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Chemoresistance is a major cause of treatment failure and poor outcome in neuroblastoma. In this study, we investigated the expression and function of dual-specificity phosphatase 26 (DUSP26), also known as mitogen-activated protein kinase phophatase-8, in human neuroblastoma. We found that DUSP26 was expressed in a majority of neuroblastoma cell lines and tissue specimens. Importantly, we found that DUSP26 promotes the resistance of human neuroblastoma to doxorubicin-induced apoptosis by acting as a p53 phosphatase to downregulate p53 tumor suppressor function in neuroblastoma cells. Inhibiting DUSP26 expression in the IMR-32 neuroblastoma cell line enhanced doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression as well as apoptosis. In contrast, DUSP26 overexpression in the SK-N-SH cell line inhibited doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression and apoptosis. Using in vitro and in vivo assays, we found that DUSP26 binds to p53 and dephosphorylates p53 at Ser20 and Ser37. In this report, we show that DUSP26 functions as a p53 phosphatase, which suppresses downstream p53 activity in response to genotoxic stress. This suggests that inhibition of this phosphatase may increase neuroblastoma chemosensitivity and DUSP26 is a novel therapeutic target for this aggressive pediatric malignancy. [ABSTRACT FROM AUTHOR]

Genetic engineering of human T lymphocytes to express tumor antigen-specific chimeric immune receptors is an attractive means for providing large numbers of effector cells for adoptive immunotherapy while bypassing major mechanisms of tumor escape from immune recognition. We have applied this strategy to the targeting of a G(D2)-positive tumor, neuroblastoma, which is the commonest extracranial solid tumor of childhood. Chimeric immune receptors were generated by joining an extracellular antigen-binding domain derived from either of the 2 ganglioside G(D2)-specific antibodies sc7A4 and sc14.G2a to a cytoplasmic signaling domain. The variable domains of hybridoma antibody 14.G2a were cloned and selected using a phage display approach. Upon coincubation with G(D2)-expressing tumor cell targets, human T lymphocytes transduced with recombinant retroviruses encoding chimeric receptors based on sc14.G2a, but not sc7A4, secreted significant levels of cytokines in a pattern comparable to the cytokine response obtained by engagement of the CD3 receptor. T cells transduced with the sc14.G2a-based chimeric T-cell receptors also displayed specific lysis of G(D2)-positive neuroblastoma cells, which was blocked in the presence of monoclonal antibody 14.G2a. In the absence of nonspecific stimulation of transduced cells, their functionality declined over time and antigenic stimulation of the chimeric receptor alone did not induce commitment to proliferation. These results support the feasibility of redirecting human T lymphocytes to a tumor-associated ganglioside epitope but emphasize that successful chimeric receptor-mediated adoptive immunotherapy will require additional strategies that overcome functional inactivation of gene-modified primary T lymphocytes., (Copyright 2001 Wiley-Liss, Inc.)

Semipermanent venous catheters remain the most commonly used access for chronic hemodialysis (HD) in pediatric patients. The recent availability of Tesio catheters in 7 and 10 F has expanded available HD catheter options for children and adolescents. We report our experience with Tesio catheter survival, complications, and effect on dialysis adequacy in comparison to standard dual-lumen (DL) catheters in our pediatric HD patients. Demographic data were similar between the two groups. Overall actuarial survival was significantly longer for Tesio versus DL catheters (46% versus 0% at 1 year; P = 0.003). A comparison of smaller catheters (7 F Tesio catheter, 8 or 10 F DL catheter) showed that smaller Tesio catheters had a significantly longer survival (median survival, 244 versus 13 catheter-days; P < 0.01). Tesio 10 F catheters also survived significantly longer than the larger 11.5 and 12 F DL catheters (P < 0.02). Catheter sepsis occurred less frequently with Tesio catheters (one episode/20 catheter-months) than DL catheters (one episode/5 catheter-months) despite the longer duration of Tesio catheters. Adequate dialysis (single-pool Kt/V > 1.2) was delivered with the same frequency, but for a longer duration with Tesio catheters (76% +/- 32% over 100 monthly measurements versus DL catheter, 57% +/- 45% over 54 monthly measurements). Our clinical practice was to replace cuffed catheters when adequate dialysis could not be delivered. We conclude that Tesio catheters provide superior performance compared with DL catheters in terms of catheter survival, infection rates, and duration of adequate performance.

