EP2 Receptor Blockade Attenuates COX-2 Upregulation During Intestinal Inflammation.

High levels of PGE2 have been implicated in the pathogenesis of intestinal inflammatory disorders such as necrotizing enterocolitis (NEC) and peritonitis. However, PGE2 has a paradoxical effect: its low levels promote intestinal homeostasis, whereas high levels may contribute to pathology. These concentration-dependent effects are mediated by four receptors, EP1-EP4. In this study, we evaluate the effect of blockade of the low affinity pro-inflammatory receptors EP1 and EP2 on expression of COX-2, the rate-limiting enzyme in PGE2 biosynthesis, and on gut barrier permeability using cultured enterocytes and three different models of intestinal injury. PGE2 upregulated COX-2 in IEC-6 enterocytes, and this response was blocked by the EP2 antagonist PF-04418948, but not by the EP1 antagonist ONO-8711 or EP4 antagonist E7046. In the neonatal rat model of NEC, EP2 antagonist and low dose of COX-2 inhibitor Celecoxib, but not EP1 antagonist, reduced NEC pathology as well as COX-2 mRNA and protein expression. In the adult mouse endotoxemia and cecal ligation/puncture models, EP2, but not EP1 genetic deficiency decreased COX-2 expression in the intestine. Our results indicate that the EP2 receptor plays a critical role in the positive feedback regulation of intestinal COX-2 by its end-product PGE2 during inflammation and may be a novel therapeutic target in the treatment of NEC.

Lactobacillus murinus HF12 colonizes neonatal gut and protects rats from necrotizing enterocolitis.

The use of lactobacilli in prevention of necrotizing enterocolitis (NEC) is hampered by insufficient knowledge about optimal species/strains and effects on intestinal bacterial populations. We therefore sought to identify lactobacilli naturally occurring in postnatal rats and examine their ability to colonize the neonatal intestine and protect from NEC. L. murinus, L. acidophilus, and L. johnsonii were found in 42, 20, and 1 out of 51 4-day old rats, respectively. Higher proportion of L. murinus in microbiota correlated with lower NEC scores. Inoculation with each of the three species during first feeding significantly augmented intestinal populations of lactobacilli four days later, indicating successful colonization. L. murinus, but not L. acidophilus or L. johnsonii, significantly protected against NEC. Thus, lactobacilli protect rats from NEC in a species- or strain-specific manner. Our results may help rationalizing probiotic therapy in NEC.

Soybean-derived recombinant human epidermal growth factor protects against experimental necrotizing enterocolitis.

BACKGROUND: Epidermal Growth Factor (EGF) reduces necrotizing enterocolitis (NEC). However, its high cost virtually prohibits clinical use. To reduce cost, soybean expressing human EGF was developed. Here we report effectiveness of soybean-derived EGF in experimental NEC. METHODS: Newborn rats were subjected to the NEC-inducing regimen of formula feeding and hypoxia. Formula was supplemented with extract from EGF-expressing or empty soybeans. NEC pathology was determined microscopically. Localization of tight junction proteins JAM-A and ZO-1 was examined by immunofluorescence and levels of mucosal COX-2 and iNOS mRNAs by real time PCR. RESULTS: Soybean extract amounts corresponding to 150µg/kg/day EGF caused considerable mortality, whereas those corresponding to 75µg/kg/day EGF were well tolerated. There was no significant difference in NEC scores between animals fed plain formula and formula supplemented with empty soybean extract. Soybean-EGF-supplemented formula at 75µg/kg/day EGF significantly decreased NEC, attenuated dissociation of JAM-A and ZO-1 proteins from tight junctions, and reduced intestinal expression of COX-2 and iNOS mRNAs. CONCLUSION: Supplementation with soybean-expressed EGF significantly decreased NEC in the rat model. Soybean-expressed EGF may provide an economical solution for EGF administration and prophylaxis of clinical NEC.

Colonization with Escherichia coli EC 25 protects neonatal rats from necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a significant cause of morbidity and mortality in premature infants; yet its pathogenesis remains poorly understood. To evaluate the role of intestinal bacteria in protection against NEC, we assessed the ability of naturally occurring intestinal colonizer E. coli EC25 to influence composition of intestinal microbiota and NEC pathology in the neonatal rat model. Experimental NEC was induced in neonatal rats by formula feeding/hypoxia, and graded histologically. Bacterial populations were characterized by plating on blood agar, scoring colony classes, and identifying each class by sequencing 16S rDNA. Binding of bacteria to, and induction of apoptosis in IEC-6 enterocytes were examined by plating on blood agar and fluorescent staining for fragmented DNA. E. coli EC 25, which was originally isolated from healthy rats, efficiently colonized the intestine and protected from NEC following introduction to newborn rats with formula at 106 or 108 cfu. Protection did not depend significantly on EC25 inoculum size or load in the intestine, but positively correlated with the fraction of EC25 in the microbiome. Introduction of EC25 did not prevent colonization with other bacteria and did not significantly alter bacterial diversity. EC25 neither induced cultured enterocyte apoptosis, nor protected from apoptosis induced by an enteropathogenic strain of Cronobacter muytjensii. Our results show that E. coli EC25 is a commensal strain that efficiently colonizes the neonatal intestine and protects from NEC.

