m6A Methylated Long Noncoding RNA LOC339803 Regulates Intestinal Inflammatory Response.

Cytokine mediated sustained inflammation increases the risk to develop different complex chronic inflammatory diseases, but the implicated mechanisms remain unclear. Increasing evidence shows that long noncoding RNAs (lncRNAs) play key roles in the pathogenesis of inflammatory disorders, while inflammation associated variants are described to affect their function or essential RNA modifications as N6-methyladenosine (m6A) methylation, increasing predisposition to inflammatory diseases. Here, the functional implication of the intestinal inflammation associated lncRNA LOC339803 in the production of cytokines by intestinal epithelial cells is described. Allele-specific m6A methylation is found to affect YTHDC1 mediated protein binding affinity. LOC339803-YTHDC1 interaction dictates chromatin localization of LOC339803 ultimately inducing the expression of NFκB mediated proinflammatory cytokines and contributing to the development of intestinal inflammation. These findings are confirmed using human intestinal biopsy samples from different intestinal inflammatory conditions and controls. Additionally, it is demonstrated that LOC339803 targeting can be a useful strategy for the amelioration of intestinal inflammation in vitro and ex vivo. Overall, the results support the importance of the methylated LOC339803 lncRNA as a mediator of intestinal inflammation, explaining genetic susceptibility and presenting this lncRNA as a potential novel therapeutic target for the treatment of inflammatory intestinal disorders.

A fast, cheap, and easy protocol for celiac disease HLA haplotype typing using buccal swabs.

Celiac disease (CeD) is a complex autoimmune disorder characterized by intestinal immune-derived injury that develops in response to dietary gluten consumption. Human Leucocyte Antigen (HLA) complex haplotype typing is one of the main tests for CeD diagnosis, together with anti-endomysium and anti-transglutaminase autoantibody detection in blood and inflammation observation in the intestine, being the former mainly used for the initial discarding of the pathogenesis. Among the many types of HLA proteins, HLA-DQ2.5 and HLA-DQ8 are considered essential for CeD development. These receptors are only expressed when specific alleles are present, which can be accurately predicted by the presence of the tagging SNPs rs2187668 and rs7454108, respectively. Taking advantage of this premise, we present here an easy workflow to assess HLA genotyping in saliva by a quick and cheap isopropanol-ethanol precipitation-based DNA extraction method followed by the genotyping of two tagging SNPs for the most frequent CeD risk-associated HLA haplotypes. All the actual diagnostic methods for CeD are performed after acquisition of intestine biopsies or blood samples by invasive techniques. Therefore, the development of non-invasive methods would be of a great improvement and advantage for patients, especially children, as an alternative method for initial CeD screening.

Gluten-induced RNA methylation changes regulate intestinal inflammation via allele-specific XPO1 translation in epithelial cells.

OBJECTIVES: Coeliac disease (CD) is a complex autoimmune disorder that develops in genetically susceptible individuals. Dietary gluten triggers an immune response for which the only available treatment so far is a strict, lifelong gluten free diet. Human leucocyte antigen (HLA) genes and several non-HLA regions have been associated with the genetic susceptibility to CD, but their role in the pathogenesis of the disease is still essentially unknown, making it complicated to develop much needed non-dietary treatments. Here, we describe the functional involvement of a CD-associated single-nucleotide polymorphism (SNP) located in the 5'UTR of XPO1 in the inflammatory environment characteristic of the coeliac intestinal epithelium. DESIGN: The function of the CD-associated SNP was investigated using an intestinal cell line heterozygous for the SNP, N6-methyladenosine (m6A)-related knock-out and HLA-DQ2 mice, and human samples from patients with CD. RESULTS: Individuals harbouring the risk allele had higher m6A methylation in the 5'UTR of XPO1 RNA, rendering greater XPO1 protein amounts that led to downstream nuclear factor kappa B (NFkB) activity and subsequent inflammation. Furthermore, gluten exposure increased overall m6A methylation in humans as well as in in vitro and in vivo models. CONCLUSION: We identify a novel m6A-XPO1-NFkB pathway that is activated in CD patients. The findings will prompt the development of new therapeutic approaches directed at m6A proteins and XPO1, a target under evaluation for the treatment of intestinal disorders.

Shaping Up Zn-Doped Magnetite Nanoparticles from Mono- and Bimetallic Oleates: The Impact of Zn Content, Fe Vacancies, and Morphology on Magnetic Hyperthermia Performance.

