Analysis and expansion of the eosinophilic esophagitis transcriptome by RNA sequencing.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder of the esophagus that is compounded by genetic predisposition and hypersensitivity to environmental antigens. Using high-density oligonucleotide expression chips, a disease-specific esophageal transcript signature was identified and was shown to be largely reversible with therapy. In an effort to expand the molecular signature of EoE, we performed RNA sequencing on esophageal biopsies from healthy controls and patients with active EoE and identified a total of 1607 significantly dysregulated transcripts (1096 upregulated, 511 downregulated). When clustered by raw expression levels, an abundance of immune cell-specific transcripts are highly induced in EoE but expressed at low (or undetectable) levels in healthy controls. Moreover, 66% of the gene signature identified by RNA sequencing was previously unrecognized in the EoE transcript signature by microarray-based expression profiling and included several long non-coding RNAs (lncRNA), an emerging class of transcriptional regulators. The lncRNA BRAF-activated non-protein coding RNA (BANCR) was upregulated in EoE and induced in interleukin-13 (IL-13)-treated primary esophageal epithelial cells. Repression of BANCR significantly altered the expression of IL-13-induced proinflammatory genes. Together, these data comprise new potential biomarkers of EoE and demonstrate a novel role for lncRNAs in EoE and IL-13-associated responses.

(99m)Tc antigranulocyte monoclonal antibody imaging for the detection and assessment of inflammatory bowel disease newly diagnosed by colonoscopy in children.

OBJECTIVE: This prospective study evaluated a (99m)Tc antigranulocyte monoclonal antibody Fab' imaging agent (Sulesomab) in children with inflammatory bowel disease (IBD) newly diagnosed by colonoscopy. MATERIALS AND METHODS: Ten children (4 boys, 6 girls; mean age 14 years) with newly diagnosed Crohn's disease (n = 6) or ulcerative colitis (n = 4) were studied. Colonoscopy was performed in all of these patients. Within 24 h after colonoscopy, they underwent scintigraphy with (99m)Tc-Sulesomab. Abdominal/pelvic images were acquired at 30 min (planar) and 2-4 h (planar and SPECT) after injection of Sulesomab. Eighty bowel segments were evaluated semi-quantitatively by the investigators, using these three sets of images. The Pediatric Disease Activity (PDA) was correlated with the erythrocyte sedimentation rate (ESR), white blood cell (WBC) count, albumin, Kirschner's score, the Sulesomab bowel segment with maximum uptake, and the sum of Sulesomab score in each segment. RESULTS: The median PDA score was 26 (range 12.5-40). Three children had normal ESR and six normal WBC counts. All patients had at least one positive mucosal biopsy for IBD. While using the Kirschner's scale, the maximal severity of colonoscopy findings was graded as none (n = 2), mild (n = 4), moderate (n = 3), or severe (n = 1). Of the 59 segments evaluated with endoscopy, 35 were found to be endoscopically abnormal. The planar images identified 17 of these abnormal segments and the SPECT images 20. Nine of these ten children had abnormal bowel uptake by scintigraphy. Thus, the sensitivity of Sulesomab per patient was 90 % and per bowel segment 57 %. The correlation coefficient between the scintigraphic score for the segment with the Sulesomab maximum activity and the PDA was 0.3 (P = 0.41). CONCLUSION: In pediatric IBD assessment, planar imaging with Sulesomab did not prove very sensitive in detecting inflammation in each bowel segment. However, SPECT detected the presence of inflammation in the majority of patients. A trial comparing (99m)Tc-HmPAO-WBC with Sulesomab in a large number of patients is required.

Bacteremia after intestinal transplantation in children correlates temporally with rejection or gastrointestinal lymphoproliferative disease.