Vigorous host immune reactivity to neuroblastoma may correlate with better prognosis, but identification of human cytotoxic T-lymphocyte (CTL) responses has been relatively unsuccessful. We generated neuroblastoma-reactive CTL lines from two human leukocyte antigen (HLA) A2+ neuroblastoma patients by stimulation of peripheral blood lymphocytes (PBLs) with irradiated autologous tumor cells pretreated with interferon-gamma in the presence of low concentrations of interleukin-2 (5 U/mL). These lines lyse autologous tumor cells but do not kill HLA mismatched allogeneic tumor cells, Epstein-Barr virus-transformed autologous B cells, or standard natural killer cell targets. Cytotoxic T lymphocytes generated from one patient recognize tumor cells from several HLA-A2 matched children, although the other patient's CTLs do not kill tumor cells from other HLA-A2+ individuals. Pretreatment of CTLs or target cells with appropriate standard monoclonal antibodies demonstrates that these CTLs are major histocompatibility complex class I (HLA-A2) restricted and that the effector cell population is CD8+. Our findings suggest that these tumor cells express at least one common HLA-A2 restricted antigen and at least one unique private epitope. Autologous tumor-specific CTLs can be readily generated from patients' PBLs and maintained in long-term culture using standard techniques.

Background/purpose: Ovarian pathology, although rare in children, must be included in the differential diagnosis of all girls who present with abdominal pain, an abdominal mass, or precocious puberty., Methods: To improve clinical appreciation of these lesions, the authors reviewed the presentation, evaluation, and outcome of all patients with ovarian pathology surgically treated at their institution since 1985., Results: One hundred two girls (aged 9.8 +/- 5.5 years; range, 2 days to 20 years) underwent 106 separate ovarian operations (43 salpingo-oophorectomies, 21 oophorectomies, 33 ovarian cystectomies, and 9 ovarian biopsies). Of those presenting with acute abdominal pain (n = 59), 25 (42%) had ovarian torsion (14 associated with a mature teratoma), and only 1 (2%) had a malignant tumor. In contrast, of those presenting with an abdominal mass (n = 23), 6 (26%) had malignancies. There was no age difference between those with benign disease (9.9 +/- 5.6 years; n = 96) and those with malignant tumors (8.6 +/- 3.9 years, n = 10). Nine children had 10 operations for presumed malignant tumors (3 dysgerminomas, 2 immature teratomas with foci of yolk sac tumor, 2 juvenile granulosa cell tumors, 1 yolk sac tumor, and 1 Sertoli-Leydig cell tumor). These patients all had unilateral salpingo-oophorectomy, 4 had chemotherapy, and all are now disease free at 8.4 +/- 4.1 years follow-up., Conclusion: Ovarian pathology remains a rare indication for surgery in girls less than 20 years of age. Because most of these lesions are benign, ovarian-preserving operations should be performed whenever feasible., (Copyright 2001 by W.B. Saunders Company.)

