Efficacy of Cranial Orthosis for Plagiocephaly Based on 2D and 3D Evaluation.

Background With the advent of cranial orthoses as therapeutic medical devices for the treatment of severe positional head deformities in Japan, an increasing number of patients are being treated with them. However, assessing the effectiveness of a treatment is often difficult due to the use of different metrics. This study aimed to evaluate the effectiveness of cranial orthoses for deformational plagiocephaly using two- (2D) and three-dimensional (3D) evaluation metrics. Methods We conducted a retrospective study of infant patients with deformational plagiocephaly who underwent cranial orthosis treatment. We evaluated the severity of deformational plagiocephaly using cranial asymmetry (CA) and the cranial vault asymmetry index (CVAI) as 2D metrics, and anterior and posterior symmetry ratios as 3D metrics. The patients were divided into 24 subgroups based on the initial severity of each outcome and their age at the start of treatment. We analyzed the changes in outcomes and correlations within improvements across the age and severity categories. Results Overall, 1,038 infants were included in this study. The mean CA, CVAI, and anterior and posterior symmetry ratios improved significantly after cranial orthosis treatment. The improvement in each score was greater in patients with more severe initial deformities and in those who underwent treatment at a younger age. Conclusion Cranial orthosis treatment was effective in correcting deformational plagiocephaly in infants, as demonstrated by improvements in both 2D and 3D metrics. Patients with more severe initial deformities and those who underwent treatment at a younger age showed greater improvement.

Design and Evaluation of Engineered Extracellular Vesicle (EV)-Based Targeting for EGFR-Overexpressing Tumor Cells Using Monobody Display.

BACKGROUND: Extracellular vesicles (EVs) are attracting interest as a new class of drug delivery vehicles due to their intrinsic nature of biomolecular transport in the body. We previously demonstrated that EV surface modification with tissue-specific molecules accomplished targeted EV-mediated DNA delivery. METHODS: Here, we describe reliable methods for (i) generating EGFR tumor-targeting EVs via the display of high-affinity monobodies and (ii) in vitro measurement of EV binding using fluorescence and bioluminescence labeling. Monobodies are a well-suited class of small (10 kDa) non-antibody scaffolds derived from the human fibronectin type III (FN3) domain. RESULTS: The recombinant protein consists of the EGFR-targeting monobody fused to the EV-binding domain of lactadherin (C1C2), enabling the monobody displayed on the surface of the EVs. In addition, the use of bioluminescence or fluorescence molecules on the EV surface allows for the assessment of EV binding to the target cells. CONCLUSIONS: In this paper, we describe methods of EV engineering to generate targeted delivery vehicles using monobodies that will have diverse applications to furnish future EV therapeutic development, including qualitative and quantitative in vitro evaluation for their binding capacity.

Sparsely methylated mitochondrial cell free DNA released from cardiomyocytes contributes to systemic inflammatory response accompanied by atrial fibrillation.

Systemic inflammation is assumed to be the consequence and the cause of atrial fibrillation (AF); however, the underlying mechanism remains unclear. We aimed to evaluate the level of cell-free DNA (cfDNA) in patients with AF and AF mimicking models, and to illuminate its impact on inflammation. Peripheral blood was obtained from 54 patients with AF and 104 non-AF controls, and cfDNA was extracted. We extracted total cfDNA from conditioned medium after rapid pacing to HL-1 cells. Nuclear and mitochondrial DNA were separately extracted and fragmented to simulate nuclear-cfDNA (n-cfDNA) and mitochondrial-cfDNA (mt-cfDNA). The AF group showed higher cfDNA concentration than the non-AF group (12.6 [9.0-17.1] vs. 8.1 [5.3-10.8] [ng/mL], p < 0.001). The copy numbers of n-cfDNA and mt-cfDNA were higher in AF groups than in non-AF groups; the difference of mt-cfDNA was particularly apparent (p = 0.011 and p < 0.001, respectively). Administration of total cfDNA and mt-cfDNA to macrophages significantly promoted IL-1ß and IL-6 expression through TLR9, whereas n-cfDNA did not. Induction of cytokine expression by methylated mt-cfDNA was lower than that by unmethylated mt-cfDNA. Collectively, AF was associated with an increased cfDNA level, especially mt-cfDNA. Sparsely methylated mt-cfDNA released from cardiomyocytes may be involved in sterile systemic inflammation accompanied by AF.

