Factors Affecting the Gut Microbiome in Pediatric Intestinal Failure.

BACKGROUND: There is little data on gut microbiome and various factors that lead to dysbiosis in pediatric intestinal failure (PIF). This study aimed to characterize gut microbiome in PIF and determine factors that may affect microbial composition in these patients. METHODS: This is a single-center, prospective cohort study of children with PIF followed at our intestinal rehabilitation program. Stool samples were collected longitudinally at regular intervals over a 1-year period. Medical records were reviewed, and demographic and clinical data were collected. Medication history including the use of acid blockers, scheduled prophylactic antibiotics, and bile acid sequestrants was obtained. Gut microbial diversity among patients was assessed and compared according to various host characteristics of interest. RESULTS: The final analysis included 74 specimens from 12 subjects. Scheduled prophylactic antibiotics, presence of central line associated bloodstream infection (CLABSI) at the time of specimen collection, use of acid blockers, and ≥50% calories delivered via parenteral nutrition (PN) was associated with reduced alpha diversity, whereas increasing age was associated with improved alpha diversity at various microbial levels ( P value <0.05). Beta diversity differed with age, presence of CLABSI, use of scheduled antibiotics, acid blockers, percent calories via PN, and presence of oral feeds at various microbial levels ( P value <0.05). Single taxon analysis identified several taxa at several microbial levels, which were significantly associated with various host characteristics. CONCLUSION: Gut microbial diversity in PIF subjects is influenced by various factors involved in the rehabilitation process including medications, percent calories received parenterally, CLABSI events, the degree of oral feeding, and age. Additional investigation performed across multiple centers is needed to further understand the impact of these findings on important clinical outcomes in PIF.

Initial Laparotomy Versus Peritoneal Drainage in Extremely Low Birthweight Infants With Surgical Necrotizing Enterocolitis or Isolated Intestinal Perforation: A Multicenter Randomized Clinical Trial.

OBJECTIVE: The aim of this study was to determine which initial surgical treatment results in the lowest rate of death or neurodevelopmental impairment (NDI) in premature infants with necrotizing enterocolitis (NEC) or isolated intestinal perforation (IP). SUMMARY BACKGROUND DATA: The impact of initial laparotomy versus peritoneal drainage for NEC or IP on the rate of death or NDI in extremely low birth weight infants is unknown. METHODS: We conducted the largest feasible randomized trial in 20 US centers, comparing initial laparotomy versus peritoneal drainage. The primary outcome was a composite of death or NDI at 18 to 22 months corrected age, analyzed using prespecified frequentist and Bayesian approaches. RESULTS: Of 992 eligible infants, 310 were randomized and 96% had primary outcome assessed. Death or NDI occurred in 69% of infants in the laparotomy group versus 70% with drainage [adjusted relative risk (aRR) 1.0; 95% confidence interval (CI): 0.87-1.14]. A preplanned analysis identified an interaction between preoperative diagnosis and treatment group (P = 0.03). With a preoperative diagnosis of NEC, death or NDI occurred in 69% after laparotomy versus 85% with drainage (aRR 0.81; 95% CI: 0.64-1.04). The Bayesian posterior probability that laparotomy was beneficial (risk difference <0) for a preoperative diagnosis of NEC was 97%. For preoperative diagnosis of IP, death or NDI occurred in 69% after laparotomy versus 63% with drainage (aRR, 1.11; 95% CI: 0.95-1.31); Bayesian probability of benefit with laparotomy = 18%. CONCLUSIONS: There was no overall difference in death or NDI rates at 18 to 22 months corrected age between initial laparotomy versus drainage. However, the preoperative diagnosis of NEC or IP modified the impact of initial treatment.

Risk models for predicting the health-related quality of life of caregivers of youth with gastrointestinal concerns.

PURPOSE: To determine the usefulness of cumulative and additive risk models in predicting the healthy-related quality of life (HRQOL) of caregivers of youth with chronic gastrointestinal conditions. METHODS: 203 caregivers (82.8% mothers; 77.3% white) of youth (M = 11.27 years; 44.3% female; 78.8% White) completed self-report questionnaires focused on potential environmental, child health, and family risk factors that could impact caregiver HRQOL. Cumulative risk models, evaluating overall combined risk level, as well as an additive risk model, exploring individual risk variables, were evaluated. RESULTS: Higher levels of cumulative risk were associated with poorer caregiver HRQOL after controlling for child and caregiver sex. A linear cumulative risk model was a better fit than a quadratic cumulative risk model for predicting caregiver HRQOL, while an additive model identified child HRQOL, child pain interference and family functioning as the most individually impactful risk variables. CONCLUSION: This study illustrates the usefulness of both additive and cumulative risk approaches in identifying caregivers at risk for poor HRQOL. Provision of appropriate referrals and interventions based on the caregiver's risk factors can help protect caregiver quality of life and, in turn, benefit the care children with chronic conditions receive at home.