The currently existing magnetic hyperthermia treatments usually need to employ very large doses of magnetic nanoparticles (MNPs) and/or excessively high excitation conditions (H × f > 1010 A/m s) to reach the therapeutic temperature range that triggers cancer cell death. To make this anticancer therapy truly minimally invasive, it is crucial the development of improved chemical routes that give rise to monodisperse MNPs with high saturation magnetization and negligible dipolar interactions. Herein, we present an innovative chemical route to synthesize Zn-doped magnetite NPs based on the thermolysis of two kinds of organometallic precursors: (i) a mixture of two monometallic oleates (FeOl + ZnOl), and (ii) a bimetallic iron-zinc oleate (Fe3-y Zn y Ol). These approaches have allowed tailoring the size (10-50 nm), morphology (spherical, cubic, and cuboctahedral), and zinc content (Zn x Fe3-x O4, 0.05 < x < 0.25) of MNPs with high saturation magnetization (≥90 Am2/kg at RT). The oxidation state and the local symmetry of Zn2+ and Fe2+/3+ cations have been investigated by means of X-ray absorption near-edge structure (XANES) spectroscopy, while the Fe center distribution and vacancies within the ferrite lattice have been examined in detail through Mössbauer spectroscopy, which has led to an accurate determination of the stoichiometry in each sample. To achieve good biocompatibility and colloidal stability in physiological conditions, the Zn x Fe3-x O4 NPs have been coated with high-molecular-weight poly(ethylene glycol) (PEG). The magnetothermal efficiency of Zn x Fe3-x O4@PEG samples has been systematically analyzed in terms of composition, size, and morphology, making use of the latest-generation AC magnetometer that is able to reach 90 mT. The heating capacity of Zn0.06Fe2.9 4O4 cuboctahedrons of 25 nm reaches a maximum value of 3652 W/g (at 40 kA/m and 605 kHz), but most importantly, they reach a highly satisfactory value (600 W/g) under strict safety excitation conditions (at 36 kA/m and 125 kHz). Additionally, the excellent heating power of the system is kept identical both immobilized in agar and in the cellular environment, proving the great potential and reliability of this platform for magnetic hyperthermia therapies.

Relative Quantification of Residue-Specific m6A RNA Methylation Using m6A-RT-QPCR.

Technological advances in high-throughput sequencing in combination with antibody enrichment and/or induced nucleotide-specific chemical modifications have accelerated the mapping of epitranscriptomic modifications. However, site-specific detection and quantification of m6A are still technically challenging. Here, we describe a simple RT-QPCR-based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues.

Highly Reproducible Hyperthermia Response in Water, Agar, and Cellular Environment by Discretely PEGylated Magnetite Nanoparticles.

Local heat generation from magnetic nanoparticles (MNPs) exposed to alternating magnetic fields can revolutionize cancer treatment. However, the application of MNPs as anticancer agents is limited by serious drawbacks. Foremost among these are the fast uptake and biodegradation of MNPs by cells and the unpredictable magnetic behavior of the MNPs when they accumulate within or around cells and tissues. In fact, several studies have reported that the heating power of MNPs is severely reduced in the cellular environment, probably due to a combination of increased viscosity and strong NP agglomeration. Herein, we present an optimized protocol to coat magnetite (Fe3O4) NPs larger than 20 nm (FM-NPs) with high molecular weight PEG molecules that avoid collective coatings, prevent the formation of large clusters of NPs and keep constant their high heating performance in environments with very different ionic strengths and viscosities (distilled water, physiological solutions, agar and cell culture media). The great reproducibility and reliability of the heating capacity of this FM-NP@PEG system in such different environments has been confirmed by AC magnetometry and by more conventional calorimetric measurements. The explanation of this behavior has been shown to lie in preserving as much as possible the magnetic single domain-type behavior of nearly isolated NPs. In vitro endocytosis experiments in a colon cancer-derived cell line indicate that FM-NP@PEG formulations with PEGs of higher molecular weight (20 kDa) are more resistant to endocytosis than formulations with smaller PEGs (5 kDa), showing quite large uptake mean-life (τ > 5 h) in comparison with other NP systems. The in vitro magnetic hyperthermia was performed at 21 mT and 650 kHz during 1 h in a pre-endocytosis stage and complete cell death was achieved 48 h posthyperthermia. These optimal FM-NP@PEG formulations with high resistance to endocytosis and predictable magnetic response will aid the progress and accuracy of the emerging era of theranostics.

