Intestinal intraepithelial lymphocyte derived angiotensin converting enzyme modulates epithelial cell apoptosis.

BACKGROUND & AIMS: Intestinal adaptation in short bowel syndrome (SBS) consists of increased epithelial cell (EC) proliferation as well as apoptosis. Previous microarray analyses of intraepithelial lymphocytes (IEL) gene expression after SBS showed an increased expression of angiotensin converting enzyme (ACE). Because ACE has been shown to promote alveolar EC apoptosis, we examined if IEL-derived ACE plays a role in intestinal EC apoptosis. METHODS: Mice underwent either a 70% mid-intestinal resection (SBS group) or a transection (Sham group) and were studied at 7 days. ACE expression was measured, and ACE inhibition (ACE-I, enalaprilat) was used to assess ACE function. RESULTS: IEL-derived ACE was significantly elevated in SBS mice. The addition of an ACE-I to SBS mice resulted in a significant decline in EC apoptosis. To address a possible mechanism, tumor necrosis factor alpha (TNF-alpha) mRNA expression was measured. TNF-alpha was significantly increased in SBS mice, and decreased with ACE-I. Interestingly, ACE-I was not able to decrease EC apoptosis in TNF-alpha knockout mice. CONCLUSIONS: This study shows a previously undescribed expression of ACE by IEL. SBS was associated with an increase in IEL-derived ACE. ACE appears to be associated with an up-regulation of intestinal EC apoptosis. ACE-I significantly decreased EC apoptosis.

The use of laparoscopic liver biopsies in pediatric patients with hepatic dysfunction following allogeneic hematopoietic stem cell transplantation.

Hepatic dysfunction following hematopoietic stem cell transplantation (HSCT) is common, but making the correct diagnosis can be challenging. Liver biopsies can serve as an important diagnostic tool when the etiology cannot be clearly determined by laboratory data, physical examination, and imaging studies. We reviewed 12 consecutive pediatric patients (seven males, five females, age 9-23 years) who received allogeneic HSCT and underwent a laparoscopic-guided liver biopsy for hepatic dysfunction of unknown etiology from 1998 to 2005. Biopsies were performed using a single-port technique with a 16 or 18 gauge, spring-loaded biopsy gun. The time from HSCT to biopsy ranged from 31 days to 821 days (median 92 days). No intra- or postoperative complications were observed. The initial clinical diagnosis was confirmed in seven patients, whereas the initial working diagnosis was inaccurate in the remaining five patients. Our results suggest that laparoscopic-guided liver biopsy is an informative and safe procedure in pediatric HSCT recipients; this approach helped delineate the true cause of hepatic dysfunction and changed our therapeutic approach in approximately 40% of the patients reviewed. While the safety record at our institution appears promising, a larger multi-institutional study would be necessary to more accurately describe the overall efficacy of this procedure in pediatric HSCT patients.

The effect of hypoxia on permeability and bacterial translocation in Caco-2 adult and I-407 fetal enterocyte cell culture models.

Hypoxia has been implicated in the breakdown of the intestinal epithelial barrier in animals, leading to bacterial translocation (BT); however, the mechanism of this hypoxic insult is unknown. To determine the effects of hypoxic injury in vitro on epithelial membrane integrity, transepithelial electrical resistance (TEER), mannitol permeability (Ma-Pm), and BT were measured in both an adult (Caco-2) and fetal (I-407) intestinal epithelial cell culture model. Caco-2 adult and I-407 fetal epithelial cell monolayers were treated with or without bacteria (1 x 10(7) Escherichia coli. C-25), and then incubated under either normoxic (5% CO(2) in room air) or hypoxic (5% CO(2) and 95% N(2)) conditions at 37 degrees C for 6 h. Hypoxia caused a 10% increase in Ma-Pm in the I-407 fetal cell model independent of the bacterial challenge. In contrast, a bacterial challenge in the Caco-2 adult model caused a 485% increase in Ma-Pm independent of hypoxia. Neither hypoxia, nor C-25 bacteria, for 6 h caused BT in either cell culture model. In the adult cell culture model, bacteria appear to mediate changes in epithelial barrier function, with hypoxia having no effect. On the other hand, hypoxia is the major factor in the loss of epithelial barrier function in fetal epithelium, but has no effect on adult epithelium. The data suggest that the breakdown of barrier function caused by a hypoxic insult is the primary stimulus for subsequent BT in neonates.