BACKGROUND: Bacteremia occurs frequently after intestinal transplantation (ITx) in children. During our initial experience with this procedure, we noted that bacteremic episodes tended to occur simultaneously with the presence of rejection and/or gastrointestinal (GI) posttransplant lymphoproliferative disease (PTLD). AIM: To document the association of bacteremia with rejection and GI PTLD in pediatric ITx recipients. METHODS: Retrospective analysis of all medical records from 62 children who underwent ITx between July 1990 and January 1998 at Children's Hospital of Pittsburgh. A bacteremic episode was defined as two positive blood cultures from different sites at the same time or from the same site at different times. Rejection and PTLD were defined using previously published criteria. RESULTS: A total of 39/62 ITx recipients had 133 blood stream infections (2.1 episodes/patient) including 121 episodes of bacteremia and 12 of fungemia. Enteric organisms were the most frequently recovered pathogens (Gram negative rods, n=76; enterococci, n=36). Enteric organisms were recovered as a single organism (n=57), with another enteric bacteria (n=23), or with coagulase negative staphylococci (CONS) (n=24). CONS were recovered as a single organism on 21 occasions. An obvious source of bacteremia was not found for 115/121 episodes. Endoscopy was performed for 107 of the 115 bacteremia episodes; an abnormal histology was identified in 74 revealing rejection (n=36), GI PTLD (n=21), or both (n=17). When endoscopy showed GI pathology, enteric organisms alone or in combination with CONS were recovered on 63/107 occasions, although CONS were recovered alone only 11 times. CONCLUSIONS: Bacteremia accompanies GI rejection and intestinal PTLD in ITx recipients. Endoscopy should be performed to inspect the allograft when bacteremia occurs without an obvious source in these patients. This is especially true for patients with bacteremia due to enteric organisms.

The spectrum of pediatric eosinophilic esophagitis beyond infancy: a clinical series of 30 children.

OBJECTIVES: Eosinophilic esophagitis, previously confused with esophageal inflammation due to gastroesophageal reflux, has recently begun to be distinguished from it. We undertook this analysis of our large series of children with the condition to clarify its spectrum: its presenting symptoms; its relation to allergy, respiratory disease, and reflux; its endoscopic and histological findings; and its diagnosis and therapy. METHODS: We analyzed the details of our clinical series of 30 children with eosinophilic esophagitis, defining it as > or =5 eosinophils per high power field in the distal esophageal epithelium. Retrospective chart review was supplemented by prospective, blinded, duplicate quantitative evaluation of histology specimens, and by telephone contact with some families to clarify subsequent course. Presentation and analysis of the series as a whole is preceded by a case illustrating a typical presentation with dysphagia and recurrent esophageal food impactions. RESULTS: Presenting symptoms encompass vomiting, pain, and dysphagia (some with impactions or strictures). Allergy, particularly food allergy, is an associated finding in most patients, and many have concomitant asthma or other chronic respiratory disease. A subtle granularity with furrows or rings is newly identified as the endoscopic herald of histological eosinophilic esophagitis. Histological characteristics include peripapillary or juxtaluminal eosinophil clustering in certain cases. Association with eosinophilic gastroenteritis occurs, but is not common. Differentiation from gastroesophageal reflux disease is approached by analyzing eosinophil density and response to therapeutic trials. Therapy encompasses dietary elimination and anti-inflammatory pharmacotherapy. CONCLUSION: Awareness of the spectrum of eosinophilic esophagitis should promote optimal diagnosis and treatment of this elusive entity, both in children and in adults.

Immunoreactive trypsinogen levels in pediatric patients with intestinal failure awaiting intestinal transplantation.

The aim of this study was to evaluate pancreatic function in total parenteral nutrition (TPN)-dependent children with permanent intestinal failure by measuring immunoreactive trypsinogen (IRT) levels. Between 1992 and 1996, 105 pediatric patients with permanent intestinal failure were referred to the Children's Hospital of Pittsburgh for small intestinal transplant evaluation. Serum samples were available from 55 of them. Ten suffered from intestinal pseudo-obstruction or microvillus inclusion disease, while 45 had short bowel syndrome (SBS). IRT levels were significantly higher (p < 0.001) in SBS patients (89.4 +/- 9.2 ng mL) compared to controls (43.4 +/- 5.6 ng/ nL) without liver, gastrointestinal, or kidney disease. IRT levels did not correlate with liver injury, length of bowel, or the cause of SBS. Five of 20 patients who underwent intestinal transplantation developed pancreatitis during a median post-operative follow up 15.4 months later. IRT levels failed to predict who would develop pancreatitis post-transplant. The data suggest that elevated plasma IRT levels are common among children with intestinal failure, but fail to identify patients at risk for pancreatitis post-transplant.

Intestinal transplantation in children with chronic intestinal pseudo-obstruction.