Background: We used phage display technology to clone human recombinant antitumor antibodies from the antibody repertoire of neuroblastoma patients immunized with cytokine-gene transduced tumor cells., Procedure: Lymphocytes obtained from neuroblastoma patients either at diagnosis or after immunization with an autologous interleukin-2 gene transduced tumor vaccine were used to construct two human single-chain Fv (scFV) phage libraries. Tumor-reactive phage were characterized using ELISA, flow cytometry, and sequencing analysis., Results: The initial screening after panning on neuroblastoma cells yielded a substantially higher proportion of selectivity tumor-binding phage clones derived from the immunized patients library (12.9%) than from the unvaccinated patients library (0.8%). The antibodies stained the cells from several additional pediatric malignancies, including Ewing sarcoma and rhabdomyosarcoma, in the absence of binding to any normal tissue cultures or epithelial tumor cell lines. The pattern of reactivity was different from that of antibodies recognizing other widely distributed neuroblastoma-associated antigens, suggesting recognition of a novel shared tumor antigen., Conclusion: The human recombinant scFV antibodies reported here appear to represent a tumor-specific B-cell response induced by autologous tumor immunization and are potentially useful targeting moieties for the treatment of selected childhood tumors., (Copyright 2000 Wiley-Liss, Inc.)

Background/purpose: To better define the indications for peritoneal drainage (PD) in premature babies with intestinal perforation, the authors reviewed their experience with this procedure in a tertiary neonatal intensive care setting., Methods: The charts of all neonates who underwent PD as initial treatment for intestinal perforation between 1996 and 1999 were reviewed. Those patients with pneumatosis intestinalis on abdominal radiograph had perforated necrotizing enterocolitis (NEC) diagnosed; whereas, those infants with no pneumatosis had isolated intestinal perforation diagnosed. The clinical characteristics and outcomes of these 2 groups were compared., Results: Twenty-one premature neonates had primary PD between 1996 and 1999, 10 for isolated intestinal perforation and 11 for perforated NEC. Patients with isolated intestinal perforation had lower birth weights (708 v 949 g; P < .05), were less likely to have started feedings (30% v 91%, P < .05), and the perforation developed at an earlier age (10.6 v 28.0 d, P < .05) compared with the patients who had perforated NEC. Only 2 of 10 infants with isolated perforation required subsequent laparotomy (at 10 weeks for stricture and 12 weeks for a persistent fistula). For these patients, the long-term survival rate was 90%. In contrast, 8 of 11 infants with perforated NEC required laparotomy, and although the 30-day survival rate was 64%, the long-term survival rate was only 27%., Conclusions: Peritoneal drainage provides successful and definitive treatment for most premature babies with isolated intestinal perforation. For neonates with perforation caused by NEC, peritoneal drainage may provide temporary stabilization, but most of these infants require subsequent laparotomy, and few survive.

The effectiveness of cell-mediated immunotherapy for cancer can be limited by loss-of-antigen mutations that occur during tumor growth. In neuroblastoma, amplification of the MYCN oncogene correlates with rapid tumor progression and a poor prognosis overall. We propose that the MYCN protein, the high-level expression of which is required for maintenance of the malignant phenotype, would be an ideal target for vaccine therapy. The MYCN-derived S9K peptide (amino acids 7-15; STMPGMICK), which contains an HLA-A1 binding motif, was used to generate CTLs from the peripheral blood lymphocytes of an HLA-A1+ healthy donor and an HLA-A1+ patient with MYCN-amplified neuroblastoma These CTL lines specifically lysed HLA-matched, MYCN-amplified neuroblastoma tumor cells. They did not lyse either HLA-mismatched, MYCN-amplified, or matched/nonmatched, non-MYCN-amplified tumor cells. The CTL activity was inhibited by a monoclonal antibody to a class I HLA monomorphic determinant but not by one specific for HLA class II, consistent with a class I-restricted mechanism of cytotoxicity. Antibodies to CD8, but not those to CD4, also inhibited CTL activity, identifying CD8+ lymphocytes as the effector cell population. These results show that MYCN-derived peptides can serve as tumor-specific antigens and suggest a rational approach to cell-mediated immunotherapy for MYCN-amplified neuroblastoma.

