Uncovering the gastrointestinal passage, intestinal epithelial cellular uptake, and AGO2 loading of milk miRNAs in neonates using xenomiRs as tracers.

BACKGROUND: Human breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiating enteral nutrition. However, this requires that relevant milk miRNA amounts survive the gastrointestinal (GI) passage, are taken up by cells, and become available to the RNA interference machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and GI passage. OBJECTIVES: Our goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine model in vivo to contribute to the discussion of whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals. METHODS: We performed cross-species profiling of miRNAs via deep sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells using adenovirus-type 5-mediated miRNA gene transfer. RESULTS: Mammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula nutrition in human preterm stool and 9 d after the onset of enteral feeding in intestinal cells (ICs) of preterm piglets. Thereafter, several xenomiRs accumulated in the ICs. Moreover, a few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in argonaute RISC catalytic component 2 (AGO2)-immunocomplexes from intestinal biopsies. CONCLUSIONS: Milk-derived miRNAs survived GI passage in human and porcine neonates. Bovine-specific miRNAs accumulated in ICs of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood concentrations of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in ICs. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract.

Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation.

BACKGROUND: Reverse transcription of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (+)RNA genome and subgenomic RNAs (sgRNAs) and subsequent quantitative polymerase chain reaction (RT-qPCR) is the reliable diagnostic gold standard for COVID-19 diagnosis and the identification of potential spreaders. Apart from clinical relevance and containment, for specific questions, it might be of interest to (re)investigate cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results, even though these cases might neither be clinically relevant nor significant for containment measures, because they might probably not be infectious. In order to expand the diagnostic bandwidth for non-routine questions, particularly for the reliable discrimination between negative and false-negative specimens associated with high CT values, we combined the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This expansion can help to confirm SARS-CoV-2 infections without the demand of confirmative antibody testing, which requires to summon patients again for blood sampling few to several weeks after symptom onset. RESULTS: We successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics. This allows a reliable interpretation of RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR. After laboratory implementation, we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 5 previously unrecognized cases of pediatric SARS-CoV-2-associated diseases, mainly exhibiting mild and heterogeneous presentation-apart from a single case of multisystem inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2, who was hospitalized in the course of the study. CONCLUSIONS: The proposed protocol allows a specific and sensitive confirmation of SARS-CoV-2 infections close to the detection limits of RT-qPCR. The tested biotinylated primers do not negatively affect the RT-qPCR pipeline and thus can be optionally applied to enable deeper inspection of RT-qPCR results by subsequent pyrosequencing. Moreover, due to the incremental transmission of SARS-CoV-2 variants of concern, we note that the used strategy can uncover (Spike) P681H allowing the pre-selection of SARS-CoV-2 B.1.1.7 candidate specimens for deep sequencing.

Late-onset Cytomegalovirus Infection Associated With Gastric Outlet Obstruction in a Preterm Twin.

The infant was born at a gestational age of 28 + 2 weeks as second twin to a 26-year-old woman, G1/P0, due to eclampsia. The patient developed well and was on full oral feeds when he started to develop nonbilious vomiting at 5 weeks. He was diagnosed with pyloric hypertrophy and underwent pylorotomy, but the condition did not improve and the patient was referred to our hospital. Here, esophagogastroduodenoscopy showed severely inflamed esophageal and gastric mucosa which was found to be due to cytomegaly virus (CMV) infection and a nonpassable pylorus. The patient underwent pyloroplasty revealing a fibrous pyloric ring. Histology showed giant cells suggestive of CMV infection which was confirmed by polymerase chain reaction. He was started on valganciclovir and discharged 4 weeks later on full enteral feeds. To our knowledge, this is the first case of gastric outlet obstruction due to CMV infection in a premature infant.

Human astrovirus infection associated with encephalitis in an immunocompetent child: a case report.

