IL-1β maintains the DNA hypermethylation of anti-inflammatory IL-10 gene in a human intestinal epithelial cell line

Intestinal inflammation is a natural process crucial to maintain gut integrity, but its deregulation is involved in the pathogenesis of severe intestinal disorders[1]. Intestinal epithelial cells play a crucial role in the inflammatory response, modulating the immune cell exposure to antigens and by their ability to secrete many inflammatory mediators. IL-1β represents a pivotal player: secreted by infiltrated leucocytes, it induces the expression of several pro-inflammatory genes. Also the anti-inflammatory IL-10, whose function is to terminate the inflammatory process, modulates the intestinal physiology[2]. Recent clinical reports showed that patients with ulcerative colitis in remission phase have significantly higher IL10 gene expression in mucosa compared with active patients and controls[3]. Moreover, in the latest years aberrant epigenetic mechanisms were put in binomial relationship with chronic inflammatory diseases[4]. Previously, we described a demethylation of pro-inflammatory IL6 and IL8 genes in human colonic Caco-2 cells differentiated into an enterocyte-like phenotype and exposed to the inflammatory action of IL-1β[5]. In the present study we evaluate whether the IL-1β treatment affected the methylation status of the anti-inflammatory IL10 gene, in the same in vitro model. Our results showed that IL-1β treatment did not change the hypermethylation status of the IL10 promoter. Moreover, in cell lysates from IL-1β-treated Caco-2 cells, we observed a dose-dependent increase of DNMTs activity and, surprisingly, a decrease of DNMT3b expression. These findings put in evidence the complexity of relationship between IL-1β and DNMTs, and may suggest a potential role of IL-1β as pleiotropic modulator of DNA methylation in Caco-2 cell line

Addition of chiral and achiral allyltrichlorostannanes to chiral±-alkoxy aldehydes

Achiral and chiral allyltrichlorostannanes reacted with chiral ±-alkoxy aldehydes to give the corresponding homoallylic alcohols with moderate to good levels of 1,4-syn-diastereoselection

Methylation decrease of BECN1 gene induced by phytochemical Indicaxantin in Caco2 cells: an epigenetic hypothesis of autophagy

Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes [1]. The role of this process in tumorigenesis and tumor progression is controversial: in the early stages, it can block tumor growth and conversely it can promote its progression in the later stages [2]. The tumor suppressor BECN1 gene, encodes the protein Beclin 1, a marker of autophagy down-regulated in several types of cancer, such as colorectal cancer [3]. There are a lot of both genetic and environmental risk factors for colorectal cancer, including diet: for this reason, in accordance with epidemiological studies, consumption of foods rich in phytochemicals is widely promoted. The betalain indicaxantin (Ind) is a phytochemical from the Opuntia Ficus-Indica fruit having several biological activities, such as antioxidant, anti-inflammatory. It showed antiproliferative and proapoptotic effects in colorectal adenocarcinoma (Caco2) cells where was able to regulate gene expression through modulation of methylation state of DNA at CpG islands [4]. For the first time, using Methylation-Sensitive Restriction Endonuclease PCR (MSRE-PCR), we report that Ind (50 e 100 µM) decreases the methylation of BECN1 promoter in Caco2 cells, to the same extent as 5-azacytidine (Zcyd, positive control). Interestingly, colorimetric detection of DNA Methyltransferases activity, indicates that Ind reduced the activity of these enzymes, like Zcyd did. These preliminary data, indicating that Ind is able to decrease the methylation of BECN1 gene, allow us to propose an epigenetic hypothesis of autophagy regulation in Caco2 cells

Extracellular microRNAs exhibit sequence-dependent stability and cellular release kinetics

Multiple studies have described extracellular microRNAs (ex-miRNAs) as being remarkably stable despite the hostile extracellular environment, when stored at 4oC or lower. Here we show that many ex-miRNAs are rapidly degraded when incubated at 37oC in the presence of serum (thereby simulating physiologically relevant conditions). Stability varied widely between miRNAs, with half-lives ranging from similar to 1.5 hours to more than 13 hours. Notably, ex-miRNA half-lives calculated in two different biofluids (murine serum and C2C12 mouse myotube conditioned medium) were highly similar, suggesting that intrinsic sequence properties are a determining factor in miRNA stability. By contrast, ex-miRNAs associated with extracellular vesicles (isolated by size exclusion chromatography) were highly stable. The release of ex-miRNAs from C2C12 myotubes was measured over time, and mathematical modelling revealed miRNA-specific release kinetics. While some ex-miRNAs reached the steady state in cell culture medium within 24 hours, the extracellular level of miR-16 did not reach equilibrium, even after 3 days in culture. These findings are indicative of miRNA-specific release and degradation kinetics with implications for the utility of ex-miRNAs as biomarkers, and for the potential of ex-miRNAs to transfer gene regulatory information between cells

Adaptation to tolerate high doses of arabinoxylan is associated with fecal levels of \u3ci\u3eBifidobacterium longum\u3c/i\u3e

