The Effect of Glutamine Supplementation on Microbial Invasion in Surgical Infants Requiring Parenteral Nutrition: Results of a Randomized Controlled Trial.

BACKGROUND: To determine whether parenteral plus enteral glutamine supplementation influences microbial invasion in surgical infants who require parenteral nutrition (PN). METHODS: An prospective double-blind randomized controlled trial studying surgical infants receiving PN for at least 5 days for congenital or acquired intestinal anomalies (2009-2012) was used. Infants were randomized to receive either glutamine supplementation (parenteral plus enteral; total 400 mg/kg/d) or isonitrogenous control. The primary end point was microbial invasion evaluated after 5 days of supplementation and defined as: (i) positive conventional blood culture, (ii) evidence of microbial DNA in blood (polymerase chain reaction), (iii) plasma endotoxin level ≥50 pg/mL, or (iv) plasma level of lipopolysaccharide binding protein ≥50 ng/mL. Data are given as median (range) and compared by logistic regression. RESULTS: Sixty infants were randomized and reached the primary end point. Twenty-five patients had intestinal obstruction, 19 had abdominal wall defects, and 13 had necrotizing enterocolitis. Thirty-six infants showed evidence of microbial invasion during the study, and 17 of these were not detected by conventional blood culture. There was no significant difference between the 2 groups in the primary outcome; evidence of microbial invasion after 5 days was found in 9/31 (control group) and 8/29 (glutamine group) (odds ratio 0.83 [0.24-2.86; P = 0.77]). CONCLUSION: More than half of surgical infants requiring PN showed evidence of microbial invasion. Approximately half of this was not detectable by conventional blood cultures. Parenteral plus enteral glutamine supplementation had no effect on incidence of microbial invasion.

Non-vascular interventional radiology in the paediatric alimentary tract.

Paediatric interventional radiology is an evolving speciality which is able to offer numerous minimally invasive treatments for gastrointestinal tract pathologies. Here we describe interventions performed by paediatric interventional radiologists on the alimentary tract from the mouth to the rectum. The interventions include sclerotherapy, stricture management by dilation, stenting and adjunctive therapies such as Mitomycin C administration and enteral access for feeding, motility assessment and administration of enemas.

5 years after an ACE: what happens then?

PURPOSE: Antegrade continence enema (ACE) revolutionised the lives of children with chronic constipation and soiling. Parents often ask how long the ACE will be required. We looked at our patients 5 years after ACE formation to answer the question. METHODS: We reviewed clinical notes of all patients undergoing ACE procedure during January 1990 to December 2010. Only patients with >5 years follow-up were included. Data are given as median (range). RESULTS: 133 patients were included with >5 years of follow-up. Primary pathology was anorectal anomaly (ARA) 64 (48%); spinal dysraphism (SD) 40 (30%); functional constipation (FC) 14 (10%); Hirschsprung's Disease (HD) 10 (8%) and others 5 (4%). Median follow-up was 7 years (5-17 years). Overall 74% still use their ACE; whilst 26% no longer access their stoma, of whom 47% recovered normal colonic function. 50% of HD patient recover colonic function. FC has the highest failure rate at 21%. CONCLUSIONS: Overall 86% achieved excellent clinical outcome with 74% of patient still using their ACE at 5 years. HD has the highest recovery rate of 50%. FC has a more unreliable clinical outcome with 21% recovered colonic function and 21% failed. Outcome varied dependent on the background diagnosis.

Effect of sodium deficiency on growth of surgical infants: a retrospective observational study.

BACKGROUND/AIM: Sodium is thought to be critical to growth. Infants who have an ileostomy may suffer from growth faltering, as sodium losses from stomas may be excessive. Urinary sodium measurements may indicate which patients could benefit from sodium supplementation; however, there is no consensus on what level of urinary sodium should be the cutoff for intervention. Our aim was to determine whether there is a relationship between urinary sodium and growth in infants undergoing ileostomy, colostomy and cystostomy. METHODS: Following audit approval, a retrospective observational study of patient notes and chemical pathology data was carried out. All infants <1 year of age that had an ileostomy, colostomy, or cystostomy procedure between February 1997 and January 2014 were included. Patients' weights, urinary and serum sodium and potassium levels and clinical variables were recorded until discharge. Weights were converted to Z-scores for analysis. RESULTS: Forty patients were identified whose notes were available for review and who had at least three urinary sodium measurements. During their hospital stay, 11 (28%) maintained weight within normal limits (Z-scores -2 to +2, 15 (38%) were moderately malnourished (-3 to -2) and 14 (35%) severely malnourished (<-3). Thirty patients had at least one urinary sodium <10 mmol/litre, six patients had their lowest recorded urinary sodium between 10 and 30 mmol/litre and only four patients had all their urinary sodium measurements >30 mmol litre. Electrolyte data were not normally distributed so that correlations between electrolytes and growth were tested using the non-parametric Spearman rank correlation coefficient. Urinary sodium levels positively correlated with growth (r = 0.3071, p < 0.0001), as did serum sodium levels (r = 0.2620, p = 0.0059) whereas there was no relationship between urine or serum potassium and growth. CONCLUSIONS: Poor growth is frequent in this group of patients and appears to be linked with sodium levels. Further work is necessary to draw up guidelines for appropriate sodium supplementation.