A Comprehensive Review of Cancer MicroRNA Therapeutic Delivery Strategies.

In the field of molecular oncology, microRNAs (miRNAs) and their role in regulating physiological processes and cancer pathogenesis have been a revolutionary discovery over the last decade. It is now considered that miRNA dysregulation influences critical molecular pathways involved in tumor progression, invasion, angiogenesis and metastasis in a wide range of cancer types. Hence, altering miRNA levels in cancer cells has promising potential as a therapeutic intervention, which is discussed in many other articles in this Special Issue. Some of the most significant hurdles in therapeutic miRNA usage are the stability and the delivery system. In this review, we cover a comprehensive update on the challenges and strategies for the development of therapeutic miRNA delivery systems that includes virus-based delivery, non-viral delivery (artificial lipid-based vesicles, polymer-based or chemical structures), and recently emerged extracellular vesicle (EV)-based delivery systems.

A case of secretory breast cancer in a 6 year-old girl: is it possible to make a correct preoperative diagnosis?

Secretory breast carcinoma constitutes the majority of breast cancers in children and young people less than 20 years of age. Noninvasive examination is particularly necessary for the diagnosis of breast carcinoma in children. Herein, we report a case of secretory breast carcinoma in a 6-year-old girl with psychomotor retardation. She was referred to our outpatient clinic for evaluation of a palpable mass in her left breast. A hard mass, rather than the increase in size typical of premature thelarche, was palpated. An excision biopsy was performed. Pathological findings revealed an invasive secretory breast carcinoma. We performed a retrospective review of the preoperative findings of this case, and compared it to the pathological diagnosis. Elastography, which can be performed without deep sedation or general anesthesia and without causing pain, resulted in a stiffness score of 4; however, the distinction between benign and malignant tumors on elastography, which is important to decide the intra-operative procedures, was not sufficient according to the Japanese breast cancer society clinical guidelines. This is the first report of secretory breast carcinoma in a child with a stiffness score determined by tissue elasticity imaging. A breast mass in a child with a high stiffness score of more than 4 on elastography should be referred for invasive diagnostic procedures, such as fine needle aspiration or excisional biopsy. According to our experience, an accurate preoperative diagnosis could be possible for malignant breast tumors in children. Such parameters as stiffness score on elastography are practical, noninvasive, and objective diagnostic tools for the accurate preoperative diagnosis of breast tumors in children.

Tracheal cartilage growth promotion by intra-tracheal administration of basic FGF.

PURPOSE: This study aimed to investigate whether intra-tracheal administration of basic fibroblast growth factor (b-FGF) promotes the growth of tracheal cartilage. METHODS: Trachea of 4-week old mice were intubated and 2.5 µg b-FGF administered (Group 4) for periods from 1 to 5 days. Cervical tracheal outer diameter and tracheal ring length were compared in Group 1 (no intervention), Group 2 (tracheal intubation), Group 3 (intra-tracheal administration of distilled water) and Group 4, at 8 weeks of age. Outer diameter and tracheal ring length in Group 4 were also compared with that in Group 1 at 12 and 16 weeks of age. RESULTS: At 8 weeks of age, tracheal ring length with b-FGF administration for more than 4 days in Group 4 was significantly increased over that following 1-day administration. At 8 weeks of age, mean outer diameter and the mean tracheal ring length in Group 4 were significantly greater than in the other groups. Mean outer diameter and mean tracheal ring length were significantly greater in Group 4 than in Group 1 at 12 and 16 weeks of age. CONCLUSION: This study has shown that intra-tracheal administration of b-FGF enlarges the tracheal lumen.

Long-term follow-up of tracheal cartilage growth promotion by intratracheal injection of basic fibroblast growth factor.

