Localisation of the SMC loading complex Nipbl/Mau2 during mammalian meiotic prophase I.

Evidence from lower eukaryotes suggests that the chromosomal associations of all the structural maintenance of chromosome (SMC) complexes, cohesin, condensin and Smc5/6, are influenced by the Nipbl/Mau2 heterodimer. Whether this function is conserved in mammals is currently not known. During mammalian meiosis, very different localisation patterns have been reported for the SMC complexes, and the localisation of Nipbl/Mau2 has just recently started to be investigated. Here, we show that Nipbl/Mau2 binds on chromosomal axes from zygotene to mid-pachytene in germ cells of both sexes. In spermatocytes, Nipbl/Mau2 then relocalises to chromocenters, whereas in oocytes it remains bound to chromosomal axes throughout prophase to dictyate arrest. The localisation pattern of Nipbl/Mau2, together with those seen for cohesin, condensin and Smc5/6 subunits, is consistent with a role as a loading factor for cohesin and condensin I, but not for Smc5/6. We also demonstrate that Nipbl/Mau2 localises next to Rad51 and γH2AX foci. NIPBL gene deficiencies are associated with the Cornelia de Lange syndrome in humans, and we find that haploinsufficiency of the orthologous mouse gene results in an altered distribution of double-strand breaks marked by γH2AX during prophase I. However, this is insufficient to result in major meiotic malfunctions, and the chromosomal associations of the synaptonemal complex proteins and the three SMC complexes appear cytologically indistinguishable in wild-type and Nipbl (+/-) spermatocytes.

The genetics of alcohol dependence: advancing towards systems-based approaches.

BACKGROUND: Personalized treatment for psychopathologies, in particular alcoholism, is highly dependent upon our ability to identify patterns of genetic and environmental effects that influence a person's risk. Unfortunately, array-based whole genome investigations into heritable factors that explain why one person becomes dependent upon alcohol and another does not, have indicated that alcohol's genetic architecture is highly complex. That said, uncovering and interpreting the missing heritability in alcohol genetics research has become all the more important, especially since the problem may extend to our inability to model the cumulative and combinatorial relationships between common and rare genetic variants. As numerous studies begin to illustrate the dependency of alcohol pharmacotherapies on an individual's genotype, the field is further challenged to identify new ways to transcend agnostic genomewide association approaches. We discuss insights from genetic studies of alcohol related diseases, as well as issues surrounding alcohol's genetic complexity and etiological heterogeneity. Finally, we describe the need for innovative systems-based approaches (systems genetics) that can provide additional statistical power that can enhance future gene-finding strategies and help to identify heretofore-unrealized mechanisms that may provide new targets for prevention/treatments efforts. Emerging evidence from early studies suggest that systems genetics has the potential to organize our neurological, pharmacological, and genetic understanding of alcohol dependence into a biologically plausible framework that represents how perturbations across evolutionarily robust biological systems determine susceptibility to alcohol dependence.

Multispecies model of cell lineages and feedback control in solid tumors.

We develop a multispecies continuum model to simulate the spatiotemporal dynamics of cell lineages in solid tumors. The model accounts for protein signaling factors produced by cells in lineages, and nutrients supplied by the microenvironment. Together, these regulate the rates of proliferation, self-renewal and differentiation of cells within the lineages, and control cell population sizes and distributions. Terminally differentiated cells release proteins (e.g., from the TGFß superfamily) that feedback upon less differentiated cells in the lineage both to promote differentiation and decrease rates of proliferation (and self-renewal). Stem cells release a short-range factor that promotes self-renewal (e.g., representative of Wnt signaling factors), as well as a long-range inhibitor of this factor (e.g., representative of Wnt inhibitors such as Dkk and SFRPs). We find that the progression of the tumors and their response to treatment is controlled by the spatiotemporal dynamics of the signaling processes. The model predicts the development of spatiotemporal heterogeneous distributions of the feedback factors (Wnt, Dkk and TGFß) and tumor cell populations with clusters of stem cells appearing at the tumor boundary, consistent with recent experiments. The nonlinear coupling between the heterogeneous expressions of growth factors and the heterogeneous distributions of cell populations at different lineage stages tends to create asymmetry in tumor shape that may sufficiently alter otherwise homeostatic feedback so as to favor escape from growth control. This occurs in a setting of invasive fingering, and enhanced aggressiveness after standard therapeutic interventions. We find, however, that combination therapy involving differentiation promoters and radiotherapy is very effective in eradicating such a tumor.

