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Minimally invasive repair has become a popular approach for pectus excavatum (PE). The bar is secured to the thoracic wall and left for approximately 2 years. The authors have noticed an intense bone formation (BF) around some of these bars at removal. A review of children undergoing bar removal was performed to better understand this BF in relation to bar placement.A retrospective review of children undergoing bar removal after PE repair since January 1998 was performed. Chart review included age at bar insertion and removal, bar insertion position (subcutaneous [SC] v submuscular [SM]), BF on Chest x-ray and at bar removal, operating time, and estimated blood loss (EBL).Thirty-six patients underwent bar removal during the study period (16 SC and 20 SM). Chest x-ray evaluation was possible in 27 patients (16 SM, 11 SC). No difference existed for length of time the bar was in place or age at insertion/removal between groups. EBL was higher in the SM (18.3 v 8.8 mL, not significant). BF was seen radiographically in 15 SM and 3 SC patients (P.001). BF was encountered at removal in 19 SM patients and a single SC patient (P.001). Operating time was statistically longer (P.01) for the SM group (30.2 v 15.6 min).Bar position during repair of PE is important. SM positioning virtually always results in BF with increased EBL and statistically longer operating time at removal. Careful placement of the bar in the SC position without violating the fascia should be used to avoid these undesirable effects.

Background/Purpose: The Adriamycin-induced rat model of esophageal atresia and tracheoesophageal fistula (EA/TEF) provides a reliable system for the study of EA/TEF pathogenesis. The authors previously hypothesized that faulty branching lung morphogenesis pathways were a critical component of its pathogenesis. The authors have found evidence for faulty fibroblast growth factor (FGF) signaling related to epithelial-mesenchymal interactions in the fistula tract. To better define FGF signaling, the differential expression of FGF ligands and their receptors between lung, fistula tract, and esophagus are described. Methods: Time-dated pregnant, Sprague-Dawley rats were injected with Adriamycin (2 mg/kg intraperitoneally) on days 6 through 9 of gestation. Tissues were processed for histology and reverse transcriptase polymerase chain reaction. FGF-1, -7 and -10 were measured from whole lung, fistula tract, and esophagus of TEF or normal embryos. Expression of FGF2RIIIb and FGF2RIIIc receptors was measured in isolated epithelium and mesenchyme of lung and fistula tract of TEF embryos as well as lung and esophagus from normal controls. Results: FGF-1 mRNA was present in the fistula tract and normal and Adriamycin-exposed lung but absent from whole esophagus. Interestingly, FGF-7 mRNA was present only in normal lung. FGF-10 was present in all tissues examined. FGF2RIIIb mRNA was absent in fistula mesenchyme but present in all other tissues examined. However, the splice variant FGF2RIIIc mRNA was present in all tissues examined. Conclusions: These findings support defective FGF signaling in the rat model of EA/TEF. Absence of FGF-7 mRNA in Adriamycin-exposed tissues suggests the primary effect of Adriamycin may be to inhibit FGF-7 expression. Moreover, absence of FGF2RIIIb in fistula mesenchyme may be caused by loss of positive feedback from FGF-7, its normal obligate ligand. Understanding these specific defects in FGF signaling may provide insight into faulty mechanisms of EA/TEF. J Pediatr Surg 38:474-477. Copyright 2003, Elsevier Science

Successful strategies for treating type 1 diabetes need to restore the function of pancreatic beta cells that are destroyed by the immune system and overcome further destruction of insulin-producing cells. Here, we infused adeno-associated virus carrying Pdx1 and MafA expression cassettes through the pancreatic duct to reprogram alpha cells into functional beta cells and normalized blood glucose in both beta cell-toxin-induced diabetic mice and in autoimmune non-obese diabetic (NOD) mice. The euglycemia in toxin-induced diabetic mice and new insulin+ cells persisted in the autoimmune NOD mice for 4 months prior to reestablishment of autoimmune diabetes. This gene therapy strategy also induced alpha to beta cell conversion in toxin-treated human islets, which restored blood glucose levels in NOD/SCID mice upon transplantation. Hence, this strategy could represent a new therapeutic approach, perhaps complemented by immunosuppression, to bolster endogenous insulin production. Our study thus provides a potential basis for further investigation in human type 1 diabetes.

The embryonic pancreas is thought to develop from pluripotent endodermal cells that give rise to endocrine and exocrine cells. A key guidance mechanism for pancreatic development has previously been found to be epithelial-mesenchymal interaction. Interactions within the epithelium, however, have not been well studied. Glucagon is the earliest peptide hormone present at appreciable levels in the developing pancreatic epithelium (embryonic day [E]-9.5 in mouse). Insulin accumulation begins slightly later (E11 in mouse), followed by a rapid accumulation during the “second wave” of insulin differentiation (∼E15). Here we found that blocking early expression and function of glucagon, but not GLP-1, an alternate gene product of preproglucagon mRNA, prevented insulin-positive differentiation in early embryonic (E11) pancreas. These results suggest a novel concept and a key role for glucagon in the paracrine induction of differentiation of other pancreatic components in the early embryonic pancreas.