BACKGROUND: Children with chronic intestinal pseudo-obstruction (CIPO) often require total parenteral nutrition (TPN) which puts them at risk of liver failure and recurrent line infections. Intestinal transplantation has become a therapeutic option for TPN dependent children with intestinal failure who are failing management with TPN. AIMS: To investigate the outcome of children with CIPO referred for intestinal transplantation. METHODS: A retrospective review was carried out of records and diagnostic studies from 27 patients with CIPO referred for intestinal transplantation. RESULTS: Five children were not listed for transplantation: two because of parental decision, two because of suspicion of Munchausen syndrome by proxy, and one because he tolerated enteral nutrition. Six are still TPN dependent and awaiting transplantation. Eight children died awaiting transplantation. Eight children underwent transplantation. Three died (two months, seven months, and four years after transplant). Five children are alive with a median follow up of 2.6 years (range two months to six years). All transplanted children were able to tolerate full enteral feedings. The postoperative course was complicated by dumping syndrome, Munchausen syndrome by proxy, narcotic withdrawal, and uncovering of achalasia. Conclusion-Intestinal transplantation may be a life saving procedure in children with CIPO. Early referral and thorough pretransplant evaluation are keys to successful transplantation.

Pediatric inflammatory bowel disease: assessment with scintigraphy with 99mTc white blood cells.

PURPOSE: To evaluate the sensitivity and specificity of scintigraphy with technetium 99m white blood cells (WBC) for detection of colonic inflammation in children with and children without inflammatory bowel disease (IBD). MATERIALS AND METHODS: In 215 patients, uptake of 99mTc WBC in 3,440 bowel segments was graded. In 137 of the 215 patients, the 99mTc WBC scans were interpreted blindly and findings compared with results at colonoscopy and endoscopic biopsy. Planar, single photon emission computed tomographic, and maximum-activity-projection images were reviewed together. In 78 children without recent endoscopic biopsy results, 99mTc WBC scan findings were compared with laboratory values, the gastroenterologist's initial clinical assessment, and findings at long-term clinical follow-up. RESULTS: In 128 of 137 children with recent biopsies, findings at histologic examination and on 99mTc WBC scans were correlated. There were seven false-negative and two false-positive studies. Sensitivity was 90%, specificity 97%, positive predictive value 97%, negative predictive value 93%, prevalence of disease 53%, and overall accuracy 93%. In 75 of 78 (96%) children without recent biopsies, 99mTc WBC scan findings were consistent with the laboratory values, gastroenterologist's clinical assessment, and long-term clinical follow-up findings. CONCLUSION: Scintigraphy with 99mTc WBC is a useful noninvasive diagnostic test to determine the extent and distribution of inflammation in children with IBD.

Intestinal transplantation in children.

Intestinal transplantation has emerged in the last decade as a lifesaving procedure for patients with intestinal failure who are suffering from complications arising from the administration of total parenteral nutrition. Indications for transplantation include irreversible liver injury and loss of vascular access. At least 50 children in the United States may benefit from intestinal transplantation every year. In this article, indications, pre- and postoperative management, and outcomes of intestinal transplantation in children are discussed.

A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.

The progressive familial intrahepatic cholestases (PFIC) are a group of inherited disorders with severe cholestatic liver disease from early infancy. A subgroup characterized by normal serum cholesterol and gamma-glutamyltranspeptidase (gammaGT) levels is genetically heterogeneous with loci on chromosomes 2q (PFIC2) and 18q. The phenotype of the PFIC2-linked group is consistent with defective bile acid transport at the hepatocyte canalicular membrane. The PFIC2 gene has now been identified by mutations in a positional candidate, BSEP, which encodes a liver-specific ATP-binding cassette (ABC) transporter, sister of p-glycoprotein (SPGP). The product of the orthologous rat gene has been shown to be an effective bile acid transporter in vitro. These data provide evidence that SPGP is the human bile salt export pump (BSEP).

Anatomic variability of rejection in intestinal allografts after pediatric intestinal transplantation.

BACKGROUND: Rejection of the allograft is a major contributor to morbidity and mortality in children who undergo a small intestinal transplant. Operational definitions for histologic rejection have been established, but little is known about the anatomic variability of the histologic abnormalities. STUDY DESIGN: Biopsy reports were reviewed retrospectively from more than 1200 endoscopies performed on the 41 children who received intestinal transplantation between 1990 and 1995. RESULTS: Biopsies were performed in the proximal jejunum and distal ileum allograft simultaneously on 248 occasions. In 168 biopsies, neither site was histologically abnormal; in 80, rejection was found. In 42, both regions were abnormal; however, in 17 only the jejunum was involved and in 21 the rejection exclusively involved the ileum. Among children whose allograft included colon, rejection was absent in colon and ileum in 34 biopsies, involved both in 6, involved ileum but not colon in another 6 and involved colon but not ileum in only one. When the allograft included stomach, rejection was absent in the stomach and jejunum 39 times, involved both sites 8 times, involved jejunum and not the stomach 10 times, but involved the stomach and not jejunum only once. Endoscopic appearance correctly predicted histologic rejection 63% of the time. CONCLUSION: Anatomic variability may exist in the rejection process. Sampling the jejunum and ileum seems to have similar sensitivity in detecting rejection, whereas sampling stomach and the colon is less sensitive. When allograft rejection is suspected on clinical grounds, sampling more than one area of the graft may be necessary for histologic confirmation.