Size-normalized Robustness of Dpp Gradient in Drosophila Wing Imaginal Disc.

Exogenous environmental changes are known to affect the intrinsic characteristics of biological organizms. For instance, the synthesis rate of the morphogen decapentaplegic (Dpp) in a Drosophila wing imaginal disc has been found to double with an increase of 5.9°C in ambient temprerature. If not compensated, such a change would alter the signaling Dpp gradient significantly and thereby the development of thewing imaginal disc. To learn how flies continue to develop "normally" under such an exogenous change, we formulate in this paper a spatially two-dimensional reaction-diffusion system of partial differential equations (PDE) that accounts for the biological processes at work in the Drosophila wing disc essential for the formation of signaling Dpp gradient. By way of this PDE model, we investigate the effect of the apical-basal thickness and antero-posterior span of the wing on the shape of signaling gradients and the robustness of wing development in an altered environment (including an enhanced morphogen synthesis rate). Our principal result is a delineation of the role of wing disc size change in maintaining the magnitude and shape of the signaling Dpp gradient. The result provides a theoretical basis for the observed robustness of wing development, preserving relative but not absolute tissue pattern, when the morphogen synthesis rate is significantly altered. A similar robustness considerqation for simultaneous changes of multiple intrinsic system characteristics is also discussed briefly.

Localized Ectopic Expression of Dpp Receptors in a Drosophila Embryo.

Receptor-mediated BMP degradation has been seen to play an important role in allowing for the formation of relatively stable P Mad patterns. To the extent that receptors act as a "sink" for BMPs, one would predict that the localized over-expression of signaling receptors would cause a net flux of freely diffused BMPs toward the ectopic, i.e., abnormally high concentration, receptor site. One possible consequence would be a depression of BMP signaling in adjacent areas since less BMPs are now available for binding with the same normal concentration of receptors at the adjacent areas. However, recent experiments designed to examine this possible effect were inconclusive. In this paper, we investigate the possibility of depression of Dpp signaling outside the area of elevated tkv in a Drosophila embryo by modeling mathematically the basic biological processes at work in terms of a system of nonlinear reaction diffusion equations with spatially varying (and possibly discontinuous) system properties. The steady state signaling morphogen gradient is investigated by the method of matched asymptotic expansions and by numerical simulations.

Membrane-associated non-receptors and morphogen gradients.

A previously investigated basic model (System B) for the study of signaling morphogen gradient formation that allows for reversible binding of morphogens (aka ligands) with signaling receptors, degradation of bound morphogens and diffusion of unbound morphogens is extended to include the effects of membrane-bound non-signaling molecules (or non-receptors for short) such as proteoglycans that bind reversibly with the same morphogens and degrade them. Our main goal is to delineate the effects of the presence of non-receptors on the existence and properties of the steady-state concentration gradient of signaling ligand-receptor complexes. Stability of the steady-state morphogen gradients is established and the time to reach steady-state behavior after the onset of morphogen production will be analyzed. The theoretical findings offer explanations for observations reported in several previous experiments on Drosophila wing imaginal discs.

Aggregation of a Distributed Source in Morphogen Gradient Formation.

In the development of a biological entity, ligands (such as Decapentaplegic (Dpp) along the anterior-posterior axis of the Drosophila wing imaginal disc) are synthesized at a localized source and transported away from the source for binding with cell surface receptors to form concentration gradients of ligand-receptor complexes for cell signaling. Generally speaking, activities such as diffusion and reversible binding with degradable receptors also take place in the region of ligand production. The effects of such morphogen activities in the region of localized distributed ligand source on the ligand-receptor concentration gradient in the entire biological entity have been modeled and analyzed as System F in [1]. In this paper, we deduce from System F, a related end source model (System A) in which the effects of the distributed ligand source is replaced by an idealized point stimulus at the border between the (posterior) chamber and the ligand production region that simulates the average effects of the ligand activities in the production zone. This aggregated end source model is shown to adequately reproduce the significant implications of System F and to contain the corresponding ad hoc point source model, System R of [2], as a special case. Because of its simpler mathematical structure and the absence of any limitation on the ligand synthesis rate for the existence of steady-state gradients, System A type models are expected to be used widely. An example of such application is the recent study of the inhibiting effects of the formation of nonsignaling ligand-nonreceptor complexes [3].