A Heterozygous RAB27A Mutation Associated with Delayed Cytolytic Granule Polarization and Hemophagocytic Lymphohistiocytosis.

Frequently fatal, primary hemophagocytic lymphohistiocytosis (HLH) occurs in infancy resulting from homozygous mutations in NK and CD8 T cell cytolytic pathway genes. Secondary HLH presents after infancy and may be associated with heterozygous mutations in HLH genes. We report two unrelated teenagers with HLH and an identical heterozygous RAB27A mutation (c.259G→C). We explore the contribution of this Rab27A missense (p.A87P) mutation on NK cell cytolytic function by cloning it into a lentiviral expression vector prior to introduction into the human NK-92 cell line. NK cell degranulation (CD107a expression), target cell conjugation, and K562 target cell lysis was compared between mutant- and wild-type-transduced NK-92 cells. Polarization of granzyme B to the immunologic synapse and interaction of mutant Rab27A (p.A87P) with Munc13-4 were explored by confocal microscopy and proximity ligation assay, respectively. Overexpression of the RAB27A mutation had no effect on cell conjugate formation between the NK and target cells but decreased NK cell cytolytic activity and degranulation. Moreover, the mutant Rab27A protein decreased binding to Munc13-4 and delayed granzyme B polarization toward the immunologic synapse. This heterozygous RAB27A mutation blurs the genetic distinction between primary and secondary HLH by contributing to HLH via a partial dominant-negative effect.

Evidence of Disturbed Sleep in Children With Eosinophilic Esophagitis and Persistent Epigastric Pain.

Objective: To determine whether the experience of persistent epigastric pain is associated with sleep disturbances in children with eosinophilic esophagitis (EoE). We hypothesized that children with EoE and persistent epigastric pain would (1) self-report greater current and worst pain severity, and (2) experience more disturbed sleep on objective and subjective measures in comparison with children with EoE but no persistent pain and healthy children. Methods: Fifty children with EoE were recruited for this cross-sectional study, of which 24 (48%) reported experiencing persistent epigastric pain. The remaining 26 (52%) children with EoE denied experiencing persistent pain. An additional 25 healthy children without EoE or persistent pain were included. All children provided severity ratings of current pain and worst pain experienced over the past week. Children then completed 12 consecutive nights of ambulatory sleep monitoring via actigraphy in the home. Caregivers provided information regarding their child's sleep patterns and internalizing symptoms. Results: Children with EoE and persistent pain reported significantly greater severity of current pain (p < .001) and worst pain over the past week (p < .001) compared with EoE without persistent pain and healthy children. Compared with the other groups, children with EoE and persistent pain also demonstrated greater actigraphic sleep disturbances, lower sleep efficiency (p = .004) and greater wake after sleep onset (p = .034). Conclusions: This study provides novel evidence that a significant proportion of children with EoE experience persistent symptoms of epigastric pain. Persistent pain was associated with significant sleep disturbances in children with EoE.

Disease-Related Predictors of Health-Related Quality of Life in Youth With Eosinophilic Esophagitis.

Objective: To evaluate relations between health-related quality of life (HRQoL) and clinical symptom presentation in youth with eosinophilic esophagitis (EoE). We hypothesized that presence of dysphagia, reflux, nausea/vomiting, and epigastric pain would be related to poorer HRQoL. In predictive models, it was hypothesized that dysphagia, reflux, nausea/vomiting, and epigastric pain would each significantly and uniquely predict poorer HRQoL. Methods: This cross-sectional, two-study design included 91 dyads comprised children with EoE and their respective caregivers across two tertiary children's hospitals, Site 1 in the Midwest (N = 47) and Site 2 in the Deep South (N = 44). Youth and their caregivers both completed questionnaires addressing HRQoL and EoE symptoms during clinic visits. Results: Per youth self-report, epigastric pain was found to be a significant predictor of poor physical and psychosocial HRQoL. Per caregiver-proxy reports, epigastric pain was found to be a significant predictor of poor physical HRQoL. Conclusions: The clinical symptoms of EoE, specifically epigastric pain, were found to be predictive of the youth's HRQoL. Targeted interventions to help youth with EoE better manage their specific symptom experiences could ultimately improve HRQoL.

Outcomes from a 12-Week, Open-Label, Multicenter Clinical Trial of Teduglutide in Pediatric Short Bowel Syndrome.