Interleukin-6 changes tight junction permeability and intracellular phospholipid content in a human enterocyte cell culture model.

Proinflammatory cytokines and secretory phospholipase A(2) (sPLA(2)) are elevated in patients with inflammatory bowel disease (IBD). We previously reported that the proinflammatory cytokine IL-6 increased the expression of sPLA(2) (a hydrolyzer of phosphatidylcholine) and decreased membrane integrity in an intestinal epithelial cell culture model. To determine the physiological effects of the IL-6 mediated increase in sPLA(2) on decreased epithelial layer integrity, we investigated alterations of intracellular/secretory phospholipid (PL) composition in a cell culture model. In addition, since other PLs may also mediate epithelial membrane activity, we investigated the effect of IL-6 on PL activity in a Caco-2 enterocyte culture model. Caco-2 cells were incubated for 72 h with IL-6 or media alone (control). Both media and cell lysate were analyzed for PL composition using thin-layer chromatography. The PL composition in the media did not show any differences between the two groups ( p>0.1). Total intracellular PL contents were also unchanged; however, IL-6 led to significant changes in PL composition including an increase in phosphatidylethanolamine (PE) and sphingomyelin (SM) and a decrease in phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) ( p<0.05). Both PE and SM are known as inflammatory signaling factors involved in human IBD. Our study suggests that the decreased membrane integrity seen with IL-6 application may occur via intracellular PL alterations, rather than through the direct effects of sPLA(2).

Recurrence after laparoscopic and open Nissen fundoplication: a comparison of the mechanisms of failure.

BACKGROUND: Laparoscopic Nissen fundoplication as treatment for gastroesophageal reflux disease (GERD) in adults has a reported recurrence rate of 2-17%. We investigated the rates and mechanisms of failure after laparoscopic Nissen fundoplication in children. METHODS: All patients who underwent a laparoscopic Nissen fundoplication for GERD and who subsequently required a redo Nissen were reviewed (n = 15). The control group consisted of the most recent 15 patients who developed recurrent GER after an open Nissen, fundoplication. RESULTS: Between 1994 and 2000, laparoscopic Nissen fundoplication was performed in 179 patients. Fifteen patients (8.7%) underwent revision. The mechanisms of failure were herniation in four patients, wrap dehiscence in four, a too-short wrap in three, a loosened wrap in two, and other reasons in two. The reoperation was performed laparoscopically in five patients (33%). The failure mechanisms were different in the open patients: eight were due to slipped wraps; three to dehiscences; and two to herniations. CONCLUSION: The failure rate after laparoscopic Nissen is acceptably low. A redo laparoscopic Nissen can be performed safely after an initial laparoscopic approach.

Total parenteral nutrition-induced apoptosis in mouse intestinal epithelium: regulation by the Bcl-2 protein family.

Apoptosis of intestinal epithelial cells (EC) plays a role in total parenteral nutrition (TPN)-induced villus atrophy. Among the mediators of apoptosis in EC are some members of the Bcl-2 family of proteins. Bcl-2 members can either be anti- (Bcl-2, Bcl-x(L), Bcl-w) or pro-apoptotic (Bax, Bak, Bid, Bad, Bcl-x(S)). To determine whether the observed increase in apoptosis induced by TPN is associated with an alteration in these Bcl-2 members' mRNA expression, mice were randomized to either TPN or oral feeding (controls). Animals were killed after 7 days and the intestine was harvested. EC were purified with magnetic beads. Apoptosis was detected by cell-surface expression of phosphatidylserine using flow cytometry. EC mRNA expression was determined by reverse-transcriptase polymerase chain reaction. Results were expressed relative to beta-actin. TPN resulted in a significant ( P < 0.05, unpaired t-test) increase in apoptosis: TPN 29.4 +/- 11.3% versus control 14.4 +/- 5.1%. The expression of the pro-apoptotic members Bax, Bak, Bid, and Bcl-x(S) was significantly ( P < 0.05) decreased after TPN. In contrast, a significant increase was observed in the anti-apoptotic member Bcl-2. mRNA expression of Bcl-w, Bad, and Bcl-x(L) was not significantly different between the control and TPN groups. Thus TPN-induced apoptosis was associated with an increased expression of anti-apoptotic factors and a decrease in pro-apoptotic factors. This contrasts with other reports where these factors showed converse effects under apoptotic conditions. Our results may demonstrate a unique regulatory pathway that may counter the observed increase in TPN-induced EC apoptosis.