A multicenter, randomized, double-blind, placebo-controlled trial of the prokinetic agent erythromycin in the postoperative recovery of infants with gastroschisis.

BACKGROUND/PURPOSE: The recovery of gut function after repair of gastroschisis is frequently prolonged, and these infants are prone to complications associated with parenteral nutrition. This trial was designed to investigate the effect of the prokinetic agent, erythromycin, on the attainment of full enteral feeding in infants after primary repair of uncomplicated gastroschisis. METHODS: A multicenter, randomized, double-blind, placebo-controlled trial was used to investigate the effect of enteral erythromycin (3 mg/kg/dose 4 times daily) compared with placebo on the attainment of full enteral feeding tolerance after primary repair of uncomplicated gastroschisis. Eleven neonatal surgical units in the United Kingdom participated in the study. The primary end-point was the time taken to achieve continuous enteral feeding at 150 mL/kg/24 hours sustained for 48 hours. RESULTS: Of 70 eligible infants, 62 were recruited and randomly divided. There were 30 patients in group I (placebo) and 32 in group II (erythromycin). The groups were comparable in terms of mean gestational age, mean birth weight, extent of evisceration, and degree of intestinal peel. There was no statistically significant difference between the 2 groups in the time taken to achieve full enteral feeding (27.2 v 28.7 days; P =.75). Similarly, no significant differences were found in the incidence of catheter-related sepsis, duration of parenteral nutrition, or time to discharge between the 2 groups. CONCLUSIONS: Enterally administered erythromycin at a dose of 3 mg/kg 4 times daily conferred no advantage in the time taken to achieve full enteral feeding after primary repair of uncomplicated gastroschisis.

Giant-cell fibroblastoma in a patient with a bone-marrow transplant.

A case of an epigastric giant-cell fibroblastoma is reported in a 6-year-old girl who had undergone a bone-marrow transplant for severe combined immunodeficiency secondary to adenosine deaminase deficiency. A small subcutaneous nodule had been excised from the epigastrium at age 12 months.

Basal forebrain cholinergic cell attachment and neurite outgrowth on organotypic slice cultures of hippocampal formation.

Distributions of somata and neurites of cholinergic neurons were studied after seeding dissociated cells onto organotypic slice cultures. Slice cultures were made from hippocampal formation and adjacent cortical regions from rats or mice. Dissociated cell suspensions of basal forebrain tissue from rat or mouse fetuses were seeded onto the slice cultures. Combined cultures were maintained for 1-21 days in vitro. Cultures processed for acetylcholinesterase (AChE) histochemistry demonstrated non-random patterns of cholinergic cells and their neurites. Labeled cells appeared most frequently in the molecular layer of the dentate gyrus, and in the deeper layers of cortical regions adjacent to the hippocampus. Neurites extending from these labeled cells appeared to target the dentate molecular layer and the cortical subplate layer. By 4 days in vitro, AChE-positive basal forebrain cells display several short and thick neurites that appear to be dendrites, and one long process that appears to be an axon. By 5 days in vitro, dendrites are well developed; by 7 days the presumed axon has extended widely over the cortical target zone. These neurites are maintained through 3 weeks in culture. Distributions of cells varied with the age of the slice. AChE-labeled cells were not seen overlying hippocampal tissue when dissociated cells were seeded on slice cultures made from day 0 rats, but a few labeled cells were seen when seeded on slices from day 2 rats. Clear non-random patterns of labeled cells and neurite outgrowth were seen on slice cultures from day 5 or older pups. The non-random distribution seen with AChE-positive neurons was not seen using other techniques that labeled all cells (non-selective fluorescent labels) or all neurons; these techniques resulted in labeled cells scattered apparently homogenously across the slice culture.These studies demonstrate a non-random pattern of attachment or differentiation of basal forebrain cholinergic neurons when these cells are seeded onto cultured cortical slices; this pattern mimics the normal patterns of basal forebrain cholinergic projections to these cortical regions. These data suggest that the factors that normally guide basal forebrain-derived cholinergic axons to their target cells in vivo are present and detectable in this model system.

Anterior resection for megarectosigmoid in congenital anorectal malformations.