BACKGROUND: Until today, classic human astroviruses have not been associated with central nervous system infections in immunocompetent patients. CASE PRESENTATION: A 16-month-old Caucasian girl presented with repetitive generalized seizures with a 4-day history of watery diarrhea, which had already gradually improved. Initially, the prolonged seizures ceased after systemic midazolam treatment and were thought to be fever associated. However, her mental status remained altered, and after seizure recurrence, she was transferred to our pediatric intensive care unit. Seizure control was achieved by a combination of high-dose levetiracetam and phenobarbital, but she remained unconscious. An electroencephalogram at this time revealed generalized high voltage theta activity. All laboratory analyses, including extended blood and cerebrospinal fluid analyses, and a brain magnetic resonance imaging were normal. On day 4, the child gradually became conscious, but was very agitated and not able to walk. Since an electroencephalogram at this time still revealed generalized high voltage theta activity, although she had not received sedative medications for 72 hours, she was diagnosed as having encephalopathy. At that time, results of diagnostic testing of the stool sample were positive for classic astrovirus infection, and we decided to analyze the initially obtained cerebrospinal fluid for astrovirus as well. Cerebrospinal fluid was also found positive for human astrovirus. Sequencing analysis revealed a classic astrovirus genotype 1 with exactly the same nucleotide sequence as in the feces. Clinically, the child gradually improved and was discharged on day 9. CONCLUSIONS: Whereas the new human astrovirus subtypes have been recently associated with central nervous system infection, this is the first case of encephalitis in an immunocompetent child due to classic human astrovirus. Considering that classic human astroviruses are the third most common etiological agents of viral gastroenteritis in children, we believe that human astroviruses as causative agents for central nervous system infections should be considered more often, especially in children and infants with preceding gastroenteritis.

[Genetic Investigations in Children with Developmental Disorders, Intellectual Disability and/or Epilepsy - The Parents' Perspective]. / Genetische Untersuchungen bei Kindern und Jugendlichen mit Entwicklungsstörung, Behinderung und Epilepsien ­ was erwarten Eltern von der Aufklärung.

BACKGROUND: Shared decision-making is indispensable when it comes to molecular genetic investigations, but data on the expectations of the parents is scarce. METHODS: Using a step-by-step approach we initially performed free in-depth-interviews with five parents on which base we developed a half standardized questionnaire. This questionnaire was then applied in interviews with 30 parents of children with intellectual disability, autism or epilepsy subject to genetic examination. RESULTS: Pre-diagnostic discussions are challenging for the parents in an intellectual as well as emotional way. The most important general aspects are diagnosis and therapy. Self-assessment of prior knowledge is very variable and many parents expressed problems in understanding. During the conversation parents rate the following specific aspects as "very important" or "important": findings of unclear relevance, incidental findings, psychic consequences, prognostic aspects, possible therapeutic interventions. 10 Parents did not have any school-degree and 20 parents were not native speakers. DISCUSSION: All parents express a high need for information covering almost all aspects of the investigation. Communicational hurdles pose additional challenges leaving a large room for improvement. Trustworthy internet-based information systems in different languages including plain language could be a first step.

Impact of an In-House Pediatric Surgery Unit and Human Milk Centered Enteral Nutrition on Necrotizing Enterocolitis.

The importance for mortality and morbidity of an in-house pediatric surgery unit for premature infants with necrotizing enterocolitis (NEC) remains undefined. Data on 389 consecutive very low birth weight infants with a birth weight <1250 g admitted between 2009 and 2014 was retrospectively analyzed in two almost identical neonatal intensive care units. Epidemiological data (n=172 and n=217, respectively) were comparable. Incidence of NEC stage II+ was significantly higher in center 1 (15.1 versus 5.5%, n=18 versus 6). This correlated with a significantly lower rate of exclusive human milk feeding compared to center 2 (24.2 versus 59.3%). Probiotic treatment did not differ. Importantly, in case of surgery the length of removed intestine (49.9 versus 19.5 cm) and the rate of severe short-bowel syndrome (38.9 versus 0 %) were significantly higher in center 1 (no in-house pediatric surgery). Furthermore, long-term morbidity assessment revealed more impaired motoric (-4.2 versus -2.2 months, p=0.21) and psychologic (-4.3 versus -1.6 months, p=0.09) development in center 1. Mortality was similar in both centers. Conclusions. Short- and possibly also long-term morbidity of NEC is clearly associated with the presence of an on-site pediatric surgery unit. Enteral nutrition with human milk seems to be a strong protective factor against NEC.

Speckle tracking stress echocardiography in children: interobserver and intraobserver reproducibility and the impact of echocardiographic image quality.