CI-994 is a substituted benzamide derivative that has demonstrated significant antitumor activity in vitro and in vivo against a broad spectrum of murine and human tumor models. Its mechanism of action is still unknown but seems to be novel compared with existing anticancer drugs. We studied the plasma and cerebrospinal fluid (CSF) pharmacokinetics of CI-994 in nonhuman primates. Three animals (total 4 doses) received an 80 mg/m2 dose of CI-994 administered over 20 min, and one animal received a dose of 100 mg/m2. Serial plasma and fourth ventricular CSF samples were obtained from 0 to 4320 min after administration of the 80-mg/m2 dose, and only plasma samples were obtained after the 100-mg/m2 dose. CI-994 was measured using a previously validated reverse-phase high-performance liquid chromatography assay. Elimination of CI-994 from plasma was triexponential (4 of 5 cases) or biexponential (1 of 5 cases), with a terminal half life (t1/2) of 7.4 +/- 2.5 h, volume of distribution of 15.5 +/- 1.8 L/m2, and clearance of 40 +/- 6 ml/min/m2. The area under the concentration-time curve (AUC) for the 80-mg/m2 dose was 125 +/- 17 microM x hr. CI-994 was first detected in CSF at the completion of the i.v. infusion. Peak concentrations of CI-994 in CSF were 3.4 +/- 0.3 microM. Elimination from CSF was monoexponential (2 of 4 cases) or biexponential (2 of 4 cases) with a terminal t1/2 in CSF of 12.9 +/- 2.5 h and AUC of 55 +/- 18 microM x hr. The AUC(CSF):AUCplasma ratio was 43 +/- 10%. This study demonstrates that there is excellent CSF penetration of CI-994 after i.v. administration. Additional studies are needed to evaluate the potential role of CI-994 in the treatment of central nervous system neoplasms.

Purpose: The aim of this study was to compare three methods of postoperative feeding after pyloromyotomy for hypertrophic pyloric stenosis (HPS)., Methods: The authors reviewed retrospectively the charts of 308 patients who underwent pyloromyotomy for HPS from 1984 to 1997. Nineteen patients had prolonged hospitalization for other reasons and were excluded from the study, leaving 289 patients for analysis. All procedures were performed by a single group of pediatric surgeons. The individual preferences of these surgeons resulted in three different feeding schedules: R, strictly regimented (>12 hours nothing by mouth, then incremental feeding over > or =24 hours), I, intermediate (>8 hours nothing by mouth, then incremental feeding over <24 hours), or A, ad lib (< or =4 hours nothing by mouth, with or without a single small feeding, then ad lib feedings)., Results: Of the 289 patients, 248 (80.5%) were boys. The average age of the patients was 5.64 weeks (range, 1 to 21 weeks). A total of 265 of 289 (92%) were full term. Thirty-nine of 289 (13.5%) had a family history positive for pyloric stenosis. A total of 104 of 289 (36%) were first-born infants, 89 of 289 (31%) were second born. The diagnosis of pyloric stenosis was made by a combination of physical examination findings and diagnostic image for most patients. An "olive" was palpated in 60.6% of the patients. Sixty percent (60.4%) of patients had an upper gastrointestinal series performed, and 42.5% were examined by ultrasonography. Overall, 53% of the patients had postoperative emesis. Only 3.5% had emesis that persisted greater than 48 hours after surgery. Patients fed ad lib after pyloromyotomy had slightly more emesis (2.2 A v. 1.2 R, and 0.7 I episodes, P = .002), but tolerated full feedings sooner than patients fed with a regimented or intermediate schedule. No patient required additional therapy or readmission after tolerating two consecutive full feedings, suggesting that this might be a suitable discharge criterion for most patients with HPS.