OBJECTIVE: To determine safety and pharmacodynamics/efficacy of teduglutide in children with intestinal failure associated with short bowel syndrome (SBS-IF). STUDY DESIGN: This 12-week, open-label study enrolled patients aged 1-17 years with SBS-IF who required parenteral nutrition (PN) and showed minimal or no advance in enteral nutrition (EN) feeds. Patients enrolled sequentially into 3 teduglutide cohorts (0.0125 mg/kg/d [n = 8], 0.025 mg/kg/d [n = 14], 0.05 mg/kg/d [n = 15]) or received standard of care (SOC, n = 5). Descriptive summary statistics were used. RESULTS: All patients experienced ≥1 treatment-emergent adverse event; most were mild or moderate. No serious teduglutide-related treatment-emergent adverse events occurred. Between baseline and week 12, prescribed PN volume and calories (kcal/kg/d) changed by a median of -41% and -45%, respectively, with 0.025 mg/kg/d teduglutide and by -25% and -52% with 0.05 mg/kg/d teduglutide. In contrast, PN volume and calories changed by 0% and -6%, respectively, with 0.0125 mg/kg/d teduglutide and by 0% and -1% with SOC. Per patient diary data, EN volume increased by a median of 22%, 32%, and 40% in the 0.0125, 0.025, and 0.05 mg/kg/d cohorts, respectively, and by 11% with SOC. Four patients achieved independence from PN, 3 in the 0.05 mg/kg/d cohort and 1 in the 0.025 mg/kg/d cohort. Study limitations included its short-term, open-label design, and small sample size. CONCLUSIONS: Teduglutide was well tolerated in pediatric patients with SBS-IF. Teduglutide 0.025 or 0.05 mg/kg/d was associated with trends toward reductions in PN requirements and advancements in EN feeding in children with SBS-IF. TRIAL REGISTRATION: ClinicalTrials.gov:NCT01952080; EudraCT: 2013-004588-30.

Third transmembrane domain of the adrenocorticotropic receptor is critical for ligand selectivity and potency.

The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, ß-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe(7) with D-Phe or D-naphthylalanine (D-Nal(2')) in ACTH(1-24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe(7) with D-Nal(2') in ACTH(1-24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe(7) with D-Phe(7) in ACTH(1-17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in D-Phe ACTH binding affinity and potency. Our results suggest that Phe(7) in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.

Quintuplets: 5 Cases of NEC. Are There Other Risk Factors?

OBJECTIVE: To review a case of quintuplets with all babies developing necrotizing enterocolitis. METHODS: A retrospective study of preterm quintuplets all developing necrotizing enterocolitis. Clinical outcomes were reviewed. RESULTS: Quintuplets were born at 24 weeks gestation. Each baby developed NEC and was treated. One baby died. Currently the remaining 4 infants are on full enteral nutrition. CONCLUSION: Further studies are needed to better understand this emerging population of multiple birth pregnancy and the frequency of NEC development.

Topical Review: Eosinophilic Esophagitis in Children: Implications for Health-Related Quality of Life and Potential Avenues for Future Research.

OBJECTIVE: To characterize the health-related quality of life (HRQoL) of children with eosinophilic esophagitis (EoE) as well as generate novel hypotheses for future research in this pediatric population. METHOD: A literature review was completed using PubMed and the keywords below. RESULTS: Research has shown that for children with EoE and their parents, symptom experiences and recommended treatments can have a negative impact on HRQoL. However, studies have yet to adequately address mechanisms that may help explain why this is. Areas of interest include sleep quality and disturbances, the experience of pain, and the presence of internalizing symptoms, all of which have the potential to uniquely and synergistically impact HRQoL. CONCLUSION: With greater understanding of the associations among sleep, pain, internalizing symptoms, and HRQoL in children with EoE may come enhanced therapies that substantially improve the quality of their health care.

Structural insight into the MC4R conformational changes via different agonist-mediated receptor signaling.