Speckle tracking echocardiography (STE) is increasingly used during functional assessments. However, reproducibility and dependence on echocardiographic image quality for speckle tracking stress echocardiography in pediatric patients have not been studied to date. 127 consecutive normotensive children without structural heart disease (mean age 13.4 ± 3.0 years, 50.4% female) underwent a stepwise semisupine cycle ergometric protocol. Left ventricular (LV) myocardial peak strain and strain rate were assessed at rest and during exercise. Interobserver and intraobserver assessments were performed and analyzed regarding echocardiographic image quality. LV peak global strain and strain rate were well reproducible with narrow limits of agreement without any significant bias both at rest and during all stages of exercise testing. Moreover, strain rate reproducibility slightly deteriorated in values between -1.5 and -3 s-1. Surprisingly, there was no significant difference in reproducibility between optimal, intermediate and poor quality of echocardiographic images. STE derived strain and strain rate measurements in children are feasible and highly reproducible during semisupine cycle ergometric stress echocardiography. Echocardiographic image quality does not seem to influence strain (rate) reproducibility. Myocardial deformation measurements in images with suboptimal visualization quality must be interpreted with caution.

Neonatal exposure to hyperoxia leads to persistent disturbances in pulmonary histone signatures associated with NOS3 and STAT3 in a mouse model.

Background: Early pulmonary oxygen exposure is one of the most important factors implicated in the development of bronchopulmonary dysplasia (BPD). Methods: Here, we analyzed short- and long-term effects of neonatal hyperoxia on NOS3 and STAT3 expression and corresponding epigenetic signatures using a hyperoxia-based mouse model of BPD. Results: Early hyperoxia exposure led to a significant increase in NOS3 (median fold change × 2.37, IQR 1.54-3.68) and STAT3 (median fold change × 2.83, IQR 2.21-3.88) mRNA levels in pulmonary endothelial cells with corresponding changes in histone modification patterns such as H2aZac and H3K9ac hyperacetylation at the respective gene loci. No complete restoration in histone signatures at these loci was observed, and responsivity to later hyperoxia was altered in mouse lungs. In vitro, histone signatures in human aortic endothelial cells (HAEC) remained altered for several weeks after an initial long-term exposure to trichostatin A. This was associated with a substantial increase in baseline eNOS (median 27.2, IQR 22.3-35.6) and STAT3α (median 5.8, IQR 4.8-7.3) mRNA levels with a subsequent significant reduction in eNOS expression upon exposure to hypoxia. Conclusions: Early hyperoxia induced permanent changes in histones signatures at the NOS3 and STAT3 gene locus might partly explain the altered vascular response patterns in children with BPD.

Speckle Tracking Stress Echocardiography Uncovers Early Subclinical Cardiac Involvement in Pediatric Patients with Inflammatory Bowel Diseases.

Inflammatory bowel disease (IBD) is an established risk factor for cardiovascular disease (CVD). However, whether cardiac consequences present early in IBD is unknown. This is the first study in children aiming to unmask altered myocardial mechanics in IBD. We enrolled 50 consecutive normotensive children with Crohn's disease (CD) (n = 28) or ulcerative colitis (UC) (n = 22). The study groups consisted of 18 patients with active inflammatory disease (mean age 14.6 ± 2.5 years) and 32 children with IBD in remission (14.3 ± 2.3 years). 60 age- and gender-matched children served as healthy controls. Speckle tracking stress echocardiography (STE) was used to assess left ventricular (LV) myocardial strain and strain rate. Circumferential strain rate was significantly decreased in children with active IBD (-1.55 ± 0.26 s-1) and IBD in remission (-1.49 ± 0.26 s-1) versus healthy controls (1.8 ± 0.4 s-1) both at rest (p < 0.001) and during exercise (p = 0.021). Moreover, longitudinal strain rate, circumferential strain and E/E' ratio were significantly impaired in IBD. Pediatric patients with IBD feature subclinical signs of LV systolic and diastolic myocardial impairment early in the course of CD and UC. This may not be reversible even when IBD is clinically controlled. Patients with IBD should be regularly screened for signs of CVD.

Nursing staff fluctuation and pathogenic burden in the NICU - effective outbreak management and the underestimated relevance of non-resistant strains.