PURPOSE: The aim of this study was to review the effectiveness of resecting dilated distal bowel in children suffering unmanageable constipation or soiling who have been operated on previously for anorectal malformations. METHODS: A retrospective review was performed of 9 children. Each child underwent excision of dilated bowel to leave normal caliber bowel anastomosed by hand to a rectal reservoir at the peritoneal reflection. The documented follow-up was reviewed. RESULTS: The 9 children had primary surgery for the following anomalies: high (n = 1), intermediate (n = 1), low (n = 3), rectal stenosis (n = 3), and anal stenosis (n = 3), Seven children had persistent fecalomas, and 7 had major problems with soiling. All were on large doses of laxatives, with 5 having regular rectal washouts and 4 having regular enemas. In all radiologic studies there was a prompt change from normal caliber bowel to dilated bowel at the upper limit of the dilatation. The mean age at operation for excision was 4 years, 11 months (range, 11 months to 9 years, 11 months). The mean period of follow-up was 4 years, 7 months (range, 2 years, 3 months to 10 years). Follow-up showed that all children improved. None had major complications. All were having between one and 3 bowel actions per day. Three continued to soil but improved. Of the remaining 6, only 2 required occasional laxatives and had regular spontaneous bowel actions without soiling. No child was having enemas or washouts. CONCLUSION: Anterior resection for the treatment of megarectosigmoid is a safe and effective procedure.

Postoperative pneumatosis intestinalis in infants does not automatically preclude enteral feeding.

BACKGROUND: A degree of feed intolerance after neonatal abdominal surgery is common but in an otherwise well baby enteral feeding usually is continued at the highest tolerated level. However, the presence of rectal bleeding, pneumatosis intestinalis, or portal vein gas seen on plain abdominal x-rays suggest the possibility of postoperative necrotising enterocolitis. When this happens feedings usually are stopped for 7 to 10 days, and intravenous antibiotics and total parental nutrition are commenced. METHODS: The authors report 12 episodes of rectal bleeding and 11 episodes of pneumatosis intestinalis in 3 infants who previously had undergone neonatal abdominal surgery for intestinal malformations. In 7 of these episodes, feedings were neither stopped nor were antibiotics given. At the time of these 7 episodes, the infants were more than 3 kg in weight, had no significant cardiac or respiratory pathology, were all clinically stable, had no evidence of peritonitis, had no thrombocytopenia, and were greater than 37 weeks postconception. RESULTS: The 3 infants were monitored closely. There were no early or late problems observed attributable to this management. CONCLUSION: Carefully selected clinically stable patients that have postoperative pneumatosis intestinalis or exhibit rectal bleeding may be successfully managed by reduced enteral feedings with no antibiotics. J Pediatr Surg 36:1820-1823.

The primary management of testicular maldescent in gastroschisis.

Gastroschisis (GS) is the commonest abdominal-wall defect in the Western world. The conventional practice has been reduction of the viscera and closure of the abdominal wall as an emergency procedure. The testis is often a part of the prolapsed viscera along with the bowel loops, stomach, fallopian tube, etc. The primary management of prolapsed (PT) (3) and intra-abdominal (5) testes (IAT) in this condition was studied in 16 consecutive male babies with GS, each was managed by simple reposition of the testes and closure of the abdominal wall. The babies were followed up for spontaneous descent of the testes. At 18-month follow-up, all five IAT had descended into the scrotum spontaneously and were palpably normal. Of the three extra-abdominal PT, two had descended into the scrotum and were normal in size and on palpation. One was palpable in the superficial inguinal pouch. Simple reposition of the testes into the abdomen and closure of the abdominal defect is the correct approach for primary management of PT or IAT in a newborn with GS.

Glypican-1 is overexpressed in human breast cancer and modulates the mitogenic effects of multiple heparin-binding growth factors in breast cancer cells.