The melanocortin-4 receptor (MC4R) plays a key role in the regulation of food intake and body weight. Previous studies indicate that α-melanocyte stimulating hormone (α-MSH) binds to MC4R and activates three signal pathways (cAMP, calcium, and mitogen-activated protein kinase pathways), whereas MC4R synthetic agonist THIQ can activate only the cAMP pathway. The molecular basis of the MC4R responsible for different ligand-mediated signaling is unknown. We hypothesize that different MC4R agonists can stabilize different MC4R conformations and result in ligand-mediated signal transduction. In this study, we examined the effect of the MC4R conformational change in cAMP signaling pathways mediated by different agonists by cross-linking two transmembrane helices (TM3 and TM6). We generated and tested 11 single and 8 double mutations that are located at the end of TM3 and beginning of TM6 in MC4R. Our results indicate that (1) single or double mutations of the MC4R did not significantly alter cAMP production induced by NDP-MSH compared to that of wild-type MC4R except single mutation 243H (the mutation 243H significantly decreased cAMP production mediated by NDP-MSH or THIQ due to a low level of receptor expression at the cell surface), (2) the mutation 247H significantly decreased THIQ-mediated cAMP production but not NDP-MSH, and (3) the receptor cAMP signaling pathway activation by THIQ is blocked in the presence of Zn(2+) with the double mutation I150/242H but activation by NDP-MSH is not, suggesting that the activated conformation of MC4R mediated by NDP-MSH and THIQ is different. This study provides insight into the molecular basis of MC4R responsible for receptor signaling mediated by different agonists.

Cytokines associated with necrotizing enterocolitis in extremely-low-birth-weight infants.

BACKGROUND: The goal was to identify cytokines associated with necrotizing enterocolitis (NEC). Based on our earlier reports of decreased tissue expression of transforming growth factor (TGF)-ß, we hypothesized that infants with NEC also have low blood TGF-ß levels. We further hypothesized that because fetal inflammation increases the risk of NEC, infants who develop NEC have elevated blood cytokine levels in early neonatal period. METHODS: Data on 104 extremely-low-birth-weight infants with NEC and 893 without NEC from 17 centers were analyzed. Clinical information was correlated with blood cytokine levels on postnatal day 1 (D1), D3, D7, D14, and D21. RESULTS: Male gender, non-Caucasian/non-African American ethnicity, sepsis, lower blood TGF-ß and interleukin (IL)-2 levels, and higher IL-8 levels were associated with NEC. The NEC group had lower TGF-ß levels than controls since D1. The diagnosis of NEC was associated with elevated IL-1ß, IL-6, IL-8, IL-10, monocyte chemoattractant protein-1/CC-motif ligand-2, macrophage inflammatory protein-1ß/CC-motif ligand-3, and C-reactive protein. CONCLUSION: Clinical characteristics, such as gender and ethnicity, and low blood TGF-ß levels are associated with higher risk of NEC. Infants who developed NEC did not start with high blood levels of inflammatory cytokines, but these rose mainly after the onset of NEC.

Molecular characterization of human melanocortin-5 receptor ligand-receptor interaction.

The melanocortin-5 receptor (MC5R) is a subtype receptor of the melanocortin receptor (MCR) family, which is expressed centrally, as well as in a variety of peripheral tissues. MC5R has been implicated in many different physiological fields such as lipid metabolism and exocrine function. However, the specific molecular determinants of MC5R responsible for ligand binding and receptor signaling are currently unknown. The aim of this study is to determine the molecular basis of human MC5R (hMC5R) responsible for ligand binding and receptor signaling. Twenty-four single mutations of hMC5R were created and tested. Our results indicate that (1) substituting charged amino acid residue E92 in transmembrane domain 2 (TM2), aspartic acid 115 (D115) and D119 in TM3, and histidine (H) 257 in TM6 with alanine dramatically reduced NDP-α-MSH binding affinity and receptor signaling and (2) substituting aromatic amino acids phenylalanine (F) 195 in TM5, F254 in TM6, and H276 in TM7 with alanine also significantly decreased NDP-α-MSH binding and receptor activity. Combining pharmacological results and computer modeling, our results suggest that D115 and D119 in TM3, F195 in TM5, and F254 in TM6 may form a binding pocket for NDP-α-MSH binding. Our results provide important information about the structural aspects of hMC5R responsible for ligand binding and receptor signaling.

Baclofen for the treatment of pediatric GERD.

BACKGROUND AND AIM: Baclofen, a γ-aminobutyric acid receptor agonist, has been shown to reduce the episodes of gastroesophageal reflux (GER) by reducing the incidence of transient lower esophageal sphincter relaxations. Although baclofen has been shown to reduce reflux symptoms in adults, data in pediatric patients are limited. The aim of the study was to evaluate the efficacy of baclofen in children with refractory GER. METHODS: Medical charts of patients 1 to 18 years of age treated with baclofen for persistent GER symptoms were reviewed retrospectively. Short-term (at first clinic visit) and long-term (12 months) clinical responses were assessed. RESULTS: A total of 53 patients were included in the final analysis. The mean duration of illness was 1.5 years and the mean age was 6.1 years. All of the patients were taking either once- (53%) or twice-daily (47%) doses of proton pump inhibitors (PPIs) at the time of initiation of baclofen. Thirty-five (66%) patients experienced a significant reduction in clinical symptoms at their first follow-up visit. In the remaining 18 patients, however, baclofen was stopped because of either no response (n = 15) or adverse events (n = 3). A total of 27 patients continued treatment and were assessed for long-term response. Of those, 22 (81%) had a sustained response to baclofen at 12 months, whereas 5 (19%) lost response. We recognized no clinical characteristic differences between those with and without a response to baclofen at either time point. CONCLUSIONS: Baclofen can be used as supplemental therapy to proton pump inhibitors in children with refractory GER; however, prospective trials are needed to further validate our results and assess safety.