Characterization of late abdominal accumulation of 99Tcm-HMPAO leukocytes in a large population of children.

We retrospectively evaluated the incidence of late accumulation of 99Tcm-HMPAO leukocytes (99Tcm-WBC) in the right lower quadrant of a large population of children and characterized some predictive patterns that would enable differentiation of active inflammation from this late occasional accumulation of 99Tcm-WBC. We reviewed the charts of 211 children. The first group evaluated consisted of 79 controls: 30 normal children with no gastrointestinal disease, but who underwent 99Tcm-WBC scanning for other medical problems, and 49 children who had non-specific gastrointestinal (GI) complaints, but had no demonstrable inflammatory bowel disease by conventional diagnostic methods. The second group consisted of 132 children with inflammatory bowel disease: 80 children with Crohn's disease (CD), 34 with ulcerative colitis (UC) and 18 with indeterminate colitis (IC). Children were imaged at 30 min and 3 h. Fifteen (19%) of the 79 controls scanned showed accumulation of 99Tcm-WBC in the right lower quadrant at 3 h and none at 30 min. Of those 15, 8 were from the control population and 7 from the group with non-specific GI complaints and negative work-ups. There was no uptake in other segments of the bowel. The accumulation was faint, of lesser intensity than in the iliac wing, and diffuse, such that identification of a specific loop of involved bowel was not possible. Migration of the 99Tcm-WBC distal to the terminal ileum was demonstrated. The other 64 children in the control group showed no accumulation of 99Tcm-WBC at any time during their scans. All 79 scans were blindly interpreted as normal studies. There were no false-positive readings encountered in the 132 children with inflammatory bowel disease (80 CD, 34 UC, 18 IC) when the aforementioned characteristics of the late accumulation of 99Tcm were used to differentiate inflammation from this physiological excretion. In conclusion, the late accumulation of 99Tcm-WBC in the right lower quadrant is characterized by (1) accumulation at no less that 3 h, (2) no accumulation in other segments of the bowel, (3) faint accumulation of lesser intensity than in the iliac wing, (4) a diffuse accumulation pattern and (5) migration of the 99Tcm-WBC into the caecum and ascending colon over time. Recognition of this excretion pattern enables differentiation of active Crohn's disease of the small bowel from migration and accumulation of 99Tcm-WBC in the right lower quadrant of the abdomen.

Brewer's yeast and Saccharomyces boulardii both attenuate Clostridium difficile-induced colonic secretion in the rat.

Saccharomyces boulardii (Sb), a nonpathogenic yeast, has been used to prevent recurrences of Clostridium difficile (C.diff) -associated diarrhea. A single report suggested that treatment with Saccharomyces cerevisiae (Sc), commonly called brewer's yeast (BY), facilitates treatment of persistent C.diff infection. We conducted this experiment to determine whether C.diff toxin A-induced colonic secretion in the rat is blunted by pretreatment with either Sb or BY. We employed closed cecal pouches in two groups of five adult male Sprague-Dawley rats fed with standard chow for five days prior to the experiment, another group whose water was supplemented with 20 x 10(9) colony-forming units (CFU) of Sb per day for five days, and another group whose water was supplemented with 20 x 10(9) CFU of Sc per day for five days. Cecal pouches were infused for 3 hr with one of the following: (1) normal saline alone for a control group, or (2) normal saline plus 5 microg of C.diff toxin A (for the other control group and for the two experimental groups). Water movement was measured by a nonabsorbable marker technique. Sodium movement and permeability to mannitol were also measured. Prior to the infusion, cecal contents were quantitatively cultured. In the three animals whose ceca were colonized with less than 10(6) CFU of either yeast per gram wet cecal content, toxin A-induced secretion could not be attenuated. In contrast, animals whose ceca were colonized with more than 10(6) CFU of either yeast per gram of wet cecal content showed significantly less secretion after toxin A application than those which were not fed yeast. S. cerevisiae reduced secretion by half (N = 5, P = 0.039 for water, 0.044 for sodium) and Sb by 75% (N = 4, P = 0.015 for water, 0.034 for sodium). Toxin-induced increases in permeability to [3H]mannitol from systemic circulation to cecum could not be blunted by either yeast. We conclude that rat ceca can be colonized by either organism and that both organisms reduce C.diff toxin A-mediated secretion. We speculate that both organisms might have benefit in human C.diff-associated enterocolitis. Further studies of their mechanisms of action as well as clinical trials for the prevention and treatment of human C.diff infections should be pursued.

