Selective hypermethylation is evident in small intestine samples from infants with necrotizing enterocolitis

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most common and lethal gastrointestinal disease affecting preterm infants. NEC develops suddenly and is characterized by gut barrier destruction, an inflammatory response, intestinal necrosis and multi-system organ failure. There is currently no method for early NEC detection, and the pathogenesis of NEC remains unclear. DESIGN: To further understand the molecular mechanisms that support NEC, we used solution phase hybridization and next-generation DNA sequencing of bisulfite converted DNA to perform targeted genome-wide analysis of DNA methylation at high read depth. RESULTS: We found that ileal samples from surgical NEC infants (n = 5) exist in a broadly hypermethylated state relative to their non-NEC counterparts (n = 9). These trends were not uniform, with hypermethylation being most consistently observed outside CpG islands and promoters. We further identified several biologically interesting gene promoters that displayed differential methylation in NEC and a number of biological pathways that appear dysregulated in NEC. We also found that DNA methylation patterns identified in ileal NEC tissue were correlated with those found and published previously in stool samples from NEC-affected infants. CONCLUSION: We confirmed that surgical NEC is associated with broad DNA hypermethylation in the ileum, and this may be detectable in stool samples of affected individuals. Thus, an epigenomic liquid biopsy of stool may have significant potential as a biomarker with respect to the diagnostic/predictive detection of NEC. Our findings, along with recent similar observations in colon, suggest that epigenomic dysregulation is a significant feature of surgical NEC. These findings motivate future studies which will involve the longitudinal screening of samples obtained prior to the onset of NEC. Our long-term goal is the development of novel screening, diagnostic and phenotyping methods for NEC

Global hypermethylation of intestinal epithelial cells is a hallmark feature of neonatal surgical necrotizing enterocolitis

BACKGROUND: Necrotizing enterocolitis (NEC) remains one of the overall leading causes of death in premature infants, and the pathogenesis is unpredictable and not well characterized. The aim of our study was to determine the molecular phenotype of NEC via transcriptomic and epithelial cell-specific epigenomic analysis, with a specific focus on DNA methylation. METHODS: Using laser capture microdissection, epithelial cell-specific methylation signatures were characterized by whole-genome bisulfite sequencing of ileal and colonic samples at the time of surgery for NEC and after NEC had healed at reanastomosis (n = 40). RNA sequencing was also performed to determine the transcriptomic profile of these samples, and a comparison was made to the methylome data. RESULTS: We found that surgical NEC has a considerable impact on the epigenome by broadly increasing DNA methylation levels, although these effects are less pronounced in genomic regions associated with the regulation of gene expression. Furthermore, NEC-related DNA methylation signatures were influenced by tissue of origin, with significant differences being noted between colon and ileum. We also identified numerous transcriptional changes in NEC and clear associations between gene expression and DNA methylation. CONCLUSIONS: We have defined the intestinal epigenomic and transcriptomic signatures during surgical NEC, which will advance our understanding of disease pathogenesis and may enable the development of novel precision medicine approaches for NEC prediction, diagnosis and phenotyping

Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of the human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes intestinal enteroids grown from surgically harvested intestinal tissue from premature infants and cocultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, simulates the predominant features of NEC, including significant upregulation of proinflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance toward a personalized medicine approach to test new therapeutics for this devastating disease

Species-specific physiological response of dinoflagellates to quantified small-scale turbulence

Turbulence has been shown to alter different aspects of the physiology of some dinoflagellates. The response appears to be species-specific and dependent on the experimental design and setup used to generate small-scale turbulence. We examined the variability of the response of three dinoflagellate species to the turbulence, following the same experimental design used by Berdalet (1992) on Akashiwo sanguinea (Hirasaka) Ge. Hansen et Moestrup (=Gymnodinium nelsonii G. W. Martin). In all experiments, turbulence was generated by an orbital shaker at 100 rpm, which corresponded on bulk average, to dissipation rates (epsilon, quantified using an acoustic Doppler velocimeter) of approximate to 2 cm(2) . s(-3). Turbulence did not appreciably affect Gymnodinium sp., a small dinoflagellate. However, Alexandrium minutum Halim and Prorocentrum triestinum J. Schiller exhibited a reduced net growth rate (33% and 28%, respectively) when shaken during the exponential growth phase. Compared to the still cultures, the shaken treatments of A. minutum and P. triestinum increased the mean cell volume (up to 1.4- and 2.5-fold, respectively) and the mean DNA content (up to 1.8- and 5.3-fold, respectively). Cultures affected by turbulence recovered their normal cell properties when returned to still conditions. The swimming speed of the cells exposed to agitation was half that of the unshaken ones. Overall, the response of A. minutum and P. triestinum was similar, but with lower intensity, to that observed previously on A. sanguinea. We found no clear trends related to taxonomy or morphology

Differences in Extracellular Vesicle Protein Cargo Are Dependent on Head and Neck Squamous Cell Carcinoma Cell of Origin and Human Papillomavirus Status