TGF-ß2 suppresses macrophage cytokine production and mucosal inflammatory responses in the developing intestine.

BACKGROUND & AIMS: Premature neonates are predisposed to necrotizing enterocolitis (NEC), an idiopathic, inflammatory bowel necrosis. We investigated whether NEC occurs in the preterm intestine due to incomplete noninflammatory differentiation of intestinal macrophages, which increases the risk of a severe mucosal inflammatory response to bacterial products. METHODS: We compared inflammatory properties of human/murine fetal, neonatal, and adult intestinal macrophages. To investigate gut-specific macrophage differentiation, we next treated monocyte-derived macrophages with conditioned media from explanted human fetal and adult intestinal tissues. Transforming growth factor-ß (TGF-ß) expression and bioactivity were measured in fetal/adult intestine and in NEC. Finally, we used wild-type and transgenic mice to investigate the effects of deficient TGF-ß signaling on NEC-like inflammatory mucosal injury. RESULTS: Intestinal macrophages in the human preterm intestine (fetus/premature neonate), but not in full-term neonates and adults, expressed inflammatory cytokines. Macrophage cytokine production was suppressed in the developing intestine by TGF-ß, particularly the TGF-ß(2) isoform. NEC was associated with decreased tissue expression of TGF-ß(2) and decreased TGF-ß bioactivity. In mice, disruption of TGF-ß signaling worsened NEC-like inflammatory mucosal injury, whereas enteral supplementation with recombinant TGF-ß(2) was protective. CONCLUSIONS: Intestinal macrophages progressively acquire a noninflammatory profile during gestational development. TGF-ß, particularly the TGF-ß(2) isoform, suppresses macrophage inflammatory responses in the developing intestine and protects against inflammatory mucosal injury. Enterally administered TGF-ß(2) protected mice from experimental NEC-like injury.

Absence of N addition facilitates B cell development, but impairs immune responses.

The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT(-/-)) and wild-type (TdT(+/+)) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT(-/-) cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP(19)CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT(-/-) bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgM(a) and congenic TdT-sufficient CB17 IgM(b) bone marrow were placed in competition. TdT(-/-) cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.

Critical role for P-glycoprotein expression in hematopoietic cells in the FVB.Mdr1a(-/-) model of colitis.

OBJECTIVE: P-glycoprotein (P-gp), the functional product of the multidrug resistance gene (MDR), is a transmembrane protein that extrudes substrates from the intracellular environment. P-gp is expressed on the apical surface of epithelial cells and on cells from the hematopoietic lineage. Human MDR polymorphisms have been associated with the increased risk of inflammatory bowel disease, and FVB/N animals deficient in mdr1a expression develop spontaneous colitis. Previous studies using adult bone marrow chimeras indicated that colitis development in this animal model was contingent on P-gp deficiency in radiation-resistant epithelial cells; however, the use of adult animals may mask the role of hematopoietic immune cells in colitis initiation, due to preexisting epithelial abnormalities. SUBJECTS AND METHODS: To assess the importance of P-gp expression in intestinal epithelial and hematopoietic-derived cells on colitis induction in FVB.mdr1a(-/-) animals, we developed a neonatal model of bone marrow reconstitution. FVB/N and FVB.mdr1a(-/-) adult and neonatal animals were lethally irradiated and reconstituted with bone marrow from FVB/N or FVB.mdr1a(-/-) donors. Animals were observed for 20 weeks. RESULTS: Adult FVB/N animals deficient in P-gp expression in hematopoietically derived immune cells developed colitis similar to adult animals deficient in P-gp expression in radiation-resistant epithelial/stromal cells. Neonatal animals deficient in P-gp expression in hematopoietically derived immune cells developed a more histologically significant colitis than those deficient in P-gp expression in epithelial tissue. CONCLUSIONS: The use of a neonatal model of bone marrow reconstitution has revealed a critical role for P-gp expression in hematopoietically derived immune cells in colitis development in the FVB.mdr1a(-/-) model.