Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.This study was supported by funds obtained from The Evelyn Trust, Crohn’s in Childhood Research Association (CICRA) and Crohn’s and Colitis in Childhood (3Cs) charity. J.K. was funded by a PhD studentship from CICRA. Funding for E.C. was provided by the Deutsche Forschungsgemeinschaft (Grant CA226/4-3) and Interne Forschungsförderung Essen (IFORES).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/mi.2015.8

Extracellular electrical signals in a neuron-surface junction: model of heterogeneous membrane conductivity

Signals recorded from neurons with extracellular planar sensors have a wide range of waveforms and amplitudes. This variety is a result of different physical conditions affecting the ion currents through a cellular membrane. The transmembrane currents are often considered by macroscopic membrane models as essentially a homogeneous process. However, this assumption is doubtful, since ions move through ion channels, which are scattered within the membrane. Accounting for this fact, the present work proposes a theoretical model of heterogeneous membrane conductivity. The model is based on the hypothesis that both potential and charge are distributed inhomogeneously on the membrane surface, concentrated near channel pores, as the direct consequence of the inhomogeneous transmembrane current. A system of continuity equations having non-stationary and quasi-stationary forms expresses this fact mathematically. The present work performs mathematical analysis of the proposed equations, following by the synthesis of the equivalent electric element of a heterogeneous membrane current. This element is further used to construct a model of the cell-surface electric junction in a form of the equivalent electrical circuit. After that a study of how the heterogeneous membrane conductivity affects parameters of the extracellular electrical signal is performed. As the result it was found that variation of the passive characteristics of the cell-surface junction, conductivity of the cleft and the cleft height, could lead to different shapes of the extracellular signals

A Practical Guide for the Diagnosis of Primary Enteric Nervous System Disorders

Objective:Primary gastrointestinal neuropathies are a heterogeneous group of enteric nervous system (ENS) disorders that continue to cause difficulties in diagnosis and histological interpretation. Recently, an international working group published guidelines for histological techniques and reporting, along with a classification of gastrointestinal neuromuscular pathology. The aim of this article was to review and summarize the key issues for pediatric gastroenterologists on the diagnostic workup of congenital ENS disorders. In addition, we provide further commentary on the continuing controversies in the field.Results:Although the diagnostic criteria for Hirschsprung disease are well established, those for other forms of dysganglionosis remain ill-defined. Appropriate tissue sampling, handling, and expert interpretation are crucial to maximize diagnostic accuracy and reduce interobserver variability. The absence of validated age-related normal values for neuronal density, along with the lack of correlation between clinical and histological findings, result in significant diagnostic uncertainties while diagnosing quantitative aberrations such as hypoganglionosis or ultrashort Hirschsprung disease. Intestinal neuronal dysplasia remains a histological description of unclear significance.Conclusions:The evaluation of cellular quantitative or qualitative abnormalities of the ENS for clinical diagnosis remains complex. Such analysis should be carried out in laboratories that have the necessary expertise and access to their own validated reference values

Single-Cell Sequencing of Developing Human Gut Reveals Transcriptional Links to Childhood Crohn's Disease.

Human gut development requires the orchestrated interaction of differentiating cell types. Here, we generate an in-depth single-cell map of the developing human intestine at 6-10 weeks post-conception. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells; distinct from LGR5-expressing cells. We propose that these cells may contribute to differentiated cell subsets via the generation of LGR5-expressing stem cells and receive signals from surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNA-seq profiles from pediatric Crohn's disease epithelium alongside matched healthy controls to reveal disease-associated changes in the epithelial composition. Contrasting these with the fetal profiles reveals the re-activation of fetal transcription factors in Crohn's disease. Our study provides a resource available at www.gutcellatlas.org, and underscores the importance of unraveling fetal development in understanding disease

Randomized controlled trials in pediatric complementary and alternative medicine: Where can they be found?