To identify potential extracellular vesicle (EV) biomarkers in head and neck squamous cell carcinoma (HNSCC), we evaluated EV protein cargo and whole cell lysates (WCL) from HPV-positive and -negative HNSCC cell lines, as well as normal oral keratinocytes and HPV16-transformed cells. EVs were isolated from serum-depleted, conditioned cell culture media by polyethylene glycol (PEG) precipitation/ultracentrifugation. EV and WCL preparations were analyzed by LC-MS/MS. Candidate proteins detected at significantly higher levels in EV compared with WCL, or compared with EV from normal oral keratinocytes, were identified and confirmed by Wes Simple Western protein analysis. Our findings suggest that these proteins may be potential HNSCC EV markers as proteins that may be (1) selectively included in EV cargo for export from the cell as a strategy for metastasis, tumor cell survival, or modification of tumor microenvironment, or (2) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. This work demonstrates that our method can be used to reliably detect EV proteins from HNSCC, normal keratinocyte, and transformed cell lines. Furthermore, this work has identified HNSCC EV protein candidates for continued evaluation, specifically tenascin-C, HLA-A, E-cadherin, EGFR, EPHA2, and cytokeratin 19

Food Insecurity and T-cell Dysregulation in Women Living With Human Immunodeficiency Virus on Antiretroviral Therapy

BackgroundFood insecurity is associated with increased morbidity and mortality in people with human immunodeficiency virus (HIV) on antiretroviral therapy, but its relationship with immune dysregulation, a hallmark of HIV infection and comorbidity, is unknown.MethodsIn 241 women participating in the Women's Interagency HIV Study, peripheral blood mononuclear cells were characterized by flow cytometry to identify cell subsets, comprising surface markers of activation (%CD38+HLADR+), senescence (%CD57+CD28-), exhaustion (%PD-1+), and co-stimulation (%CD57- CD28+) on CD4+ and CD8+ T cells. Mixed-effects linear regression models were used to assess the relationships of food insecurity with immune outcomes, accounting for repeated measures at ≤3 study visits and adjusting for sociodemographic and clinical factors.ResultsAt the baseline study visit, 71% of participants identified as non-Hispanic Black, 75% were virally suppressed, and 43% experienced food insecurity. Food insecurity was associated with increased activation of CD4+ and CD8+ T cells, increased senescence of CD8+ T cells, and decreased co-stimulation of CD4+ and CD8+ T cells (all P < .05), adjusting for age, race/ethnicity, income, education, substance use, smoking, HIV viral load, and CD4 count. In stratified analyses, the association of food insecurity with CD4+ T-cell activation was more pronounced in women with uncontrolled HIV (viral load >40 copies/mL and CD4 <500 cells/mm3) but remained statistically significant in those with controlled HIV.ConclusionsFood insecurity may contribute to the persistent immune activation and senescence in women with HIV on antiretroviral therapy, independently of HIV control. Reducing food insecurity may be important for decreasing non-AIDS-related disease risk in this population

Trauma-focused psychodynamic therapy and STAIR Narrative Therapy of post-traumatic stress disorder related to childhood maltreatment: trial protocol of a multicentre randomised controlled trial assessing psychological, neurobiological and health economic outcomes (ENHANCE)

Introduction Success rates of psychotherapy in post-traumatic stress disorder related to childhood maltreatment (PTSD-CM) are limited.Methods and analysis Observer-blind multicentre randomised clinical trial (A-1) of 4-year duration comparing enhanced methods of STAIR Narrative Therapy (SNT) and of trauma-focused psychodynamic therapy (TF-PDT) each of up to 24 sessions with each other and a minimal attention waiting list in PTSD-CM. Primary outcome is severity of PTSD (Clinician-Administered PTSD Scale for DSM-5 total) assessed by masked raters. For SNT and TF-PDT, both superiority and non-inferiority will be tested. Intention-to-treat analysis (primary) and per-protocol analysis (secondary). Assessments at baseline, after 10 sessions, post-therapy/waiting period and at 6 and 12 months of follow-up. Adult patients of all sexes between 18 and 65 years with PTSD-CM will be included. Continuing stable medication is permitted. To be excluded: psychotic disorders, risk of suicide, ongoing abuse, acute substance related disorder, borderline personality disorder, dissociative identity disorder, organic mental disorder, severe medical conditions and concurrent psychotherapy. To be assessed for eligibility: n=600 patients, to be e randomly allocated to the study conditions: n=328. Data management, randomisation and monitoring will be performed by an independent European Clinical Research Infrastructure Network (ECRIN)-certified data coordinating centre for clinical trials (KKS Marburg). Report of AEs to a data monitoring and safety board. Complementing study A-1, four inter-related add-on projects, including subsamples of the treatment study A-1, will examine (1) treatment integrity (adherence and competence) and moderators and mediators of outcome (B-1); (2) biological parameters (B-2, eg, DNA damage, reactive oxygen species and telomere shortening); (3) structural and functional neural changes by neuroimaging (B-3) and (4) cost-effectiveness of the treatments (B-4, costs and utilities).Ethics and dissemination Approval by the institutional review board of the University of Giessen (AZ 168/19). Following the Consolidated Standards of Reporting Trials statement for non-pharmacological trials, results will be reported in peer-reviewed scientific journals and disseminated to patient organisations and media.Trial registration number DRKS 00021142