Parenteral nutrition-associated cholestasis.

Cholestasis is a major complication that occurs frequently in patients with the short bowel syndrome and accounts for the majority of morbidity and mortality in this group of patients. The exact cause of this condition is not known and the etiology is likely multifactorial. Many new mechanistic insights into this disease are discussed and have paved the way for future investigation. For now, prompt recognition, early initiation of enteral feeding, prevention of overfeeding with parenteral nutrition, and agents that induce bile flow may be useful to prevent this catastrophic morbidity.

Inferior vena cavectomy for nonexcisable Wilms' tumor thrombus.

Wilms' tumor with tumor extension into the vena cava is a not uncommon variant of tumor presentation. Typically, this extension is nonadherent to the endothelium and readily lends itself to removal either before or after chemotherapy. This case illustrates an unusual variant in which the tumor within the cava was tightly adherent to the venous wall and required the complete excision of the vena cava, left renal vein, and a portion of the iliac system.

Congenital cystic adenomatoid malformation: impact of prenatal diagnosis and changing strategies in the treatment of the asymptomatic patient.

OBJECTIVE: This study was designed to assess the effect of prenatal sonographic diagnosis on the treatment of congenital cystic adenomatoid malformation of the lung. MATERIALS AND METHODS: The medical records of 27 patients with pathologically proven congenital cystic adenomatoid malformations were retrospectively reviewed. Patients were divided into four groups based on mode of presentation: with or without abnormal findings on prenatal sonography and with or without symptoms at birth. Age at diagnosis, age at surgical intervention, complications, and length of hospital stay were recorded for each group. RESULTS: Twenty-seven patients with 31 proven congenital cystic adenomatoid malformations were included. Eleven patients underwent prenatal sonography establishing the diagnosis (6 asymptomatic at birth, 5 symptomatic), and 16 did not have a prenatal diagnosis (10 asymptomatic at birth, 6 symptomatic). In the symptomatic populations, prenatal diagnosis had no impact on age at surgery, length of stay, or surgical complication rate (p = 0.78-0.83). In the asymptomatic population, prenatal diagnosis allowed early diagnosis (p < 0.001) and resection in the asymptomatic period. It was also associated with a shorter length of stay at the time of surgical resection (mean time, 4.2 days for patients with prenatal diagnosis versus 12.9 days for those without it;p < 0.001) and with a trend toward lower serious complication rate (3 patients without prenatal diagnosis versus 1 patient with it). CONCLUSION: Prenatal sonography provides the radiologist a means to identify congenital cystic adenomatoid malformations in a population of infants who are asymptomatic at birth. Surgical intervention in the asymptomatic infant is associated with a shorter length of stay, a trend toward fewer complications, and decreased medical cost compared with intervening after symptoms develop.

A decade of experience with the primary pull-through for hirschsprung disease in the newborn period: a multicenter analysis of outcomes.

OBJECTIVE: To determine whether use of a primary pull-through would result in equivalent perioperative and long-term complications compared with the two-stage approach. SUMMARY BACKGROUND DATA: During the past decade, the authors have advanced the use of a primary pull-through for Hirschsprung disease in the newborn, and preliminary results have suggested excellent outcomes. METHODS: From May 1989 through September 1999, 78 infants underwent a primary endorectal pull-through (ERPT) procedure at four pediatric surgical sites. Data were collected from medical records and a parental telephone interview (if the child was older than 3 years) to assess stooling patterns. A similar group of patients treated in a two-stage fashion served as a historical control. RESULTS: Mean age at the time of ERPT was 17.8 days of life. Comparing primary ERPT with a two-stage approach showed a trend toward a higher incidence of enterocolitis in the primary ERPT group compared with those with a two-stage approach (42.0% vs. 22.0%). Other complications were either lower in the primary ERPT group or similar, including rate of soiling and development of a bowel obstruction. Median number of stools per day was two at a mean follow-up of 4.1 +/- 2.5 years, with 83% having three or fewer stools per day. CONCLUSIONS: Performance of a primary ERPT for Hirschsprung disease in the newborn is an excellent option. Results were comparable to those of the two-stage procedure. The greater incidence of enterocolitis appears to be due to a lower threshold in diagnosing enterocolitis in more recent years.

