Intestinal alkaline phosphatase administration in newborns is protective of gut barrier function in a neonatal necrotizing enterocolitis rat model.

BACKGROUND: Previously, we have shown that supplementation of intestinal alkaline phosphatase (IAP) decreased severity of necrotizing enterocolitis (NEC)-associated intestinal injury. We hypothesized that IAP administration is protective of intestinal epithelial barrier function in a dose-dependent manner. METHODS: Control rat pups were vaginally delivered and breast-fed. Premature rats were divided into 4 groups: formula fed with lipopolysaccharide and hypoxia (NEC) or additional daily bovine IAP 40, 4, or 0.4 U/kg (NEC + IAP 40 U, IAP 4 U, or IAP 0.4 U). RESULTS: Necrotizing enterocolitis is associated with decreased IAP protein expression and activity. Supplemental IAP increases IAP activity in intestinal homogenates and decreased NEC injury score in a dose-dependent manner. Intestinal injury as measured by fluorescein isothiocyanate-dextran flux from ileal loops showed increased permeability vs control, but supplemental IAP reversed this. Tight junction proteins claudin-1, claudin-3, occludin, and zonula occludin 1 were elevated in the NEC and IAP-treated groups with differences in expression patterns. No differences in messenger RNA levels were observed on postinjury day 3. Intestinal alkaline phosphatase administration decreases intestinal NEC injury in a dose-dependent manner. CONCLUSION: Early enteral supplemental IAP may reduce NEC-related injury and may be useful for preserving the intestinal epithelial barrier function.

Making a definitive diagnosis: successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease.

PURPOSE: We report a male child who presented at 15 months with perianal abscesses and proctitis, progressing to transmural pancolitis with colocutaneous fistulae, consistent with a Crohn disease-like illness. The age and severity of the presentation suggested an underlying immune defect; however, despite comprehensive clinical evaluation, we were unable to arrive at a definitive diagnosis, thereby restricting clinical management. METHODS: We sought to identify the causative mutation(s) through exome sequencing to provide the necessary additional information required for clinical management. RESULTS: After sequencing, we identified 16,124 variants. Subsequent analysis identified a novel, hemizygous missense mutation in the X-linked inhibitor of apoptosis gene, substituting a tyrosine for a highly conserved and functionally important cysteine. X-linked inhibitor of apoptosis was not previously associated with Crohn disease but has a central role in the proinflammatory response and bacterial sensing through the NOD signaling pathway. The mutation was confirmed by Sanger sequencing in a licensed clinical laboratory. Functional assays demonstrated an increased susceptibility to activation-induced cell death and defective responsiveness to NOD2 ligands, consistent with loss of normal X-linked inhibitor of apoptosis protein function in apoptosis and NOD2 signaling. CONCLUSIONS: Based on this medical history, genetic and functional data, the child was diagnosed as having an X-linked inhibitor of apoptosis deficiency. Based on this finding, an allogeneic hematopoietic progenitor cell transplant was performed to prevent the development of life-threatening hemophagocytic lymphohistiocytosis, in concordance with the recommended treatment for X-linked inhibitor of apoptosis deficiency. At >42 days posttransplant, the child was able to eat and drink, and there has been no recurrence of gastrointestinal disease, suggesting this mutation also drove the gastrointestinal disease. This report describes the identification of a novel cause of inflammatory bowel disease. Equally importantly, it demonstrates the power of exome sequencing to render a molecular diagnosis in an individual patient in the setting of a novel disease, after all standard diagnoses were exhausted, and illustrates how this technology can be used in a clinical setting.

Dynamic Lkb1-TORC1 signaling as a possible mechanism for regulating the endoderm-intestine transition.

The intestinal epithelium arises from undifferentiated endoderm via a developmental program known as the endoderm-intestine transition (EIT). Previously we found that the target of rapamycin complex 1 (TORC1) regulates intestinal growth and differentiation during the EIT in zebrafish. Here we address a possible role for the tumor-suppressor kinase Lkb1 in regulating TORC1 in this context. We find that TORC1 activity is transiently upregulated during the EIT in both zebrafish and mouse. Concomitantly, Lkb1 becomes transiently localized to the nucleus, suggesting that these two phenomena may be linked. Morpholino-mediated knockdown of lkb1 stimulated intestinal growth via upregulation of TORC1, and also induced precocious intestine-specific gene expression in the zebrafish gut epithelium. Knockdown of tsc2, which acts downstream of lkb1, likewise induced early expression of intestine-specific genes. These data suggest that programmed localization of Lkb1 could represent a novel mechanism for regulating the EIT during intestinal development in vertebrates.

RBM19 is essential for preimplantation development in the mouse.

BACKGROUND: RNA-binding motif protein 19 (RBM19, NCBI Accession # NP_083038) is a conserved nucleolar protein containing 6 conserved RNA recognition motifs. Its biochemical function is to process rRNA for ribosome biogenesis, and it has been shown to play a role in digestive organ development in zebrafish. Here we analyzed the role of RBM19 during mouse embryonic development by generating mice containing a mutation in the Rbm19 locus via gene-trap insertion. RESULTS: Homozygous mutant embryos failed to develop beyond the morula stage, showing defective nucleologenesis, activation of apoptosis, and upregulation of P53 target genes. A unique feature of RBM19 is its localization to the cytoplasm in morula stage-embryos, whereas most other nucleolar proteins are localized to the nucleolar precursor body (NPB). The nucleoli in the Rbm19 mutant embryos remain immature, yet they can carry out rRNA synthesis. The timing of developmental arrest occurs after expression of the inner cell mass markers OCT3/4 and NANOG, but prior to the specification of trophectoderm as reflected by CDX2 expression. CONCLUSION: The data indicate that RBM19 is essential for preimplantation development, highlighting the importance of de novo nucleologenesis during this critical developmental stage.

Rbm19 is a nucleolar protein expressed in crypt/progenitor cells of the intestinal epithelium.

Intestinal development and homeostasis rely on the coordination of proliferation and differentiation of the epithelium. To better understand this process, we are studying Rbm19, a gene expressed in the gut epithelium that is essential for intestinal morphogenesis and differentiation in the zebrafish (Development 130, 3917). Here we analyzed the expression of Rbm19 in several biological contexts that feature proliferation/differentiation cell fate decisions. In the undifferentiated embryonic gut tube, Rbm19 is expressed throughout the epithelium, but then becomes localized to the crypts of Lieberkühn of the adult intestine. Consistent with its expression in adult crypt/progenitor cells, expression is widespread in human colorectal carcinomas and dividing Caco-2 cells. Its expression in Caco-2 cells recapitulates the in vivo pattern, declining when the cells undergo confluence-induced arrest and differentiation. Rbm19 protein localizes to the nucleolus during interphase and to the perichromosomal sheath during mitosis, in accordance with the pattern described for other nucleolar proteins implicated in ribosome biogenesis. Interestingly, the loss of nucleolar rbm19, nucleolin/C23, and nucleophosmin/B23 in confluent Caco-2 cells did not signify loss of nucleoli as detected by electron microscopy. Taken together, these data point to the nucleolus as a possible locus for regulating the proliferation/differentiation cell fate decision in the intestinal epithelium.