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.

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.

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.

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.

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.

Human ß-defensin 2 expression in ELBW infants with severe necrotizing enterocolitis.

BACKGROUND: The aim of this study was to analyze whether the mucosal innate immune response of extremely-low-birth-weight (ELBW) infants might play a role in the development of necrotizing enterocolitis (NEC). METHODS: Between April 2008 and December 2009 antimicrobial peptides were prospectively measured in fecal samples of ELBW infants. In cases requiring abdominal surgery, full-thickness gut biopsies were analyzed for expression of human ß-defensin 2 (hBD2), interleukin-8 (IL-8), villin, MD2, and Toll-like receptor 4 (TLR4). RESULTS: Fecal hBD1 concentrations were consistently low in all patients, whereas hBD2 concentrations were high in meconium, particularly in clinical chorioamnionitis, and then dropped, followed by a steady increase after day 14. Infants with moderate NEC showed significantly increased fecal hBD2 concentrations before clinical symptoms, in contrast to infants developing severe NEC. Analysis of intestinal resection material obtained from patients with severe NEC revealed low hBD2 mRNA and protein levels, and increased expression of the inflammatory cytokine IL-8. CONCLUSION: High hBD2 concentrations, reflecting strong intestinal immune responses, were associated with moderate courses of the disease. In severe NEC, low hBD2 expression was accompanied by low TLR4/MD2 expression, suggesting an inadequate response to luminal bacteria, possibly predisposing those infants to the development of NEC.

The nonviral episomal replicating vector pEPI-1 allows long-term inhibition of bcr-abl expression by shRNA.

The inhibition of gene expression by RNA interference harbors a high potential for application in the therapy of human diseases. However, while exogenous application of siRNAs efficiently inhibits gene expression, these effects are only transient in mammalian cells. We designed a short hairpin RNA-expression cassette to target the bcr-abl oncogene that was then introduced into the nonviral vector system pEPI-1, which replicates episomally in the absence of selection in the bcr-abl-positive cell line K562. Forty-two days after transfection the bcr-abl- but not the cytokine-dependent growth rate was found to be drastically reduced in K562 cells. Western analysis revealed a more than 90% reduction in the expression of the fusion protein bcr-abl while the expression of the bcr protein remained unaffected. In addition, we show that the level of bcr-abl mRNA was specifically reduced in these cells for more than 90%. These results demonstrate that the vector system pEPI-1 allows specific and efficient long term gene suppression by using a short hairpin RNA transcription unit.