Altered development of intestinal intraepithelial lymphocytes in P-glycoprotein-deficient mice.

Intraepithelial lymphocytes (IEL) that reside in the intestinal epithelium are known to exhibit phenotypic and functional characteristics that are distinct from other T cells. We have recently shown that peripheral T cells exclusively express an isoform of P-glycoprotein (P-gp) encoded by the mdr1a gene, but do not require mdr1a expression for normal proliferative, cytokine, or cytotoxic responses. In the present study, we have used mdr1-type knockout (KO) mice to demonstrate that IEL also utilize mdr1a, but only preferentially, in that the mdr1b isoform can be expressed in the absence of mdr1a expression. We also report that a high level of P-gp activity appears to be necessary for the normal development of certain IEL subpopulations. In specific, while the total number of IEL was relatively unaffected by the absence of mdr1a expression, the proportions of CD8 alpha beta and TCR alpha beta+ IEL increased significantly in mdr1a and mdr1a/b KO mice at the expense of CD8 alpha alpha and TCR gamma delta+ IEL, respectively. Moreover, these subset alterations also appeared to have functional consequences, in that proliferative, IL-2, and IFN-gamma responses of IEL from KO mice were distinct from those of normal IEL. In summary, our data suggest that mdr1a expression is required for the development of certain IEL subpopulations, most notably TCR gamma delta+ cells, and thereby indirectly influences the balance of T cell subsets in the intestinal epithelium.

Long-term follow-up of redo pull-through procedures for Hirschsprung's disease: efficacy of the endorectal pull-through.

BACKGROUND/PURPOSE: The purpose of this study was to review the authors' 25-year experience with redo pull-through procedures for Hirschsprung's disease including surgical technique and long-term outcome. METHODS: From 1974 to now, over 325 patients with Hirschsprung's disease have been treated at C.S. Mott Children's Hospital. This includes 30 patients referred after an unsuccessful pull-through at another hospital and 2 patients with an unsuccessful pull-through from C.S. Mott. All redo pull-throughs (n = 19) were performed in these patients, and their clinical courses are reviewed. RESULTS: Twelve patients required reoperation secondary to a mechanical problem with their first pull-through. The other 7 patients had evidence of residual segments of dilated colon leading to functional failure of their initial operation including 5 patients with documented aganglionic bowel present at the second pull-through. Ten of the patients requiring reoperation initially had an endorectal pull-through (ERPT), 5 had a Duhamel procedure, 3 had a Swenson procedure, and 1 had a Rehbein procedure. Choice of revision was an ERPT in 8 patients in whom an adequate rectal cuff could be developed. Additional redo procedures included a Duhamel in 8 patients and a Swenson in 3 patients. Follow-up ranges from 3 months to 23 years (mean, 13.8 years). There were no deaths in the series, and 1 patient required a third pull-through. All patients who are not neurologically impaired and are over age 3 are continent except one (94%). Stools per day range from 1 to 10 (mean, 3.2). CONCLUSIONS: Redo pull-through operations for Hirschsprung's disease appear to be as effective as primary procedures in terms of continence and stooling frequency. Distinct from other series, we found an ERPT to be the procedure of choice if an adequate rectal cuff was present.

Recent advances in the management of Hirschsprung's disease.

BACKGROUND: Major advances have occurred in the management of Hirschsprung's disease since Swenson described his definitive operation in 1948. These advances have occurred in the following areas: genetics, neurophysiology, definitive management in the newborn, total colonic aganglionosis (TCA), Hirschsprung's-associated enterocolitis (HAEC), intestinal neuronal dysplasia (IND), and laparoscopic and perineal approaches for definitive pull-through and redo pull-through operations. METHODS: This paper will focus on the definitive management of the newborn, TCA, and HAEC, areas in which we have had considerable experience at our institution. RESULTS: We have treated almost 90 newborns with the definitive pull-through with minimum morbidity. We have managed 25 patients with TCA, of whom 5 had total intestinal involvement and died. The remaining 20 have undergone a total colectomy and endorectal pull-through (ERPT), with zero mortality and a very acceptable stooling pattern and continence rate. Our experience with more than 350 patients with Hirschsprung's disease over the past 25 years has demonstrated an incidence of HAEC of between 20% and 30%. During this period, we have performed 19 redo pull-through operations, the majority of which were ERPTs, with results comparable with those seen with a primary pull-through operation. CONCLUSIONS: The major advances that have occurred in the management of Hirschsprung's disease include the definitive management of the newborn, our understanding of Hirschsprung's-associated enterocolitis and the treatment of this entity, and the recent successful management of the very complex form of this disease, total colonic aganglionosis.