Retrospective analysis of alternate-day prednisone maintenance therapy for Crohn's disease.

We reviewed the medical records of 98 children with Crohn's disease followed at Children's Hospital of Pittsburgh from 1983 to 1993 to evaluate the merits of alternate-day prednisone (AD) maintenance therapy once initial remission was achieved. Of the 98 children, 35 had adequate data recorded for eligibility to the study. Of these, 11 were in the AD group and 24 were in a group whose maintenance regimen did not include prednisone (NO). The dependent variables were frequency of flares and linear growth over time. AD therapy reduced mean symptomatic flares (0.23 +/- 0.1 vs 0.69 +/- 0.14 flares/patient/year; p = 0.04) over a 2-year follow-up period but did not delay significantly the onset of a flare after remission was achieved (16.5 +/- 3.4, vs 13.4 +/- 1.8 months; p = 0.4). Site of disease involvement had no impact on frequency of flares. Fewer patients in the AD group experienced flares, but this finding did not achieve statistical significance (4/11, 36%, vs 17/24, 71%; p = 0.07). Linear growth, measured in height percentile and growth velocity (cm/year), was not significantly reduced by the second year of either therapy. This small retrospective study suggests that AD prednisone therapy may be effective in reducing symptomatic flares in Crohn's patients without a resultant inhibition of linear growth.

Endoscopies in pediatric small intestinal transplant recipients: five years experience.

OBJECTIVE: Intestinal transplantation has become an option as a treatment for permanent intestinal failure. Endoscopy is an essential tool in assessing the intestinal allograft after intestinal transplantation. The aim of this study was to analyze our experience using endoscopy in intestinal transplant recipients. METHODS: This was a retrospective review of endoscopic and histological reports in 41 children who received an intestinal transplant between 1990 and 1995 at Children's Hospital of Pittsburgh. RESULTS: A total of 1273 endoscopies was performed of which 760 were ileoscopies via allograft ileostomy, 273 were upper endoscopies, and 240 were colonoscopies. One hundred four rejection episodes were documented histologically in 32 patients, 6 days to >4 yr after transplantation. Most episodes were mild and easily treated with increased immunosuppression; however, severe rejection with mucosal exfoliation was seen in nine patients. Rejection sometimes involved only part of the allograft. Endoscopic appearance alone without biopsies was sensitive enough to diagnose only 63% of the rejection episodes. Epstein-Barr and cytomegalovirus infections occurred in 11 and eight patients, respectively, and involved both native bowel and allograft in some. Complications of endoscopy were few: one perforation, three episodes of bleeding, and three episodes of transient respiratory compromise. CONCLUSIONS: Endoscopy is an essential tool in the postoperative assessment of intestinal transplant recipients. Frequent surveillance ileoscopies with biopsies should be performed after transplantation. If patients clinically deteriorate with fever, diarrhea, bacteremia, or gastrointestinal bleeding and a clear cause is not elucidated by ileoscopy, an upper endoscopy with biopsies is indicated.

Neonatal hemochromatosis: outcomes of pharmacologic and surgical therapies.

BACKGROUND: Neonatal hemochromatosis (NH), also known as perinatal hemochromatosis or neonatal iron storage disease, is a disorder in fetuses and newborn infants. A retrospective study was conducted to report management of patients with NH. METHODS: Retrospective analysis was conducted by chart review and by review of histologic material from patients with NH. RESULTS: Neonatal hemochromatosis was diagnosed in 14 patients between 1985 and 1995. All were considered for orthotopic liver transplantation (OLTX). From 1993 onward, all patients were treated with an antioxidant-chelation "cocktail," consisting of deferoxamine, vitamin E, N-acetylcysteine, selenium, and prostaglandin-E1. Of 6 patients with NH diagnosed before 1993, 4 underwent OLTX; only 1 is still alive. Of 8 patients with NH diagnosed after 1993 and treated with the cocktail, 7 expired before OLTX. One stabilized on therapy, but having never recovered full synthetic liver function, underwent OLTX and is now alive and well. CONCLUSION: Neonatal hemochromatosis carries a grim prognosis; however, successful OLTX is curative. The use of an antioxidant-chelation cocktail did not improve outcome in the patients studied. Earlier (perinatal) diagnosis may be required for optimal results. Further study of other interventions, including antenatal diagnosis and earlier institution or modification of cocktail therapy appears warranted.