Dietary fiber supplements are a strategy to close the ‘fiber gap’ and induce targeted modulations of the gut microbiota. However, higher doses of fiber supplements cause gastrointestinal (GI) symptoms that differ among individuals. What determines these inter-individual differences is insufficiently understood. Here we analyzed findings from a six-week randomized controlled trial that evaluated GI symptoms to corn bran arabinoxylan (AX; n = 15) relative to non-fermentable microcrystalline cellulose (MCC; n = 16) at efficacious supplement doses of 25 g/day (females) or 35 g/day (males) in adults with excess weight. Self-reported flatulence, bloating, and stomach aches were evaluated weekly. Bacterial taxa involved in AX fermentation were identified by bioorthogonal non-canonical amino acid tagging. Associations between GI symptoms, fecal microbiota features, and diet history were systematically investigated. AX supplementation increased symptoms during the first three weeks relative to MCC (p \u3c 0.05, Mann-Whitney tests), but subjects ‘adapted’ with symptoms reverting to baseline levels toward the end of treatment. Symptom adaptations were individualized and correlated with the relative abundance of Bifidobacterium longum at baseline (rs = 0.74, p = 0.002), within the bacterial community that utilized AX (rs = 0.69, p = 0.006), and AX-induced shifts in acetate (rs = 0.54, p = 0.039). Lower baseline consumption of animal-based foods and higher whole grains associated with less severity and better adaptation. These findings suggest that humans do ‘adapt’ to tolerate efficacious fiber doses, and this process is linked to their microbiome and dietary factors known to interact with gut microbes, providing a basis for the development of strategies for improved tolerance of dietary fibers

Investigation of selected genomic deletions and duplications in a cohort of 338 patients presenting with syndromic obesity by multiplex ligation-dependent probe amplification using synthetic probes

Background: Certain rare syndromes with developmental delay or intellectual disability caused by genomic copy number variants (CNVs), either deletions or duplications, are associated with higher rates of obesity. Current strategies to diagnose these syndromes typically rely on phenotype-driven investigation. However, the strong phenotypic overlap between syndromic forms of obesity poses challenges to accurate diagnosis, and many different individual cytogenetic and molecular approaches may be required. Multiplex ligation-dependent probe amplification (MLPA) enables the simultaneous analysis of multiple targeted loci in a single test, and serves as an important screening tool for large cohorts of patients in whom deletions and duplications involving specific loci are suspected. Our aim was to design a synthetic probe set for MLPA analysis to investigate in a cohort of 338 patients with syndromic obesity deletions and duplications in genomic regions that can cause this phenotype.Results: We identified 18 patients harboring copy number imbalances; 18 deletions and 5 duplications. the alterations in ten patients were delineated by chromosomal microarrays, and in the remaining cases by additional MLPA probes incorporated into commercial kits. Nine patients showed deletions in regions of known microdeletion syndromes with obesity as a clinical feature: in 2q37 (4 cases), 9q34 (1 case) and 17p11.2 (4 cases). Four patients harbored CNVs in the DiGeorge syndrome locus at 22q11.2. Two other patients had deletions within the 22q11.2 'distal' locus associated with a variable clinical phenotype and obesity in some individuals. the other three patients had a recurrent CNV of one of three susceptibility loci: at 1q21.1 'distal', 16p11.2 'distal', and 16p11.2 'proximal'.Conclusions: Our study demonstrates the utility of an MLPA-based first line screening test to the evaluation of obese patients presenting with syndromic features. the overall detection rate with the synthetic MLPA probe set was about 5.3% (18 out of 338). Our experience leads us to suggest that MLPA could serve as an effective alternative first line screening test to chromosomal microarrays for diagnosis of syndromic obesity, allowing for a number of loci (e.g., 1p36, 2p25, 2q37, 6q16, 9q34, 11p14, 16p11.2, 17p11.2), known to be clinically relevant for this patient population, to be interrogated simultaneously.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Inst Biosci, Dept Genet & Evolutionary Biol, Human Genome & Stem Cell Ctr, São Paulo, BrazilUniv São Paulo, Sch Med, Children Inst, Genet Unit,Dept Pediat, São Paulo, BrazilUniv São Paulo, Sch Med, Dept Med Genet, Neurogenet Unit, BR-14049 Ribeirao Preto, BrazilUniversidade Federal de São Paulo, Ctr Med Genet, Dept Morphol, São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Med Genet, Dept Morphol, São Paulo, BrazilFAPESP: 09/52523-1FAPESP: 1998/14254-2CNPq: 304381/2007-1Web of Scienc

Chronic Airway Infection/Inflammation Induces a Ca 2+ i -dependent Hyperinflammatory Response in Human Cystic Fibrosis Airway Epithelia

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung

The Cytotoxic Necrotizing Factor of Yersinia pseudotuberculosis (CNFy) is Carried on Extracellular Membrane Vesicles to Host Cells

In this study we show Yersinia pseudotuberculosis secretes membrane vesicles (MVs) that contain different proteins and virulence factors depending on the strain. Although MVs from Y. pseudotuberculosis YPIII and ATCC 29833 had many proteins in common (68.8% of all the proteins identified), those located in the outer membrane fraction differed significantly. For instance, the MVs from Y. pseudotuberculosis YPIII harbored numerous Yersinia outer proteins (Yops) while they were absent in the ATCC 29833 MVs. Another virulence factor found solely in the YPIII MVs was the cytotoxic necrotizing factor (CNFy), a toxin that leads to multinucleation of host cells. The ability of YPIII MVs to transport this toxin and its activity to host cells was verified using HeLa cells, which responded in a dose-dependent manner; nearly 70% of the culture was multinucleated after addition of 5 mu g/ml of the purified YPIII MVs. In contrast, less than 10% were multinucleated when the ATCC 29833 MVs were added. Semi-quantification of CNFy within the YPIII MVs found this toxin is present at concentrations of 5 -10 ng per mu g of total MV protein, a concentration that accounts for the cellular responses see