An analysis of the morbidity and mortality of short-bowel syndrome in the pediatric age group.

Twenty-two children with short-bowel syndrome (SBS) were treated at the C. S. Mott Children's Hospital in the University of Michigan Medical Center between June 1983 and May 1993. Definition of SBS was loss of 70% or more of the total small bowel. Seventeen of these children are currently alive, a 77% survival rate. Patients were followed for a mean of 1,148 days. The mean age of SBS development was 71 days of life. The only predictive indicator of patient survival was direct bilirubin levels. Sixty-seven percent of the children died if they had a direct bilirubin of > 4 mg/dl > or = 6 months duration.

Specialty networking in pediatric surgery: a paradigm for the future of academic surgery.

OBJECTIVE: To review retrospectively a 4-year experience with pediatric surgical networking at a major academic medical center in the Midwest. BACKGROUND: The growth of managed care in the United States during the past decade has had a major impact on the practice of medicine in general, but especially on academic medicine. In some academic medical centers, the loss of market share has not only affected clinical activity but has also compromised the educational and research missions of these institutions. METHODS: At the authors' institution, a networking strategy in pediatric surgery was established in 1993 and implemented on July 1, 1994. In 1994, one new satellite practice was established; over the next 4 years, four additional practices were added, including one in another state. To assess the impact on financial status, clinical activity, education, and academic productivity, the following parameters were analyzed: gross and net revenue, surgical cases, clinic visits, ranking of the pediatric surgery residency, publications, grant support, and development and endowment funds. RESULTS: Gross and net revenue increased from $3,273,000 and $302,000 in 1993 to $10,087,000 and $2,826,000, respectively, in 1998. Surgical cases and clinic visits increased from 1240 and 3751 in 1993 to 5872 and 11,604, respectively, in 1998. At the medical center's children's hospital, surgical cases and clinic visits increased from 1240 and 3751 to 2592 and 4729 during the same time period. During this 4-year period, the faculty increased from 4 to 11. Since 1997, the National Resident Matching Program has provided data on how pediatric surgery residency candidates ranked a training program. In 1997, this program received the second-most one to five rankings; in 1998, it tied for first. This exceeds the faculty's perception of previous years' rankings. Publications increased from 26 in 1993 to a peak number of 62 in 1996; in 1997 and 1998 the publications were 48 and 37, respectively. External grant support increased from $139,882 in 1993 to a total of $6,109,971 in 1998. Development and endowment funds increased from $103,559 in 1993 to $2,702,2777 in 1998. CONCLUSIONS: Pediatric surgical networking at the authors' institution has had a markedly positive impact on finances, clinical activity, education, and academic productivity during a 4-year period. The residency training program appears to have improved in popularity among candidates, probably because of the increased referral of complex cases to the medical center from the various networking satellites. External grant support and basic laboratory research significantly increased, most likely because of the greater number of faculty with protected time for research recruited. Development and endowment funds dramatically grew because of the excellent fiscal health of the pediatric surgical program. This experience may serve as a model for other academic surgical specialties.

Prenatal diagnosis of congenital cystic adenomatoid malformation and its postnatal presentation, surgical indications, and natural history.