BACKGROUND: The safety and effectiveness of CAM interventions are of great relevance to pediatric health care providers. The objective of this study is to identify sources of reported randomized controlled trials (RCTs) in the field of pediatric complementary and alternative medicine (CAM). METHODS: Reports of RCTs were identified by searching Medline and 12 additional bibliographic databases and by reviewing the reference lists of previously identified pediatric CAM systematic reviews. RESULTS: We identified 908 reports of RCTs that included children under 18 and investigated a CAM therapy. Since 1965, there has been a steady growth in the number of these trials that are being published. The four journals that published the most reported RCTs are The American Journal of Clinical Nutrition, Pediatrics, Journal of Pediatrics, and Lancet. Medline, CAB Health, and Embase were the best database sources for identifying these studies; they indexed 93.2%, 58.4% and 42.2 % respectively of the journals publishing reports of pediatric CAM RCTs. CONCLUSIONS: Those working or interested in the field of pediatric CAM should routinely search Medline, CAB Health and Embase for literature in the field. The four core journals identified above should be included in their collection

APOBEC mutagenesis is a common process in normal human small intestine

APOBEC mutational signatures SBS2 and SBS13 are common in many human cancer types. However, there is an incomplete understanding of its stimulus, when it occurs in the progression from normal to cancer cell and the APOBEC enzymes responsible. Here we whole-genome sequenced 342 microdissected normal epithelial crypts from the small intestines of 39 individuals and found that SBS2/SBS13 mutations were present in 17% of crypts, more frequent than most other normal tissues. Crypts with SBS2/SBS13 often had immediate crypt neighbors without SBS2/SBS13, suggesting that the underlying cause of SBS2/SBS13 is cell-intrinsic. APOBEC mutagenesis occurred in an episodic manner throughout the human lifespan, including in young children. APOBEC1 mRNA levels were very high in the small intestine epithelium, but low in the large intestine epithelium and other tissues. The results suggest that the high levels of SBS2/SBS13 in the small intestine are collateral damage from APOBEC1 fulfilling its physiological function of editing APOB mRNA. Whole-genome sequencing of healthy human epithelial crypts from the small intestines of 39 individuals highlights APOBEC enzymes as a common contributor to the overall mutational burden in this tissue.Peer reviewe

Use of complementary and alternative medicine in Germany – a survey of patients with inflammatory bowel disease

BACKGROUND: Previous studies have suggested an increasing use of complementary and alternative medicine (CAM) in patients with inflammatory bowel disease (IBD). The aim of our study was to evaluate the use of CAM in German patients with IBD. METHODS: A questionnaire was offered to IBD patients participating in patient workshops which were organized by a self-help association, the German Crohn's and Colitis Association. The self-administered questionnaire included demographic and disease-related data as well as items analysing the extent of CAM use and satisfaction with CAM treatment. Seven commonly used CAM methods were predetermined on the questionnaire. RESULTS: 413 questionnaires were completed and included in the analysis (n = 153 male, n = 260 female; n = 246 Crohn's disease, n = 164 ulcerative colitis). 52 % of the patients reported CAM use in the present or past. In detail, homeopathy (55%), probiotics (43%), classical naturopathy (38%), Boswellia serrata extracts (36%) and acupuncture/Traditional Chinese Medicine (TCM) (33%) were the most frequently used CAM methods. Patients using probiotics, acupuncture and Boswellia serrata extracts (incense) reported more positive therapeutic effects than others. Within the statistical analysis no significant predictors for CAM use were found. 77% of the patients felt insufficiently informed about CAM. CONCLUSION: The use of CAM in IBD patients is very common in Germany, although a large proportion of patients felt that information about CAM is not sufficient. However, to provide an evidence-based approach more research in this field is desperately needed. Therefore, physicians should increasingly inform IBD patients about benefits and limitations of CAM treatment

High efficient electrical stimulation of hippocampal slices with vertically aligned carbon nanofiber microbrush array