A novel RT-QPCR-based assay for the relative quantification of residue specific m6A RNA methylation.

N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3'UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic ß-cells exposed to inflammatory stimuli.

The methylome of the celiac intestinal epithelium harbours genotype-independent alterations in the HLA region.

The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation.

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

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.

A long noncoding RNA associated with susceptibility to celiac disease.

Recent studies have implicated long noncoding RNAs (lncRNAs) as regulators of many important biological processes. Here we report on the identification and characterization of a lncRNA, lnc13, that harbors a celiac disease-associated haplotype block and represses expression of certain inflammatory genes under homeostatic conditions. Lnc13 regulates gene expression by binding to hnRNPD, a member of a family of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). Upon stimulation, lnc13 levels are reduced, thereby allowing increased expression of the repressed genes. Lnc13 levels are significantly decreased in small intestinal biopsy samples from patients with celiac disease, which suggests that down-regulation of lnc13 may contribute to the inflammation seen in this disease. Furthermore, the lnc13 disease-associated variant binds hnRNPD less efficiently than its wild-type counterpart, thus helping to explain how these single-nucleotide polymorphisms contribute to celiac disease.

Detection of atomic scale changes in the free volume void size of three-dimensional colorectal cancer cell culture using positron annihilation lifetime spectroscopy.

Positron annihilation lifetime spectroscopy (PALS) provides a direct measurement of the free volume void sizes in polymers and biological systems. This free volume is critical in explaining and understanding physical and mechanical properties of polymers. Moreover, PALS has been recently proposed as a potential tool in detecting cancer at early stages, probing the differences in the subnanometer scale free volume voids between cancerous/healthy skin samples of the same patient. Despite several investigations on free volume in complex cancerous tissues, no positron annihilation studies of living cancer cell cultures have been reported. We demonstrate that PALS can be applied to the study in human living 3D cell cultures. The technique is also capable to detect atomic scale changes in the size of the free volume voids due to the biological responses to TGF-ß. PALS may be developed to characterize the effect of different culture conditions in the free volume voids of cells grown in vitro.

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

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.

Angiogenesis-related gene expression analysis in celiac disease.

Celiac disease (CD) involves disturbance of the small-bowel mucosal vascular network, and transglutaminase autoantibodies (TGA) have been related to angiogenesis disturbance, a complex phenomenon probably also influenced by common genetic variants in angiogenesis-related genes. A set of genes with "angiogenesis" GO term identified in a previous expression microarray experiment (SCG2, STAB1, TGFA, ANG, ERBB2, GNA13, PML, CASP8, ECGF1, JAG1, HIF1A, TNFSF13 and TGM2) was selected for genetic and functional studies. SNPs that showed a trend for association with CD in the first GWAS were genotyped in 555 patients and 541 controls. Gene expression of all genes was quantified in 15 pairs of intestinal biopsies (diagnosis vs. GFD) and in three-dimensional HUVEC and T84 cell cultures incubated with TGA-positive and negative serum. A regulatory SNP in TNFSF13 (rs11552708) is associated with CD (p = 0.01, OR = 0.7). Expression changes in biopsies pointed to TGM2 and PML as up-regulated antiangiogenic genes and to GNA13, TGFA, ERBB2 and SCG2 as down-regulated proangiogenic factors in CD. TGA seem to enhance TGM2 expression in both cell models, but PML expression was induced only in T84 enterocytes while GNA13 and ERBB2 were repressed in HUVEC endothelial cells, with several genes showing discordant effects in each model, highlighting the complexity of gene interactions in the pathogenesis of CD. Finally, cell culture models are useful tools to help dissect complex responses observed in human explants.

Upregulation of KIR3DL1 gene expression in intestinal mucosa in active celiac disease.