In the course of a hospital management takeover, a microbial outbreak took place in a tertiary neonatal intensive care unit (NICU). Here, we characterize the outbreak and its management. About 4 months prior to takeover, there was a sharp increase in positive isolates for MSSA and multidrug-resistant organisms (MDROs). Simultaneously, the nursing staff sick leave rate increased dramatically which directly correlated with the number of infection/colonization per week (r2 = 0.95, p = 0.02). During the following months we observed several peaks in positive isolates of methicillin-sensitive staphylococcus aureus (MSSA), MDROs and subsequently a vancomycin-resistant enterococcus (VRE) outbreak. Interventional outbreak management measures were only successful after substantial recruitment of additional nursing staff. None of the VRE, but 44% (n = 4) of MDRO and 32% (n = 23) of MSSA colonized infants developed symptomatic infections (p = 0.02). Among the latter, 35% suffered from serious consequences such as osteomyelitis. The most important risk factors for colonization-to-infection progression were low gestational age and birth weight. Nursing staff fluctuation poses a substantial risk for both bacterial colonization and infection in neonates. Comprehensive outbreak management measures are only successful if adequate nursing staff is available. Non resistant strains account for most neonatal infections - possibly due to their limited perception as being harmful.

A longitudinal, randomized, and prospective study of nocturnal monitoring in children and adolescents with epilepsy: Effects on quality of life and sleep.

OBJECTIVE: Most studies on seizure detection systems focus more on the effectiveness of devices than on their practicability in and impact on everyday life. Our study investigated the impact of a technical monitoring system on subjective quality of sleep and the lives of affected families. Furthermore, we evaluated the impact of anxiety levels on seizure monitoring and vice versa. METHODS: Forty-three patients with newly diagnosed epilepsy were included. Initially, the families decided whether they did (group 1, n=27) or did not (group 2, n=16) want to use a monitoring device. In group 1, patients were randomly assigned to using Epi-Care® (group 1A, n=14) or an audio baby monitor (group 1B, n=13). Quality of life was assessed at two points (t1, at the start of the study and t2, at 5-7months of follow-up) using the SF-12, Kindl-R, and "Familien-Belastungs-Fragebogen" (German version of the "Impact on Family Scale"). In addition, parental anxiety was measured using the State-Trait Anxiety-Inventory, and subjective quality of sleep was measured using the Pittsburgh Sleep Quality Index. Statistical analysis focused on the possible differences between groups 1 and 2 that may influence parents' decisions and the effects of the presence and types of technical monitoring over time. RESULTS: Anxiety levels were not significantly different between the groups with and without monitoring (group 1 vs. group 2). We also found no statistically significant, substantial baseline differences between the Epi-Care® and audio baby monitor groups, with at least medium effect sizes (group 1A vs. group 1B). Parents' health-related mental quality of life measured via the SF-12 increased significantly over time in all groups. By tendency, the fear of further seizures as well as the frequency of cosleeping arrangements in the monitoring group decreased during the study and approached the stable values of the control group. SIGNIFICANCE: Individual parental anxiety levels are not crucial in the decision regarding the use of a monitoring device. A monitoring system may help some families in certain aspects of daily life. During the first months following a diagnosis of epilepsy, quality of life increases independently of the use of a monitoring system.

Subclinical Alterations of Cardiac Mechanics Present Early in the Course of Pediatric Type 1 Diabetes Mellitus: A Prospective Blinded Speckle Tracking Stress Echocardiography Study.

Diabetic cardiomyopathy substantially accounts for mortality in diabetes mellitus. The pathophysiological mechanism underlying diabetes-associated nonischemic heart failure is poorly understood and clinical data on myocardial mechanics in early stages of diabetes are lacking. In this study we utilize speckle tracking echocardiography combined with physical stress testing in order to evaluate whether left ventricular (LV) myocardial performance is altered early in the course of uncomplicated type 1 diabetes mellitus (T1DM). 40 consecutive asymptomatic normotensive children and adolescents with T1DM (mean age 11.5 ± 3.1 years and mean disease duration 4.3 ± 3.5 years) and 44 age- and gender-matched healthy controls were assessed using conventional and quantitative echocardiography (strain and strain rate) during bicycle ergometer stress testing. Strikingly, T1DM patients had increased LV longitudinal (p = 0.019) and circumferential (p = 0.016) strain rate both at rest and during exercise (p = 0.021). This was more pronounced in T1DM patients with a longer disease duration (p = 0.038). T1DM patients with serum HbA1c > 9% showed impaired longitudinal (p = 0.008) and circumferential strain (p = 0.005) and a reduced E/A-ratio (p = 0.018). In conclusion, asymptomatic T1DM patients have signs of hyperdynamic LV contractility early in the course of the disease. Moreover, poor glycemic control is associated with early subclinical LV systolic and diastolic impairment.