BACKGROUND: Intratracheal injection of basic fibroblast growth factor (b-FGF) has been shown to enlarge the tracheal lumen 4 weeks after treatment. The objective of this study was to investigate the long-term effect of tracheal cartilage growth promotion by intratracheal injection of b-FGF. MATERIALS AND METHODS: New Zealand white rabbits were classified into four groups to receive either distilled water alone (Group 1; n = 16; control), 40 µg (Group 2; n = 10), 100 µg (Group 3; n = 13), or 200 µg (Group 4; n = 16) of b-FGF dissolved in water. The treatment was injected into the posterior wall of the cervical trachea using a tracheoscope. The animals were sacrificed 4 or 12 weeks later. RESULTS: Four weeks after treatment, the mean luminal areas of tracheas for Groups 1, 2, 3, and 4 were 27.2, 25.6, 32.2, and 36.2 mm2, respectively. At 12 weeks, these were 29.3, 37.9, 42.5, and 56.0 mm2, respectively. The levels of glycosaminoglycan at 12 weeks were 93.9, 152.5, 123.2, and 210.6 µg/mg, respectively. At 12 weeks, the levels of type II collagen were 77.2, 133.1, 99.2, and 148.9 µg/mg, respectively. CONCLUSION: Twelve weeks after a single injection of b-FGF, the mean luminal area of the trachea continued to increase.

Tracheal cartilage growth by intratracheal injection of basic fibroblast growth factor.

BACKGROUND/PURPOSE: We have previously shown that intratracheal injection of slowly released (in gelatin) basic fibroblast growth factor (bFGF) significantly enlarged the tracheal lumen by a slight margin. This study aimed to investigate differences in tracheal cartilage growth by the intratracheal injection of bFGF doses in a rabbit model. METHODS: Water (group 1; n=7; control) or 100µg (group 2; n=8) or 200µg (group 3; n=8) of bFGF dissolved in water was injected into the posterior wall of the cervical trachea of New Zealand white rabbits using a tracheoscope. All animals were sacrificed four weeks later. RESULTS: The mean circumferences of cervical tracheas for groups 1, 2, and 3 were 18.8±0.83, 21.1±2.0, and 22.1±1.3mm, respectively. A significant difference was found between groups 1 and 2 (P=0.034) and groups 1 and 3 (P=0.004). The mean luminal areas of cervical tracheas for groups 1, 2, and 3 were 27.0±2.1, 32.2±4.8, and 36.3±4.6mm2, respectively. A significant difference was found between groups 1 and 3 (P=0.001). CONCLUSION: Intratracheal injection of bFGF in the dose range used significantly promoted the growth of tracheal cartilage in a rabbit model. LEVELS OF EVIDENCE: Level II at treatment study (animal experiment).

Promotion of muscle regeneration by myoblast transplantation combined with the controlled and sustained release of bFGFcpr.

Although myoblast transplantation is an attractive method for muscle regeneration, its efficiency remains limited. The efficacy of myoblast transplantation in combination with the controlled and sustained delivery of basic fibroblast growth factor (bFGF) was investigated. Defects of thigh muscle in Sprague-Dawley (SD) rats were created, and GFP-positive myoblasts were subsequently transplanted. The rats were divided into three groups. In control group 1 (C1) only myoblasts were transplanted, while in control group 2 (C2) myoblasts were introduced along with empty gelatin hydrogel microspheres. In the experimental group (Ex), myoblasts were transplanted along with bFGF incorporated into gelatin hydrogel microspheres. Four weeks after transplantation, GFP-positive myoblasts were found to be integrated into the recipient muscle and to contribute to muscle fibre regeneration in all groups. A significantly higher expression level of GFP in the Ex group demonstrated that the survival rate of transplanted myoblasts in Ex was remarkably improved compared with that in C1 and C2. Furthermore, myofibre regeneration, characterized by centralization of the nuclei, was markedly accelerated in Ex. The expression level of CD31 in Ex was higher than that in both C1 and C2, but the differences were not statistically significant. A significantly higher expression level of Myogenin and a lower expression level of MyoD1 were both observed in Ex after 4 weeks, suggesting the promotion of differentiation to myotubes. Our findings suggest that the controlled and sustained release of bFGF from gelatin hydrogel microspheres improves the survival rate of transplanted myoblasts and promotes muscle regeneration by facilitating myogenesis rather than angiogenesis.

Patch tracheoplasty in body tissue engineering using collagenous connective tissue membranes (biosheets).