Background/purpose: For over 50 years there has been debate over how to manage the contralateral groin in children who present with a unilateral inguinal hernia. Many preoperative and intraoperative tools to diagnose a contralateral patent processus vaginalis or true inguinal hernia have been described. In 1992 laparoscopy was introduced as a new diagnostic test. Although multiple series have assessed this new tool, none of them have been able to statistically show that laparoscopy is effective in assessing the contralateral groin. By combining all published studies and using the technique of meta-analysis, intraoperative laparoscopy can be shown to be effective in diagnosing a contralateral patent processus vaginalis in children undergoing unilateral inguinal herniorrhaphy., Methods: All available studies of children with a unilateral hernia who had exploration of the contralateral groin by laparoscopy were reanalyzed. Sensitivity and specificity of laparoscopy was determined using open exploration or development of a metachronous hernia as the gold standard., Results: Nine hundred sixty-four patients were suitable for analysis. A contralateral hernia was seen on laparoscopy in 376 patients. All of these patients underwent open contralateral exploration. A patent processus vaginalis or true hernia sac was found in 373. The sensitivity of laparoscopy was 99.4% (95% confidence interval 97.87 to 99.91). Five hundred eighty-eight patients had a laparoscopy with negative results. Sixty-two of these patients then had open contralateral exploration. In one case, a patent processus vaginalis was found; the other 61 patients underwent exploration with negative results. In the remaining 526 laparoscopy-negative patients, follow-up (1 month to 3 years) was used to see if a contralateral hernia developed. A metachronous hernia developed in one of the 526 patients. The specificity of laparoscopy was 99.5% (95% confidence interval 98.39 to 99.87). Laparoscopy added an average of 6 minutes to the surgical time and was accurate regardless of the technique. There were two minor complications related to laparoscopy and no deaths., Conclusions: Laparoscopy may be the ideal tool to diagnose a contralateral patent processus vaginalis intraoperatively. It is sensitive, specific, fast, and safe. Although the presence of a patent processus does not imply that the patient will go on to develop a metachronous hernia, identifying and ligating a patent processus should certainly prevent the development of an indirect inguinal hernia.

Ventricular tachycardia has been reported after blunt cardiac trauma in both children and adults. However, to the best of our knowledge, there are no reports of sudden onset of ventricular tachycardia at the time of surgical closure of gastroschisis. This case describes a patient with gastroschisis who developed a medically resistant ventricular tachycardia upon reduction of the gastroschisis.

Giant omphalocele is a major clinical challenge for pediatric surgeons. Whereas small- to medium-sized defects can be repaired primarily, larger omphaloceles cannot be closed at birth because the liver and small bowel have lost the right of domain to the abdomen. Two divergent strategies have evolved for treating these giant defects: (1) use of a silon chimney with gradual reduction of the contents of the sac, and (2) initial nonoperative management (epithelialization) of the omphalocele followed by repair of the residual ventral hernia. In an 18-year retrospective study, we have reviewed our experience with these treatment methods. Ninety-four infants underwent treatment for omphalocele between 1975 and 1993. Primary closure (PC) was possible in 55 patients, silon chimney (SC) was used in 15, and 7 had nonoperative management (NM) with epithelialization. In the remaining 17 infants, surgery was believed to be inappropriate because of the lethality of their associated anomalies. Major (but potentially survivable) anomalies were present in 26% of PC, 13% of SC, and 71% of the NM group patients. The majority of the liver was present in 73% of SC- and 86% of NM-treated omphaloceles. There was a decrease in length of stay, time to enteral feeding, and mortality over the 18-year period. However, those patients whose defects could not be closed primarily had consistently longer hospital stays. This was particularly true for the SC patients. The decreased use of total parenteral nutrition seems to reflect a shift from SC to NM rather than a decrease in the interval to full enteral feeding in any given treatment group over time.(ABSTRACT TRUNCATED AT 250 WORDS)

Congenital cystic adenomatoid malformation (CCAM), pulmonary sequestration, congenital lobar emphysema (CLE), and bronchogenic cysts are all congenital malformations of the lung that present in imaging studies as abnormal air, air/fluid, or fluid-filled cysts. The embryology, histology, clinical presentation, and treatment of these lesions are discussed based on world literature and our experience with 22 operative resections of congenital lung cysts over the past 10 years. The roles of prenatal diagnosis and fetal surgery in the management of certain lung cysts are considered. Computed tomography has emerged as an extremely useful tool in the differential diagnosis of these lesions. Surgical excision by segmentectomy or lobectomy for intrapulmonary lesions and simple excision for extralobar sequestrations and bronchogenic cysts are safe and have a low incidence of complications. Asymptomatic patients with CLE may not require resection. Overall, with accurate diagnosis and preoperative planning these rare but fascinating anomalies can be treated safely and effectively, with excellent results.