BACKGROUND/PURPOSE: Regression of a cystic adenomatoid malformation (CAM) in a fetus is well described. Little, however, is known about the postnatal course of these infants. This study attempts to correlate the prenatal course of CAMs with postnatal symptoms, radiological manifestations, and need for surgery. METHODS: The clinical course of patients with a CAM diagnosed prenatally were retrospectively reviewed. Inclusion in the study required a prenatal ultrasound scan documenting a CAM. RESULTS: Over 10 years, 14 patients with a CAM were diagnosed prenatally. Six (43%) showed a partial in utero regression. Four patients were symptomatic at birth and underwent a resection as newborns. Ten patients were asymptomatic at birth, and eight of these had normal chest x-rays. Elective resection has been performed in 3 of these 10, and two additional children are scheduled to undergo an excision near 1 year of age. The remaining five patients have undergone follow-up nonoperatively for a mean of 36 +/- 15 months. Of the seven asymptomatic patients not undergoing immediate surgery, only one has shown a slight postnatal regression, despite five of these showing regression in utero. None have become symptomatic. CONCLUSIONS: The results suggest that regression of a CAM on prenatal ultrasound scan is common, but this process does not continue after birth. A normal chest x-ray does not indicate complete regression of a CAM; a computed tomography (CT) scan is required to evaluate such patients, and will generally demonstrate a CAM. Asymptomatic patients with a CAM may be followed up nonoperatively with no apparent adverse effects. The decision and timing of an excision in an asymptomatic patient remains controversial among pediatric surgeons.

Alteration of the intestinal intraepithelial lymphocytes during total parenteral nutrition.

Total parenteral nutrition (TPN) may cause increased rates of bacterial translocation (BT), possibly due to a loss of epithelial integrity. Cultured epithelial cells have been shown to lose tight junction integrity with interferon gamma (INF-gamma) an action which may be blocked by transforming growth factor beta (TGF-beta). Because intraepithelial lymphocytes (IEL) are a rich source of these cytokines in the epithelium, we hypothesized that changes in the IEL, while mice were receiving TPN, may be responsible for the mediation of such cytokine responses. C57BL/6 mice were randomized to a Control group which received intravenous saline and mouse chow, or a TPN group which received intravenous TPN with no oral feeding. At 7 days mice were assessed for BT. Isolated IEL were stained for CD4, CD8, and CD44 (as a marker for memory T-cells) and flow cytometry was performed. mRNA was extracted from remaining IEL for cytokine expression. Reverse transcriptase polymerase chain reaction was performed to detect TGF-beta1 and INF-gamma mRNA expression. Densities were standardized to beta-actin expression. The incidence of BT to mesenteric lymph nodes was 40 and 12.5%, for the TPN and Control groups, respectively. TPN led to statistically significant decreases in the CD4+, CD8-; CD4+, CD8+; and the CD8+, CD44+ IEL subpopulations (P < 0.05). mRNA expression for INF-gamma was increased by 53% (P < 0.05), and TGF-beta1 mRNA expression was decreased by 75% (P = 0.1) in the IEL of TPN mice when compared with Controls. TPN led to significant changes in the IEL. Such alterations of the IEL phenotype and function may be a critical mechanism by which epithelial integrity is lost.

Latex sensitization in general pediatric surgical patients: a call for increased screening of patients.

BACKGROUND/PURPOSE: Latex sensitization is a well-documented occurrence in children with myelodysplastic and urologic anomalies. The incidence of latex allergy in general pediatric surgical patients, however, has not been previously addressed. The purpose of this study was to examine the risk of perioperative latex reactions in a general pediatric surgical practice over a 1-year period. METHODS: This study examined the occurrence of latex sensitization using two methods. First, the preoperative anesthesia records of patients that have undergone surgery from October 1995 through September 1996 at Mott Children's Hospital were reviewed retrospectively. Second, all patients who had intraoperative anaphylaxis attributable to latex sensitization, including those from three additional hospitals, were evaluated. RESULTS: During a 12-month period, 1,523 pediatric general surgical operations were performed at the C.S. Mott Children's Hospital. Of these, only 11 operations on five patients were performed under latex precautions. All of these patients had a preoperative diagnosis of latex sensitivity. During the same period, intraoperative anaphylactic reactions caused by latex allergy occurred in two of the general surgical patients (0.13%) at the C.S. Mott Hospital. Four additional cases are also reported from other study hospitals. None of these patients were suspected, based on current screening methods, of having a latex allergy before their surgery. CONCLUSIONS: Latex allergy is a potentially life-threatening condition in the pediatric general surgical population. Further study is needed to develop criteria to preoperatively identify patients at risk for latex sensitization.