Proliferation and differentiation of intestinal epithelial cells (IECs) occur in part through precise regulation of key transcription factors, such as SOX9. MicroRNAs (miRNAs) have emerged as prominent fine-tuners of transcription factor expression and activity. We hypothesized that miRNAs, in part through the regulation of SOX9, may mediate IEC homeostasis. Bioinformatic analyses of the SOX9 3′-UTR revealed highly conserved target sites for nine different miRNAs. Of these, only the miR-30 family members were both robustly and variably expressed across functionally distinct cell types of the murine jejunal epithelium. Inhibition of miR-30 using complementary locked nucleic acids (LNA30bcd) in both human IECs and human colorectal adenocarcinoma-derived Caco-2 cells resulted in significant up-regulation of SOX9 mRNA but, interestingly, significant down-regulation of SOX9 protein. To gain mechanistic insight into this non-intuitive finding, we performed RNA sequencing on LNA30bcd-treated human IECs and found 2440 significantly increased genes and 2651 significantly decreased genes across three time points. The up-regulated genes are highly enriched for both predicted miR-30 targets, as well as genes in the ubiquitin-proteasome pathway. Chemical suppression of the proteasome rescued the effect of LNA30bcd on SOX9 protein levels, indicating that the regulation of SOX9 protein by miR-30 is largely indirect through the proteasome pathway. Inhibition of the miR-30 family led to significantly reduced IEC proliferation and a dramatic increase in markers of enterocyte differentiation. This in-depth analysis of a complex miRNA regulatory program in intestinal epithelial cell models provides novel evidence that the miR-30 family likely plays an important role in IEC homeostasis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency of neonates. Epithelial tight junction (TJ) proteins, such as claudins, are essential for regulation and function of the intestinal barrier. Rho kinase (ROCK) affects cellular permeability and TJ regulation. We hypothesized that TJ protein changes would correlate with increased permeability in experimental NEC, and ROCK inhibitors would be protective against NEC by regulation of key claudin proteins. We tested this hypothesis using an in vivo rat pup model, an in vitro model of experimental NEC, and human intestinal samples from patients with and without NEC. Experimental NEC was induced in rats via hypoxia and bacteria-containing formula, and in Caco-2 cells by media inoculated with LPS. The expression of claudins was measured by gene and protein analysis. Experimental NEC in rat pups and Caco-2 cells had increased permeability compared to controls. Gene and protein expression of claudin 2 was increased in experimental NEC. Sub-cellular fractionation localized increased claudin 2 protein to the cytoskeleton. ROCK inhibition was associated with normalization of these alterations and decreased severity of experimental NEC. Co-immunoprecipitation of caveolin-1 with claudin 2 suggests that caveolin-1 may act as a shuttle for the internalization of claudin 2 seen in experimental NEC. In conclusion, NEC is associated with intestinal permeability and increased expression of claudin 2, increased binding of caveolin-1 and claudin 2, and increased trafficking of claudin 2 to the cytoskeleton.

No abstract

No abstract

Introduction The burden of critical illness in low- and middle-income countries (LMICs) is high; however, there is a paucity of data describing pediatric critical care outcomes in this setting. Methods We performed a prospective observational study of the pediatric (≤18 years) intensive care population in Malawi, from August 2016 to May 2018. Data collected include patient demographics and clinical data, admission criteria and outcome. A multivariate Poisson regression was performed to determine risk factors for mortality. Results Over the study period, 499 patients were admitted to the intensive care unit (ICU) and 105 (21.0%) were children. The average age was 10.6 ± 5.4 years. Primary indications for ICU admission were sepsis (n = 30, 30.3%) and traumatic brain injury (TBI, n = 23, 23.2%). Of those who died, sepsis (n = 18, 32.7%), acute respiratory failure (n = 11, 20.0%) and TBI (n = 11, 20.0%) were the primary admission diagnoses. Overall, ICU mortality was 54.3% (n = 57). Multivariate regression for increased ICU mortality revealed: age ≤5 years [risk ratio (RR) 1.96, 95% CI 1.10–2.26, p < 0.001], hemoglobin < 10 g/dl (RR 1.58, 95% CI 1.08—2.01, p = 0.01) and shock requiring epinephrine support (RR 2.76, 95% CI 1.80–4.23, p < 0.001). Conclusions Pediatric ICU mortality is high. Predictors of mortality were age ≤5 years, anemia at ICU admission and the need for epinephrine support. Training of pediatric intensive care specialists and increased blood product availability may attenuate the high mortality for critically ill children in Malawi.