Glypicans are a family of glycosylphosphatidylinositol-anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. Here we show that glypican-1 is strongly expressed in human breast cancers, whereas expression of glypican-1 is low in normal breast tissues. In contrast, the expression of glypican-3 and -4 is only slightly increased in breast cancers by comparison with normal breast tissues, and glypican-2 and -5 are below the level of detection by Northern blotting in both normal and cancer samples. Treatment of MDA-MB-231 and MDA-MB-468 breast cancer cells with phosphoinositide-specific phospholipase-C abrogated the mitogenic response to two heparin-binding growth factors, heparin-binding epidermal growth factor-like growth factor and fibroblast growth factor 2. Stable transfection of these cells with a glypican-1 antisense construct markedly decreased glypican-1 protein levels and the mitogenic response to the same heparin-binding growth factors, as well as that to heregulin alpha, heregulin beta, and hepatocyte growth factor. Syndecan-1 was also expressed at high levels in both breast cancer tissues and breast cancer cells when compared with normal breast tissues. There was a good correlation between glypican-1 and syndecan-1 expression in the tumors. However, clones expressing the glypican-1 antisense construct did not exhibit decreased syndecan-1 levels, indicating that loss of responsiveness to heparin-binding growth factors in these clones was not due to altered syndecan-1 expression. Furthermore, 8 of 10 tumors with stage 2 or 3 disease exhibited high levels of glypican-1 by Northern blot analysis. In contrast, low levels of glypican-1 mRNA were evident in 1 of 10 tumors with stage 2 or 3 disease and in 9 of 10 tumors with stage 1 disease. Taken together, these data suggest that glypican-1 may play a pivotal role in the ability of breast cancer cells to exhibit a mitogenic response to multiple heparin-binding growth factors and may contribute to disease progression in this malignancy.

Cell surface glypicans are low-affinity endostatin receptors.

Endostatin, a collagen XVIII fragment, is a potent anti-angiogenic protein. We sought to identify its endothelial cell surface receptor(s). Alkaline phosphatase- tagged endostatin bound endothelial cells revealing two binding affinities. Expression cloning identified glypican, a cell surface proteoglycan as the lower-affinity receptor. Biochemical and genetic studies indicated that glypicans' heparan sulfate glycosaminoglycans were critical for endostatin binding. Furthermore, endostatin selected a specific octasulfated hexasaccharide from a sequence in heparin. We have also demonstrated a role for endostatin in renal tubular cell branching morphogenesis and show that glypicans serve as low-affinity receptors for endostatin in these cells, as in endothelial cells. Finally, antisense experiments suggest the critical importance of glypicans in mediating endostatin activities.

Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a common malignant tumour worldwide, and its differential diagnosis from benign lesions of the liver is often difficult yet of great clinical importance. In the present study, we analysed whether glypican-3 is useful in differentiating between benign and malignant liver diseases and whether it influences the growth behaviour of HCC. METHODS: Northern blot analysis and in situ hybridisation. RESULTS: Northern blot analysis indicated that expression of glypican-3 mRNA was either low or absent in normal liver, in focal nodular hyperplasia (FNH), and in liver cirrhosis. In contrast, expression of glypican-3 mRNA was markedly increased in 20 of 30 and moderately increased in five of 30 HCC samples. The average increase in glypican-3 mRNA expression in HCC was significant compared with expression in normal liver (21.7-fold increase, p<0.01). In comparison with FNH or liver cirrhosis, glypican-3 mRNA expression in HCC was increased 7.2- (p<0.05) and 10.8-fold (p<0.01), respectively. In addition, pushing HCCs exhibited significantly higher glypican-3 mRNA expression than invading tumours (p<0.05). In situ hybridisation analysis demonstrated weak expression of glypican-3 mRNA in normal hepatocytes and bile ductular cells, and weak to occasionally moderate signals in hepatocytes forming nodules of liver cirrhosis and in regenerated hepatic nodules of FNH. In contrast, glypican-3 in situ hybridisation signals were intense in hepatic cancer cells with even higher levels in pushing HCCs than in invading HCCs. CONCLUSIONS: These findings suggest that glypican-3, in many cases, has the potential to differentiate between benign and malignant liver diseases.

Mechanisms underlying preferential assembly of heparan sulfate on glypican-1.