Assembly of class I major histocompatibility complex (MHC) molecules involves the interaction of two distinct polypeptides (the heavy and light chains) with peptide antigen. Cell lines synthesizing both chains but expressing low levels of MHC class I molecules on their surface as a result of a failure in assembly and transport have been identified. We now report that although the apparent steady-state distribution in these cells of class I molecules is in the endoplasmic reticulum (ER), the molecules in fact are recycled between the ER and Golgi, rather than retained in the ER. This explains the failure of class I molecules to negotiate the secretory pathway. Class I molecules do not seem to be modified by Golgi enzymes, suggesting that the proteins do not reach the Golgi apparatus during recycling. But morphological and subcellular fractionation evidence indicates that they pass through the cis Golgi or a Golgi-associated organelle, which we postulate to be the recycling organelle. This compartment, which we call the 'cis-Golgi network', would thereby be a sorting organelle that selects proteins for return to the ER.

Models for antigen presentation have divided the world of antigens into two categories, endogenous and exogenous, presented to T cells by class I and class II major histocompatibility complex (MHC) encoded molecules, respectively. Exogenous antigens are though to be taken up into peripheral endosomal compartments where they are processed for binding to class II MHC molecules. Endogenous antigens are either synthesized or efficiently delivered to the cytoplasm before being partially degraded in an as yet undefined way, and complexed with class I MHC molecules. A useful phenotypic distinction between the two pathways has been the sensitivity to weak bases, such as chloroquine, which is a property only of the exogenous pathway. The fungal antibiotic brefeldin A (BFA), which blocks protein transport from the endoplasmic reticulum to the Golgi network, also blocks class I-restricted antigen-presentation, providing us with the corresponding marker of the endogenous pathway. Experiments with influenza virus antigens have supported the view that class II MHC molecules can present exogenous but not endogenous antigen, whereas the observation that class II MHC molecules present measles virus non-membrane antigens by a chloroquine-insensitive pathway suggests that this is not always the case. We show here that influenza A matrix protein can be effectively presented to class II-restricted T cells by two pathways: one of which is chloroquine-sensitive, BFA-insensitive, the other being chloroquine-insensitive and BFA-sensitive. Our results indicate that both class I and class II molecules can complex with antigenic peptides in a pre-Golgi compartment and favour a unified mechanism for MHC-restricted endogenous antigen presentation.

Most antigens must be processed intracellularly before they can be presented, in association with major histocompatibility complex (MHC) molecules at the cell surface, for recognition by the antigen-specific receptor of T cells. This processing appears to involve cleavage of protein antigens to smaller peptides. Only certain fragments of any protein can serve as T-cell epitopes and this is, at least in part, determined by the requirement that peptides be able to bind the MHC molecules. Class I restricted antigens are derived from proteins, such as viral antigens, that are synthesized within the presenting cell. Many of these antigens are cytosolic proteins and recent evidence suggests that it is in the cytosol that these proteins are processed to produce either the antigenic peptides or processed intermediates. How and where these processed cytosolic antigens cross the membrane of the vacuolar system and bind to the extracellular domain of the class I molecule is not known but one obvious site for this process is the endoplasmic reticulum (ER), because this organelle is specialized to translocate proteins across the membrane from the cytosol into the secretory system. Based on this model, we reasoned that if we could pharmacologically block the movement of proteins out of the ER, endogenous antigen presentation would cease. An agent which causes such an effect is available--the fungal antibiotic Brefeldin A (BFA). Consistent with the above hypothesis, we report that BFA completely abolishes the ability of a cell to present endogenously synthesized antigens to class I restricted cytotoxic T cells.