BACKGROUND: Collagenous connective tissue membranes (biosheets) are useful for engineering cardiovascular tissue in tissue engineering. The aim was to evaluate the use of biosheets as a potential tracheal substitute material in vivo in a rabbit model. METHODS: Group 1: Rectangular-shaped Gore-Tex (4×7mm) was implanted into a 3×6mm defect created in the midventral portion of the cervical trachea. Group 2: Rectangular-shaped dermis was implanted into a tracheotomy of similar size. Group 3: Biosheets were prepared by embedding silicone moulds in dorsal subcutaneous pouches in rabbits for 1month. Rectangular-shaped biosheets were implanted into a tracheotomy of similar size in an autologous fashion. All groups (each containing 10 animals) were sacrificed 4weeks after implantation. MAIN RESULTS: All materials maintained airway structure for up to 4weeks after implantation. Regenerative cartilage in implanted Biosheets in group 3 was confirmed by histological analysis. Tracheal epithelial regeneration occurred in the internal lumen of group 3. There were significant differences in the amounts of collagen type II and glycosaminoglycan between group 3 and group 1 or 2. CONCLUSION: We confirm that cartilage can self-regenerate onto an airway patch using Biosheets.

Tailor-made treatment combined with proton beam therapy for children with genitourinary/pelvic rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas among children. Patients who developed genitourinary/pelvic rhabdomyosarcoma (GU/P-RMS) have a higher complication ratio and relatively poorer event free survival, with local therapy being very important. While proton beam therapy (PBT) is expected to reduce co-morbidity, especially for children, this lacks firm evidence and analysis. We analyzed GU/P-RMS children who had undergone multimodal therapy combined with PBT at a single institution. METHOD: We retrospectively reviewed charts of children with GU/P-RMS treated from January 2007 to May 2013 at the University of Tsukuba Hospital who had undergone multimodal therapy with PBT. RESULTS: There were 5 children and their median age at diagnosis was 2.8 years (0.6-4.4 years). Primary sites were the bladder (2) and the prostate (3). All received neo-adjuvant chemotherapy and 3 underwent chemotherapy during PBT (Group Cx). All patients of Group Cx developed leukocytopenia (WBC <1000/µL). The median dose of PBT was 47.7 GyE (41.4-50.4 GyE). All patients survived by their last hospital visit (median, 36 months). CONCLUSIONS: We analyzed multimodal treatment combined with PBT applied for GU/P-RMS. PBT was well tolerated and could be a plausible choice instead of photon therapy for this population.

Tracheoplasty with cartilage-engineered esophagus environments.

PURPOSE: Our objective was to investigate the feasibility of engineering cartilage on the esophagus layer and outside the esophagus. Moreover, we investigated the feasibility of tracheoplasty with cartilage engineered on the esophagus in rabbits. METHODS: Chondrocytes were isolated from auricular cartilages. 1. Engineered cartilage formation by histological findings on/into the esophageal layer was compared with that of injectable scaffold and preformed scaffold with chondrocytes. 2. Chondrocytes adhered to gelatin+vicryl mesh™ and b-FGF, were implanted on the outer esophageal surface. Four weeks after seeding, we found that cartilage was implanted in the midposterior portion of the cervical trachea (n=5), and it was retrieved 8weeks after seeding. RESULTS: 1. A gelatin sponge incorporating ß-TCP with vicryl mesh™ showed the best performance for fabricating engineered cartilage on the outer side of the esophagus. 2. Two of 5 rabbits died due to obstructed esophagus. Cartilage engineered outside the esophagus by a composite scaffold as the main material in the gelatin sponge, maintained the airway structure for up to 1month after implantation. Tracheal epithelial regeneration occurred in the internal lumen of this engineered cartilage. CONCLUSION: Tracheoplasty with cartilage engineered outside the esophagus may be useful for reconstructing airways.

Slow release of basic fibroblast growth factor (b-FGF) enhances mechanical properties of rat trachea.

AIM: Severe tracheomalacia is a life-threatening disease, but symptoms usually improve with growth. The aims of this study were to investigate how slow release basic-Fibroblast Growth Factor (b-FGF) acts on tracheal cartilage, and whether growth-promoted trachea is more resistant against an increase in externally-applied pressure. METHODS: Biodegradable gelatin hydrogel sheets soaked in 10 µl of distilled water (sham) or 0.5 or 5 µg/10 µl of b-FGF solution were inserted behind the cervical trachea of three-week-old male Wistar rats. The cervical trachea was harvested 4 weeks later. Extratracheal pressure was increased from 0 to 40 cmH2O in a chamber, while video-recording the internal lumen. The luminal area at each pressure was expressed as a proportion to that at 0 cmH2O. The amounts of collagen type II and glycosaminoglycan were measured by ELISA. RESULTS: The luminal areas at 40 cmH2O in the control (no intervention), sham, and each of the b-FGF groups were 0.65, 0.62, 0.72, and 0.73, respectively. The amounts of collagen type II and glycosaminoglycan in each group were 127, 136, 193, 249 µg/mg, respectively, and 15, 16, 19, 33 µg/mg, respectively. There were significant differences between the control group and the FGF 5 group (P=0.02, 0.01, 0.01, for luminal area, collagen, and glycosaminoglycan, respectively). CONCLUSION: 5 µg of slow-release b-FGF promotes matrix production (collagen type II and glycosaminoglycan). The growth-enhanced trachea was more resistant to collapse, suggesting that slowly released b-FGF might be useful in patients with severe tracheomalacia.