Glypicans are major cell surface heparan sulfate proteoglycans, the structures of which are characterized by the presence of a cysteine-rich globular domain, a short glycosaminoglycan (GAG) attachment region, and a glycosylphosphatidylinositol membrane anchor. Despite strong evolutionary conservation of the globular domains of glypicans, no function has yet been attributed to them. By using a novel quantitative approach for assessing proteoglycan glycosylation, we show here that removal of the globular domain from rat glypican-1 converts the proteoglycan from one that bears approximately 90% heparan sulfate (HS) to one that bears approximately 90% chondroitin sulfate. Mutational analysis shows that sequences at least 70 amino acids away from the glypican-1 GAG attachment site are required for preferential HS assembly, although more nearby sequences also play a role. The effects of the glypican-1 globular domain on HS assembly could also be demonstrated by fusing this domain to sequences representing the GAG attachment sites of other proteoglycans or, surprisingly, simply by expressing the isolated globular domain in cells and analyzing effects either on an exogenously expressed glypican-1 GAG attachment domain or on endogenous proteoglycans. Quantitative analysis of the effect of the globular domain on GAG addition to proteoglycan core proteins suggested that preferential HS assembly is achieved, at least in part, through the inhibition of chondroitin sulfate assembly. These data identify the glypican-1 globular domain as a structural motif that potently influences GAG class determination and suggest that an important role of glypican globular domains is to ensure a high level of HS substitution of these proteoglycans.

Regulation of neurite outgrowth by integrin activation.

During late-embryonic development, retinal neurons lose the ability to attach and extend neurites on the extracellular matrix molecule laminin-1 (LN-1), despite the fact that they retain expression of integrin receptors for LN-1. Here we show that the developmental loss of responsiveness to LN-1 can be reversed by treatments that increase the activation state of integrins. Both extracellular application of Mn(2+) (at micromolar concentrations) and viral-mediated neuronal expression of a constitutively active form of the ras-related GTPase R-ras (R-ras(38V)) potently promoted late-embryonic retinal neurite outgrowth on LN-1 substrata. In both cases, outgrowth was mediated by integrin alpha6beta1 and not alpha3beta1, even though these neurons express alpha3beta1 and use it for outgrowth on other laminin isoforms, as well as on LN-1 that has been proteolytically or conformationally activated (Ivins et al., 1998). Mn(2+)-and to a much lesser extent R-ras(38V)-also reversed the developmental loss of retinal neuron responsiveness to type IV collagen, by promoting the function of integrin alpha1beta1. Interestingly, the responses of other late-embryonic CNS neurons to LN-1 were also enhanced by treatments that activate integrin function, but those of peripheral nervous system neurons (dorsal root ganglion neurons) were either not enhanced (embryonic neurons) or only modestly improved (adult neurons). These results suggest that a developmental decline occurs in the activation state of neuronal integrins, particularly among CNS neurons. Such a decline may underlie some of the intrinsic loss of regenerative ability sustained by CNS neurons during development and may be a valid target for therapeutic intervention.

Pyloric atresia: five new cases, a new association, and a review of the literature with guidelines.

BACKGROUND/PURPOSE: Pyloric atresia is an uncommon condition occurring in 1 of 100,000 live births. When occurring in isolation, the clinical course usually is uncomplicated after surgical treatment. However, it may occur in association with other congenital abnormalities. The authors present 5 new cases, 3 of associated abnormalities including 1 of esophageal atresia and 2 of agenesis of the gall bladder and malrotation. Agenesis of the gall bladder has not been described previously in combination with pyloric atresia. The literature has been reviewed and guidelines are suggested for the management. METHODS: The case records of 4 neonates who presented to the author's institution between January 1998 and June 1999 and 1 who presented at another center in 1991 were reviewed. A Medline literature search was performed, and guidelines were developed for the management of this condition based on our cases and the literature review. RESULTS: Patients 1 and 5 had no associated anomalies. Patient 2 had associated esophageal atresia, tracheoesophageal fistula, atrial septal defect, crossed renal ectopia, malrotation, and absent gall bladder. Patient 3 had a rectovestibular fistula, vaginal atresia, atrial septal defect, malrotation absent gallbladder, and absent extrahepatic portal vein. Patient 4 had epidermolysis bullosa. Patients 2 and 5 had unremarkable recoveries, patients 2 and 3 had markedly delayed gastric emptying that responded to cisapride. Patient 3 has portal hypertension and remains under close follow-up. Patient 4 died at 22 days of age of pseudomonas sepsis. CONCLUSIONS: Based on our cases and literature review, we have adopted the following guidelines: (1) All children with pyloric atresia should be screened for multiple anomalies. (2) Delayed gastric emptying should be considered early and may respond to prokinetic agents. (3) Association with Epidermolysis bullosa should not preclude surgical treatment. (4) A skin biopsy specimen should be taken at the time of surgery for electron microscopy if there is a family history of epidermolysis bullosa.