Limiting chest computed tomography in the evaluation of pediatric thoracic trauma.

BACKGROUND: Computed tomography (CT) of the chest (chest CT) is overused in blunt pediatric thoracic trauma. Chest CT adds to the diagnosis of thoracic injury but rarely changes patient management. We sought to identify a subset of blunt pediatric trauma patients who would benefit from a screening chest CT based on their admission chest x-ray (CXR) findings. We hypothesize that limiting chest CT to patients with an abnormal mediastinal silhouette identifies intrathoracic vascular injuries not otherwise seen on CXR. METHODS: All blunt trauma activations that underwent an admission CXR at our Level 1 pediatric trauma center from 2005 to 2013 were retrospectively reviewed. Patients who had a chest CT were evaluated for added diagnoses and change in management after CT. RESULTS: An admission CXR was performed in 1,035 patients. One hundred thirty-nine patients had a CT, and the diagnosis of intra-thoracic injury was added in 42% of patients. Chest CT significantly increased the diagnosis of contusion or atelectasis (30.3% vs 60.4%; p < 0.05), pneumothorax (7.2% vs 18.7%; p < 0.05), and other fractures (4.3% vs 10.8%; p < 0.05) on CXR compared to chest CT. Chest CT changed the management of only 4 patients (2.9%). Two patients underwent further radiologic evaluation that was negative for injury, one had a chest tube placed for an occult pneumothorax before exploratory laparotomy, and one patient had a thoracotomy for repair of aortic injury. Chest CT for select patients with an abnormal mediastinal silhouette on CXR would have decreased CT scans by 80% yet still identified patients with an intrathoracic vascular injury. CONCLUSIONS: The use of chest CT should be limited to the identification of intrathoracic vascular injuries in the setting of an abnormal mediastinal silhouette on CXR. LEVEL OF EVIDENCE: Therapeutic study, level IV; diagnostic study, level III.

Roles of nitric oxide and intestinal microbiota in the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis remains one of the most vexing problems in the neonatal intensive care unit. Risk factors for NEC include prematurity, formula feeding, and inappropriate microbial colonization of the GI tract. The pathogenesis of NEC is believed to involve weakening of the intestinal barrier by perinatal insults, translocation of luminal bacteria across the weakened barrier, an exuberant inflammatory response, and exacerbation of the barrier damage by inflammatory factors, leading to a vicious cycle of inflammation-inflicted epithelial damage. Nitric oxide (NO), produced by inducible NO synthase (iNOS) and reactive NO oxidation intermediates play a prominent role in the intestinal barrier damage by inducing enterocyte apoptosis and inhibiting the epithelial restitution processes, namely enterocyte proliferation and migration. The factors that govern iNOS upregulation in the intestine are not well understood, which hampers efforts in developing NO/iNOS-targeted therapies. Similarly, efforts to identify bacteria or bacterial colonization patterns associated with NEC have met with limited success, because the same bacterial species can be found in NEC and in non-NEC subjects. However, microbiome studies have identified the three important characteristics of early bacterial populations of the GI tract: high diversity, low complexity, and fluidity. Whether NEC is caused by specific bacteria remains a matter of debate, but data from hospital outbreaks of NEC strongly argue in favor of the infectious nature of this disease. Studies in Cronobacter muytjensii have established that the ability to induce NEC is the property of specific strains rather than the species as a whole. Progress in our understanding of the roles of bacteria in NEC will require microbiological experiments and genome-wide analysis of virulence factors.

The non-diagnostic ultrasound in appendicitis: is a non-visualized appendix the same as a negative study?

PURPOSE: The purpose of this retrospective study was to investigate outcomes in children who underwent a non-diagnostic ultrasound (US) evaluating for appendicitis and to identify predictors of a negative diagnosis. METHODS: An IRB-approved retrospective chart review was performed on patients age 0-18, who underwent an abdominal US evaluating for acute appendicitis from 2004 through 2013. Clinical data and specified outcomes were recorded, and exams were categorized into non-diagnostic studies and further separated into studies where the appendix was non-visualized. RESULTS: Of the 1383 studies included for analysis, 876 were non-diagnostic for acute appendicitis (63.34%) with 777 specifically because the appendix was non-visualized. Seven hundred forty of the 876 non-diagnostic studies and 671 of the 777 non-visualized studies were ultimately considered true negatives, corresponding to a negative predictive value (NPV) of 84.47 and 86.36%, respectively. In patients with WBC <7.5×10(9)/L, the NPV of non-diagnostic and non-visualized studies increased to 97.12 and 98.86%, respectively. Patients with WBC <11.0×10(9)/L have similarly high NPVs of 95.59 and 96.99% (non-diagnostic and non-visualized). CONCLUSION: Based on the high NPV of a non-diagnostic US in children without leukocytosis, these patients may safely avoid further diagnostic imaging for the workup of suspected appendicitis.