Slow release of basic fibroblast growth factor (b-FGF) promotes growth of tracheal cartilage.

PURPOSE: Tracheomalacia is a major cause of morbidity in conditions such as oesophageal atresia. However, symptoms usually improve with age. A more rapid growth of tracheal cartilage can be induced by basic-Fibroblast Growth Factor (b-FGF). This study aimed to investigate whether slow-release b-FGF could act as a novel treatment for tracheomalacia. METHODS: Biodegradable gelatin hydrogel sheets incorporating 0.5, 5, or 50 µg/20 µl of b-FGF solution were inserted between the cervical trachea and esophagus of rats. No intervention was performed in rats in a control group. All animals were sacrificed 4 weeks later, and the luminal area of the cervical trachea and the thickness of the cartilage were measured. RESULTS: The mean luminal areas in the control group and in the b-FGF groups were 3.1, 3.2, 3.8, and 2.6mm(2), respectively, and showed a peak area at 5 µg of b-FGF. A significant difference was seen only between the control group and the b-FGF 5 µg group (p<0.05). The mean thickness of the tracheal cartilage was 0.12, 0.13, 0.19, and 0.32 mm in the control and the b-FGF groups, respectively, and showed a dose-dependent increase, which was statistically significant between the b-FGF 5 µg or 50 µg groups and the control group (p<0.01). CONCLUSION: This study showed that slow-release b-FGF enlarges the tracheal lumen and thickens the cartilage in a dose-dependent fashion.

Management of cervicomediastinal neuroblastoma presenting with life-threatening tracheal obstruction in infancy.

The authors report on 3 infants below 6 months of age at diagnosis, with cervicomediastinal neuroblastoma who presented with life-threatening tracheal obstruction as an oncologic emergency. These neuroblastomas were characterized by favorable biology and chemoresistance. All initially grew rapidly before spontaneously regressing. Nerve injuries occurred in all patients as a result of tumor location. Maintenance of the airway until the expected spontaneous regression was a critical component in the management of these patients.

Epithelial-mesenchymal transition-related gene expression as a new prognostic marker for neuroblastoma.

Neuroblastoma (NB) is a highly metastatic tumor in children. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. This study investigated the role of EMT in the progression of NB. Using EMT assays on samples from 11 tumors, we identified 14 genes that were either differentially expressed between tumors of different stages or highly upregulated in NB. Quantitative RT­PCR of these genes was conducted in 96 NB tumors and their expression levels were compared between stages and between tumors with the presence and absence of MYCN amplification. The association of survival rate with differential gene expression was investigated. Expression of KRT19 was significantly decreased in stage 3 or 4 NB as well as stage 4S NB compared with stage 1 or 2 NB. Expression levels of KRT19 and ERBB3 were significantly low, and expression levels of TWST1 and TCF3 were high in MYCN­amplified NB. The patients with low expression of KRT19 or ERBB3 showed significantly worse overall survival. Furthermore, the correlation between high invasive ability and low expression of KRT19 and ERBB3 was suggested in vitro using six NB cell lines. The authors conclude that downregulation of KRT19 is highly associated with tumor progression in NB and metastasis in localized primary NB and that low expression of ERBB3 is also associated with progression of NB.

Massive pneumatic expansion of lymphatic vessel resulting in cystic lesions in the pulmonary parenchyma: a rare case of persistent interstitial pulmonary emphysema in a non-ventilated infant.