The influence of real-time blood glucose levels on left ventricular myocardial strain and strain rate in pediatric patients with type 1 diabetes mellitus - a speckle tracking echocardiography study.

BACKGROUND: Echocardiographic myocardial performance parameters such as strain and strain rate are increasingly used to assess systolic and diastolic function in patients with diabetes mellitus and several other clinical and scientific scenarios. While long-term metabolic marks such as HbA1C are inherently assessed in diabetic patients, the actual blood glucose level at the very moment of the echocardiographic study has not yet been taken into account for the assessment of cardiac mechanics. The aim of this study was to investigate the influence of real-time blood glucose levels on left ventricular (LV) myocardial strain and strain rate in pediatric patients with type 1 diabetes mellitus (T1DM). METHODS: We performed speckle tracking echocardiography on 39 normotensive pediatric patients with uncomplicated type 1 diabetes mellitus (mean age 11.5 ± 3.5 years, 40 % female) and 44 sex- and age-matched healthy controls (mean age 11.4 ± 2.9 years, 45 % female). T1DM patients were sub-categorized according to their blood sugar levels (with a cutoff of 150 mg/dL) at the moment of the echocardiographic exam. Investigators were blinded to the participants' study group status. RESULTS: Interestingly, diabetic patients with higher blood sugar levels demonstrated significantly increased LV circumferential strain (p = 0.003) and strain rate (p = 0.005) as well as global longitudinal strain rate (p = 0.002) in comparison to T1DM patients with lower blood sugar levels or healthy controls. CONCLUSIONS: For the investigation of myocardial performance with sensitive methods such as speckle tracking echocardiography in diabetic study populations real-time blood sugar levels should be taken into account. Further studies are needed to verify these findings in large-scale patient cohorts and serial intra-individual measurements in different metabolic states.

Operative management of cryptorchidism: guidelines and reality--a 10-year observational analysis of 3587 cases.

BACKGROUND: Undescended testis (UDT) is the most common disorder in pediatric surgery and one of the most important risk factors for malignancy and subfertility. In 2009 local guidelines were modified and now recommend treatment to be completed by the age of 1. Aim of this study was to analyze age distribution at the time of orchidopexy, whether the procedure is performed according to guideline recommendations and to assess primary care pediatricians' attitude regarding their treatment approach. METHODS: We retrospectively analyzed 3587 patients with UDT regarding age at orchidopexy between 2003 and 2012 in 13 German hospitals. Furthermore, we conducted an anonymized nation-wide survey among primary care pediatricians regarding their attitude toward management of UDT. RESULTS: Before modification of the guideline 78% (n = 1245) of the boys with UDT were not operated according to guideline recommendations. After the modification that number rose to 95% (n = 1472). 42% of the orchidopexies were performed on patients aged 4 to 17 years. 46% of the primary care pediatricians were not aware of this discrepancy and 38% would only initiate operative management after the first year of life. In hospitals with pediatric surgery departments significantly more patients received orchidopexy in their first year of life (p < .001). CONCLUSION: The guideline for UDT in Germany has not yet been implemented sufficiently. Timing of orchidopexy must be optimized in order to improve long-term prognosis. Both primary care providers and parents should be educated regarding the advantages of early orchidopexy in UDT. Prospective studies are needed to elucidate the high rate of late orchidopexies.

Introducing enteral feeding induces intestinal subclinical inflammation and respective chromatin changes in preterm pigs.