Long-term neuroprostheses for functional electrical stimulation must efficiently stimulate tissue without electrolyzing water and raising the extracellular pH to toxic levels. Comparison of the stimulation efficiency of tungsten wire electrodes (W wires), platinum microelectrode arrays (PtMEA), as-grown vertically aligned carbon nanofiber microbrush arrays (VACNF MBAs), and polypyrrole coated (PPy-coated) VACNF MBAs in eliciting field potentials in the hippocampus slice indicates that, at low stimulating voltages that preclude the electrolysis of water, only the PPy-coated VACNF MBA is able to stimulate the CA3 to CA1 pathway. Unlike the W wires, PtMEA, as-grown VACNF MBA, and the PPy-coated VACNF MBA elicit only excitatory postsynaptic potentials (EPSPs). Furthermore, the PPy-coated VACNF MBA evokes somatic action potentials in addition to EPSPs. These results highlight the PPy-coated VACNF’s advantages in lower electrode impedance, ability to stimulate tissue through a biocompatible chloride flux, and stable vertical alignment in liquid that enables access to spatially confined regions of neuronal cells

Zollinger-Ellison syndrome associated with neurofibromatosis type 1: a case report

BACKGROUND: Neurofibromatosis type 1 is an autosomal dominant neurocutaneous disorder with characteristic features of skin and central nervous system involvement. Gastrointestinal involvement is rare, but the risk of malignancy development is considerable. Zollinger-Ellison syndrome is caused by gastrin-secreting tumors called gastrinomas. Correct diagnosis is often difficult, and curative treatment can only be achieved surgically. CASE PRESENTATION: A 41-year-old female affected by neurofibromatosis type 1 presented with a history of recurrent epigastric soreness, diarrhea, and relapsing chronic duodenal ulcer. Her serum fasting gastrin level was over 1000 pg/mL. An abdominal CT scan revealed a 3 × 2-cm, well-enhanced mass adjacent to the duodenal loop. She was not associated with multiple endocrine neoplasia type 1. Operative resection was performed and gastrinoma was diagnosed by immunohistochemical staining. The serum gastrin level decreased to 99.1 pg/mL after surgery, and symptoms and endoscopic findings completely resolved without recurrences. CONCLUSION: Gastrinoma is difficult to detect even in the general population, and hence symptoms such as recurrent idiopathic peptic ulcer and diarrhea in neurofibromatosis type 1 patients should be accounted for as possibly contributing to Zollinger-Ellison syndrome

Lipid Alterations in Experimental Murine Colitis: Role of Ceramide and Imipramine for Matrix Metalloproteinase-1 Expression

BACKGROUND:Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD). Therefore, we analysed alterations of bioactive lipids in experimental models of colitis and examined the functional consequence of the second messenger ceramide in inflammatory pathways leading to tissue destruction. METHODOLOGY/PRINCIPAL FINDINGS:Chronic colitis was induced by dextran-sulphate-sodium (DSS) or transfer of CD4(+)CD62L(+) cells into RAG1(-/-)-mice. Lipid content of isolated murine intestinal epithelial cells (IEC) was analysed by tandem mass spectrometry. Concentrations of MMP-1 in supernatants of Caco-2-IEC and human intestinal fibroblasts from patients with ulcerative colitis were determined by ELISA. Imipramine was used for pharmacologic inhibition of acid sphingomyelinase (ASM). Ceramide increased by 71% in chronic DSS-induced colitis and by 159% in the transfer model of colitis. Lysophosphatidylcholine (LPC) decreased by 22% in both models. No changes were detected for phosphatidylcholine. Generation of ceramide by exogenous SMase increased MMP-1-protein production of Caco-2-IEC up to 7-fold. Inhibition of ASM completely abolished the induction of MMP-1 by TNF or IL-1beta in Caco-2-IEC and human intestinal fibroblasts. CONCLUSIONS/SIGNIFICANCE:Mucosal inflammation leads to accumulation of ceramide and decrease of LPC in the intestinal epithelium. One aspect of ceramide generation is an increase of MMP-1. Induction of MMP-1 by TNF or IL-1beta is completely blocked by inhibition of ASM with imipramine. Therefore, inhibition of ASM may offer a treatment strategy to reduce MMP-1 expression and tissue destruction in inflammatory conditions