Isolation of esophageal stem cells with potential for therapy.

PURPOSE: Long-gap esophageal atresia represents a significant challenge for pediatric surgeons and current surgical approaches are associated with significant morbidity. A tissue-engineered esophagus, comprising cells seeded onto a scaffold, represents a therapeutic alternative. In this study, we aimed to determine the optimal techniques for isolation and culture of mouse esophageal epithelial cells and to isolate CD34-positive esophageal epithelial stem cells from cadaveric mouse specimens. METHODS: Primary epithelial cells were isolated from mouse esophagi by enzymatic dissociation from the mucosal layer (Dispase, Trypsin/EDTA) using three different protocols. In protocol A, isolated mucosa was minced and incubated with trypsin once. In protocol B, intact mucosal sheets underwent two trypsin incubations yielding a single-cell suspension. In protocol C, intact mucosa explants were plated epithelial side down. Epithelial cells were cultured on collagen-coated wells. RESULTS: Initial findings showed that Protocol B gave the best results in terms of yield, viability, and least contamination with different cell types and microbes. Esophageal epithelial cells isolated using Protocol B were stained for CD34 and sorted using fluorescence-activated cell sorting (FACS). Of the total cells sorted, 8.3% (2-11.3) [%median (range)] were CD34 positive. CONCLUSIONS: Our results demonstrate that mouse esophageal epithelial cells can be successfully isolated from fresh mouse esophagi using two consecutive trypsin incubations of intact mucosal sheets. Furthermore, the cells obtained using this method were successfully stained for CD34, a putative esophageal epithelial stem cell marker. Further research into the factors necessary for the successful proliferation of CD34 positive stem cell lines is needed to progress toward clinical application.

Currarino's syndrome in twins presenting as neonatal intestinal obstruction--identical presentation in non-identical twins.

We report a case of non-identical twins who presented with identical neonatal intestinal obstruction with features of anorectal stenosis, presacral mass and sacral anomaly consistent with Currarino's syndrome or triad. Plain sacral radiograph, contrast enema and MRI were diagnostic. Initial management involved a defunctioning colostomy followed by a posterior sagittal anorectoplasty with excision of the teratoma ± anterior sacral meningocele and finally closure of colostomy in a staged multidisciplinary approach. The twins' father is also affected with features of Currarino's syndrome but was diagnosed during family screening. Currarino's syndrome presenting with identical neonatal low intestinal obstruction in a non-identical set of twins is rare and interesting. Antenatal diagnosis of Currarino's syndrome is difficult and may prove to be a challenge even in the postnatal period. Sacral spine radiograph, contrast enema and MRI are diagnostic. Management requires high index of suspicion, low threshold for MRI and multidisciplinary staged approach.

Human mid-trimester amniotic fluid stem cells cultured under embryonic stem cell conditions with valproic acid acquire pluripotent characteristics.

Human mid-trimester amniotic fluid stem cells (AFSC) have promising applications in regenerative medicine, being broadly multipotent with an intermediate phenotype between embryonic (ES) and mesenchymal stem cells (MSC). Despite this propluripotent phenotype, AFSC are usually cultured in adherence in a serum-based expansion medium, and how expansion in conditions sustaining pluripotency might affect their phenotype remains unknown. We recently showed that early AFSC from first trimester amniotic fluid, which endogenously express Sox2 and Klf4, can be reprogrammed to pluripotency without viral vectors using the histone deacetylase inhibitor valproic acid (VPA). Here, we show that mid-trimester AFSC cultured under MSC conditions contained a subset of cells endogenously expressing telomerase, CD24, OCT4, C-MYC, and SSEA4, but low/null levels of SOX2, NANOG, KLF4, SSEA3, TRA-1-60, and TRA-1-81, with cells unable to form embryoid bodies (EBs) or teratomas. In contrast, AFSC cultured under human ESC conditions were smaller in size, grew faster, formed colonies, upregulated OCT4 and C-MYC, and expressed KLF4 and SOX2, but not NANOG, SSEA3, TRA-1-60, and TRA-1-81. Supplementation with VPA for 5 days further upregulated OCT4, KLF4, and SOX2, and induced expression of NANOG, SSEA3, TRA-1-60, and TRA-1-81, with cells now able to form EBs and teratomas. We conclude that human mid-trimester AFSC, which may be isolated autologously during pregnancy without ethics restriction, can acquire pluripotent characteristics without the use of ectopic factors. Our data suggest that this medium-dependant approach to pluripotent mid-trimester AFSC reflects true reprogramming and not the selection of prepluripotent cells.

Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach.

Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.

Clot dissolution: a novel treatment of midgut volvulus.

Midgut volvulus due to malrotation may result in loss of the small bowel. Until now, after derotation of the volvulus, pediatric surgeons do not deal with the mesenteric thrombosis, which causes continuing ischemia of the intestine. On occasion, a "second look" laparotomy is performed in the hope that some improvement in blood supply to the intestine has occurred. We describe a new combined treatment to restore intestinal perfusion based on digital massage of the superior mesenteric vessels after derotation and systemic infusion of tissue-type plasminogen activator. This new therapy has been successful in 2 neonates with severe intestinal ischemia due to volvulus.