AIM: To analyze how enteral food introduction affects intestinal gene regulation and chromatin structure in preterm pigs. MATERIALS & METHODS: Preterm pigs were fed parenteral nutrition plus/minus slowly increasing volumes of enteral nutrition. Intestinal gene-expression and chromatin structure were analyzed 5 days after birth. RESULTS: Enteral feeding led to differential upregulation of inflammatory and pattern recognition receptor genes, including IL8 (median: 5.8, 95% CI: 3.9-7.8 for formula; median: 2.2, 95% CI: 1.3-3.3 for colostrum) and TLR4 (median: 3.7, 95% CI: 2.6-4.8 for formula; no significant differences for colostrum) with corresponding decondensed chromatin configurations. On histology this correlated with mild mucosal lesions, particularly in formula-fed pigs. In CaCo-2 cells, histone hyperacetylation led to a marked increase in TLR4 mRNA and increased IL8 expression upon stimulation with lipopolysaccharide (median: 7.0; interquartile range: 5.63-8.85) compared with naive cells (median 4.2; interquartile range: 2.45-6.33; p = 0.03). CONCLUSION: Enteral feeding, particular with formula, induces subclinical inflammation in the premature intestine and more open chromatin structure in key inflammatory genes. This may increase the susceptibility for necrotizing enterocolitis.

Prevalence of Epileptiform Discharges in Healthy Infants.

Most studies on epileptiform discharges in young children were performed using analog electroencephalographic (EEG) recording systems and a limited numbers of electrodes that might have a lower detection rate for epileptiform discharges than modern digital recording systems. Knowing the prevalence of epileptiform discharges in healthy children is critical for a valid interpretation of findings in patients with a suspected epileptic disorder. We reviewed EEG recordings of 393 otherwise healthy children aged 12 to 60 months using digital EEG recording with respect to epileptiform discharges. We found epileptiform discharges in 3 children aged 12, 34 and 55 months resulting in a prevalence of epileptiform discharges in our cohort of 0.76% (95% confidence interval 0.0% to 1.62%). The prevalence of epileptiform discharges in children younger than 5 years is by far lower than in older children, and the digital findings are in accordance with previous data of conventional EEG.

Differential expression of mucosal trefoil factors and mucins in pediatric inflammatory bowel diseases.

In the intestinal mucosa trefoil factors (TFF) and mucins (Muc) - primarily produced by goblet cells - are thought to play a major role in providing barrier function during infection and inflammation. To investigate their role in pediatric Crohn's disease (CD) and ulcerative colitis (UC) we obtained mucosal biopsies of children with CD, UC and healthy controls and analyzed genetic expression. Levels of TFF2 mRNA were lower in inflamed mucosal samples (terminal ileum (TI) and duodenum) of children with CD, but higher in non-inflamed mucosal samples when compared to healthy controls (p < 0.05). Similarly, TFF2 levels in the TI were significantly lower in inflamed UC tissue. Adjustment for goblet cell density revealed slightly less marked, yet significantly different gene expression in IBD and controls. Furthermore, TI expression of TFF2 and Muc2 was inversely correlated with interleukin-8 expression in CD (p = 0.027). In Summary, our data demonstrate significant changes in Muc and TFF mRNA expression in pediatric patients with IBD suggesting a role in mucosal healing. Further studies are needed to elucidate a potential use as biomarkers for disease progression.

DNA methylation analysis in the intestinal epithelium-effect of cell separation on gene expression and methylation profile.

BACKGROUND: Epigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore desirable for all epigenetic studies. However, to date little information is available on whether separation protocols might influence epigenetic and/or gene expression signatures and hence might be less beneficial. We investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (IECs) from 6 healthy individuals. MATERIALS AND METHODS: Epithelial cells were isolated from small bowel (i.e. terminal ileum) biopsies using EDTA/DTT and enzymatic release followed by magnetic bead sorting via EPCAM labeled microbeads. Effects on gene/mRNA expression were analyzed using a real time PCR based expression array. DNA methylation was assessed by pyrosequencing of bisulfite converted DNA and methylated DNA immunoprecipitation (MeDIP). RESULTS: While cell purity was >95% using both cell separation approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable and only revealed subtle differences. Comparison of DNA methylation of the epithelial cell marker EPCAM in unseparated whole biopsy samples with separated epithelium (i.e. EPCAM positive and negative fraction) demonstrated significant differences in DNA methylation between all three tissue fractions indicating cell type specific methylation patterns can be masked in unseparated tissue samples. CONCLUSIONS: Taken together, our data highlight the importance of considering the potential effect of cell separation on gene expression as well as DNA methylation signatures. The decision to separate tissue samples will therefore depend on study design and specific separation protocols.