Killer cell immunoglobulin-like receptors (KIRs) modulate natural killer (NK) and T-cell function by human leukocyte antigen class I interaction and have been implicated in celiac disease (CD). Qualitative expression of 16 KIR genes was determined in biopsies from 22 CD patients at diagnosis and after >2 years on a gluten-free diet (GFD). Quantitative expression analysis of KIR2DL4, KIR3DL1, KIR3DL3, and KLRC2 (a marker of an NK-reprogrammed T-cell subpopulation augmented in CD) was performed in 35 additional CD biopsy pairs and 14 non-CD control biopsies. No specific KIR expression profile was observed in CD. KIR3DL1 was more frequently expressed in active CD compared with GFD (p = 0.0312) and controls (p = 0.0008), with slightly increased levels in active disease. KLRC2 was overexpressed in active (p = 0.0037) and GFD (p = 0.0469) patients compared with non-CD controls and coexpressed with KIR3DL1. Results suggest the participation of KIR3DL1 overexpression in the overall immune activation seen in CD mucosa, which could be partly explained by the NK-like T-cell subpopulation increase.

A regulatory single nucleotide polymorphism in the ubiquitin D gene associated with celiac disease.

An aberrant immune response triggered by dietary gluten is the main driving force underlying celiac disease (CD), but other biologic pathways that are dysregulated also participate in disease development. Genetic variation within these pathways might influence expression, contributing to susceptibility to CD. We have investigated the implication of ubiquitin D (UBD), a member of the ubiquitin-proteasome system that is strongly upregulated in the intestinal mucosa of active CD. Reverse transcriptase-polymerase chain reaction analysis of intestinal biopsy sample pairs (at diagnosis vs treated) from 30 CD patients confirmed overexpression of UBD in active disease tissue (fold change = 8.3; p = 0.0022). In silico prediction tools identified rs11724 as a putative regulatory single nucleotide polymorphism and association analysis of 468 CD patients and 459 controls revealed that the minor rs11724*C allele was more frequent among patients (minor allele frequency = 0.44 vs 0.39; odds ratio [OR] = 1.23; p = 0.028) and suggested a dominant allele effect (OR = 1.49; p = 0.0045). Correlation of the rs11724 genotype and UBD mRNA levels (OR = 0.76; p = 0.0021) further supports its implication in disease development.

TH17 (and TH1) signatures of intestinal biopsies of CD patients in response to gliadin.

Celiac disease (CD) is an immunological disorder caused by intolerance to ingested gliadin and other cereal prolamins that has been included in the T(H)1-dominated group of diseases, where IL-12 induced IFNgamma is the major proinflamatory signal. Recently, another linage of T cells has been described, namely T(H)17, characterized by production of IL-17, that differentiate in response to TGFbeta and IL-6 and participate in the pathogenesis of several autoimmune diseases. Using RT-PCR analysis of gene expression, we analyzed the presence of T(H)1 (IL-12 and IFNgamma) and T(H)17 (TGFbeta, IL-6, IL-17A, IL-17F and IL-23) related cytokines in intestinal biopsies from CD patients with active disease compared to remission and from treated patients after acute, in vitro re-exposure to gliadin. Potent T(H)1 and T(H)17 responses were present in the active stage of the disease, whereas short incubation of normalized biopsies with gliadin did not increase the expression of the effector cytokines, although a tendency of upregulation for both T(H)1 and T(H)17 promoting factors was observed, suggestive of a reactivation of proinflammatory pathways. These results place CD into the group of autoimmune disorders in which T(H)17 cells also participate, although the relative importance of each T cell response and their role in the initial events of the disease need further investigation.

Combined functional and positional gene information for the identification of susceptibility variants in celiac disease.

BACKGROUND & AIMS: Celiac disease is a complex, immune-mediated disorder of the intestinal mucosa with a strong genetic component. HLA-DQ2 is the major determinant of risk, but other minor genes, still to be identified, also are involved. METHODS: We designed a strategy that combines gene expression profiling of intestinal biopsy specimens, linkage region information, and different bioinformatics tools for the selection of potentially regulatory single-nucleotide polymorphisms (SNPs) involved in the disease. We selected 361 SNPs from 71 genes that fulfilled stringent functional (changes in expression level) and positional criteria (located in regions that have been linked to the disease, other than HLA). These polymorphisms were genotyped in 262 celiac patients and 214 controls. RESULTS: We detected strong evidence of association with several SNPs (the most significant were rs6747096, P = 2.38 x 10(-5); rs7040561, P = 6.55 x 10(-5); and rs458046, P = 1.35 x 10(-4)) that pinpoint novel candidate determinants of predisposition to the disease in previously identified linkage regions (eg, SERPINE2 in 2q33, and PBX3 or PPP6C in 9q34). CONCLUSIONS: Our study shows that the combination of function and position is a valid strategy for the genetic dissection of complex traits.