We report the case of 2-week-old female infant with cystic lung disease who presented with mild tachypnea and had no history of mechanical ventilation. Chest CT demonstrated multiple air-filled cystic lesions in right upper lobe, and the patient subsequently underwent a right upper lobectomy. Histology revealed cystic lesions located in the pulmonary parenchyma and showed that the lesions were lined by lymphatic endothelium and were communicating with dilated lymphatic vessels in the interstitium. Additionally, multinucleated foreign body giant cells were attached to the lumen of the cyst. On the basis of these findings, we considered this a case of persistent interstitial pulmonary emphysema (PIPE) with massive pneumatic expansion of the lymphatic vessels, resulting in cystic lesions with lymphatic endothelium in the pulmonary parenchyma. While PIPE is extremely rare in term non-ventilated infants, our case demonstrated that this disease should be added to the differential diagnosis of cystic lung diseases with lymphatic endothelium even in infants without mechanical ventilation. When cystic lesions and symptoms persist despite conservative treatment, open or thoracoscopic resection is an appropriate option for diagnosis and treatment.

A pediatric case of an unusual type of annular pancreas presenting with duodenopancreatic reflux.

Pancreatitis, a late complication of an annular pancreas (AP), results from coexisting pancreaticobiliary malformations including pancreas divisum (PD), and pancreaticobiliary maljunction (PBM). The authors report the case of a 3-year-old boy with an unusual type of AP in which the dorsal anlage encircled the duodenum. The patient developed duodenal obstruction as well as duodenopancreatic reflux with resulting hyperamylasemia and hyperlipasemia. This type of AP associated with duodenopancreatic reflux in AP has not been reported previously. The patient was successfully treated by duodenoduodenostomy, which, by correcting the duodenopancreatic reflux, prevented the later development of pancreatitis.

Clinical characteristics of liver fibrosis in patients with choledochal cysts.

PURPOSE: The aim of the study was to identify the clinical characteristics and outcome of patients with liver fibrosis in choledochal cyst (CC). METHODS: Forty patients with CC who underwent liver biopsy were included. Liver fibrosis was classified as follows: grade 0, no fibrosis; grade 1, mild fibrosis localized in the portal area; grade 2, moderate fibrosis with occasional bridging; and grade 3, severe fibrosis with diffuse bridging. RESULTS: Fourteen patients (35%) had liver fibrosis. Patients in the fibrosis group were significantly younger (1.2 vs 2.7 years) and had higher total bilirubin (5.3 vs 2.6 mg/dL). Severity of liver fibrosis was inversely correlated with age (P = .044). Amylase and lipase in bile were significantly lower in the fibrosis group (amylase, 531 vs 15,000 U/L; lipase, 783 vs 23,100 U/L). Postoperative serum analysis demonstrated no differences between the two groups. Most patients in both groups had normal aspartate aminotransferase, alanine aminotransferase, total bilirubin, and γ-glutamyl transpeptidase regardless of severity of fibrosis. Postoperative biliary complication or cholangiocarcinoma was not found in the fibrosis group. CONCLUSIONS: Our data suggest that liver fibrosis is mainly influenced by obstructive cholangiopathy rather than refluxed pancreatic secretion. Prognosis of patients with CC and liver fibrosis was as good as that of patients without fibrosis.

Bile duct duplication as a cause of distal bowel gas in neonatal duodenal obstruction.

PURPOSE: The aim of this study is to investigate the clinical characteristics of cases of duodenal atresia (DA) which present with bowel gas distal to a typical double-bubble sign through an anomalous bile duct conduit. METHODS: Medical records of 57 neonates with duodenal obstruction (atresia or stenosis), presenting with a double-bubble sign and treated at our institute from 1978 to 2010, were retrospectively reviewed. RESULTS: Thirteen (23%) of 57 neonates presented with bowel gas distal to the double-bubble sign. Passage of gas through the duodenal stenosis may have occurred in 3 cases, whereas in 9 cases, gas may have bypassed the atresia through an anomalous bifurcated bile duct termination and through the pancreatic duct from the accessory to the main pancreatic duct in one case. A preoperative upper gastrointestinal series was performed in 9 cases, and an anomalous bifurcated bile duct conduit was demonstrated in 5 cases. Severe and prolonged cholestasis necessitating evaluation for biliary atresia was found in 2 patients with anomalous bile duct anatomy. CONCLUSIONS: Neonatal DA presenting with distal bowel gas via an anomalous bifurcated bile duct conduit is more common than initially thought and occurs more frequently than duodenal stenosis. These patients might be at risk for cholestasis, possibly owing to duodeno-biliary reflux through the ampulla.