set of uncultured active and treated CD and control biopsies, and in cultured biopsy series challenged with gliadin, the NFκB modulator, both compounds and none. Methylation of eight genes involved in NFκB signaling was analyzed by conventional pyrosequencing. Groups were compared and Pearson's correlation matrixes were constructed to check for coexpression and co-methylation. Our results confirm the upregulation of the NFκB pathway and show that constitutively altered genes usually belong to the core of the pathway and have central roles, whereas genes overexpressed only in active CD are more peripheral. Additionally, this is the first work to detect methylation level changes in celiac intestinal mucosa. Coexpression is very common in controls, whereas gliadin challenge and especially chronic inflammation present in untreated CD result in the disruption of the regulatory equilibrium. In contrast, co-methylation occurs more often in active CD. Importantly, NFκB modulation partially restores coregulation, opening the door to future therapeutic possibilities and targets. 

Coregulation and modulation of NFκB-related genes in celiac disease: uncovered aspects of gut mucosal inflammation.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of inflammation. To further understand the interaction between inflammatory signaling pathways and lncRNAs, we characterized the function of cardiac and apoptosis-related lncRNA (Carlr), an lncRNA expressed in both mouse and human cells of diverse tissues. Carlr expression is increased following NF-κB signaling in macrophages, with concomitant translocation to, and enrichment of, the transcript in the cytoplasm. Knockdown of Carlr results in impaired expression of NF-κB pathway genes and influences the interaction between macrophages and intestinal cells in an inflammatory environment. In human celiac disease patient samples, increased levels of the Carlr transcript were detected in the cytoplasm, alongside elevated expression of NF-κB pathway genes. These findings suggest that increased Carlr expression and/or cytoplasmic localization is required for efficient NF-κB signaling and is associated with the inflamed tissue state observed in human celiac disease.

Cytoplasmic Form of Carlr lncRNA Facilitates Inflammatory Gene Expression upon NF-κB Activation.

To identify additional celiac disease associated loci in the Major Histocompatibility Complex independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level.

Ainara Castellanos-Rubio

Department of Genetics,
Physical Anthropology and Animal Physiology,
Department of Genetics, Physical Anthropology and Animal Physiology

University of the Basque Country (UPV-EHU), Biocruces Health Research Institute

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

Ainara Castellanos-Rubio

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Genetics,Physical Anthropology and Animal Physiology,Department of Genetics, Physical Anthropology and Animal Physiology

University of the Basque Country (UPV-EHU), Biocruces Health Research Institute

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

Department of Genetics,
Physical Anthropology and Animal Physiology,
Department of Genetics, Physical Anthropology and Animal Physiology