Background: Laparoscopic adrenalectomy (LA) is now being recognized as the standard in the management of benign adrenal pathology in adult patients. Few reports have described the use of this technique in pediatric patients. This study combines experience from 2 institutions with lateral transperitoneal LA in children to analyze our results and the clinical and biochemical response to laparoscopic adrenalectomy in patients with hormonally active adrenal tumors. Methods: A bi-institutional retrospective review of all patients undergoing LA between January 1997 and January 2001 was performed. Clinical and biochemical data were obtained during routine follow-up. Results: Seventeen laparoscopic adrenalectomies were performed during this period. The average operating time was 120 minutes, mean estimated blood loss was 25 mL, the mean size of the adrenal lesion was 4.8 cm, and the mean length of hospitalization was 35 hours. Resolution of clinical and biochemical parameters of adrenal hyperfunction was accomplished in all patients with adrenocortical hyperplasia and pheochromocytoma in postoperative follow-up. Conclusions: Laparoscopic adrenalectomy can be performed safely and effectively with a short hospital stay, minimal blood loss, and excellent functional outcome in this age group. The authors believe laparoscopic adrenalectomy is an excellent approach for the management of benign pediatric

The embryonic pancreatic epithelium, and later the ductal epithelium, is known to give rise to the endocrine and exocrine cells of the developing pancreas, but no specific surface marker for these cells has been identified. Here, we utilized Dolichos Biflorus Agglutinin (DBA) as a specific marker of these epithelial cells in developing mouse pancreas. From the results of an immunofluorescence study using fluorescein-DBA and pancreatic specific cell markers, we found that DBA detects specifically epithelial, but neither differentiating endocrine cells nor acinar cells. We further applied this marker in an immunomagnetic separation system (Dynabead system) to purify these putative multi-potential cells from a mixed developing pancreatic cell population. This procedure could be applied to study differentiation and cell lineage selections in the developing pancreas, and also may be applicable to selecting pancreatic precursor cells for potential cellular engineering.

Background/Purpose: Many studies in pediatric surgical research use a quantitative analysis of gene expression in microscopic quantities of tissue. The authors describe an analysis of the beta-tubulin mRNA content of the embryonic pancreas, which contains abundant endogenous RNases. A detailed analysis of this RNase-containing system will provide a good template for analysis of other potentially simpler systems. Methods: Embryonic mouse pancreases were harvested at serial gestational ages. DAPI nuclear staining allowed for counting of cells. cDNA was amplified using a fluoresceinated primer and the normalized fluorescence determined. Known numbers of molecules were amplified in parallel as a standard control. Results: The number of cells increased from 38,000 to 2,700,000 between embryonic day 10.5 (E10.5) and E18.5. mRNA for beta-tubulin did not increase proportionately. Assuming a yield of 100% at E10.5 when no RNases are present, the yield of expected mRNA was 65.3% at E12.5, 13.8% at E15.5, and 0.9% at E18.5, presumably because of the appearance of RNases. Conclusions: Several parameters must be considered in performing semiquantitative reverse transcription polymerase chain reaction: (1) the yield of RNA based on the projected amount of mRNA, (2) the number of cells in the tissue, and (3) a known number of template molecules amplified in parallel.

Background Purpose: Intestinal atresia occurs in approximately 10% to 20% of children with gastroschisis and may be missed at the initial closure if a thick peel obscures the bowel. Some investigators have identified intestinal atresia as a significant contributor to morbidity and mortality. The authors reviewed their experience with gastroschisis and intestinal atresia in an attempt to answer the following questions. What is the incidence of this association? How often is the intestinal atresia unrecognized as a result of the peel? What is the optimal management for infants with atresia and gastroschisis, and does the atresia affect morbidity or mortality? Methods: The hospital charts and medical records of all patients with gastroschisis treated at our institution from 1969 to present were reviewed thoroughly. Parameters analyzed included gestational age (GA), birth weight (BW), antenatal diagnosis, mode of delivery, type of closure, era of repair, presence of other major anomalies, and development of necrotizing enterocolitis. Morbidity and mortality rates were examined. Characteristics of patients with and without atresia were compared. Chi-squared was used for crosstabular analysis. Sample parameters were compared with Student’s t test. P values of less than .05 were considered significant. Results: A total of 199 babies had gastroschisis and 25 (12.6%) had intestinal atresia. Intestinal atresia was initially unrecognized in 3 patients. Most patients (80%) underwent primary closure of the abdominal wall. Initial stoma formation and delayed anastomosis was performed in 12 (48%) patients, none of whom required prosthetic material for abdominal wall closure. Initial stomas were avoided in 5 patients who required SILASTIC® (Dow Corning, Midland, MI) silos. Skin closure alone was used in 2 babies. The level of the atresia was most commonly jejunoileal (20 of 25, 80%). Mean hospital stay was increased in babies with intestinal atresia, 36.2 versus 63.1 days (P , .001). Conclusions: Although patients with intestinal atresia did have feeding delays, an increased incidence of adhesive intestinal obstruction, and prolonged hospitalization, neither x 2 nor logistic regression analysis showed any correlation with mortality. Intestinal repair at the first operation is sometimes possible and depends on the severity of the peel. Delayed repair of the atresia after a period of bowel decompression and parenteral nutrition is preferred, but in certain situations (colonic atresia, necrotic intestine, complicated atresia) may not be possible. The combination of stomas and prosthetic material can be avoided in almost all patients. A management algorithm for patients with atresia and gastroschisis is discussed. J Pediatr Surg 36:1542-1545. Copyright © 2001 by W.B.

Background/Purpose: The fistula tract in esophageal atresia with tracheoesophageal fistula (EA-TEF) appears to arise from a trifurcation of the embryonic lung bud. Subsequently, it does not branch like the other bronchi, which also arise from the lung bud. Previous results have implied that aberrant mesenchymal-epithelial signaling in the developing foregut, possibly involving fibroblast growth factors, may allow for the nonbranching growth of the fistula, and the ultimate development of the fistula tract in TEF. Methods: Adriamycin injections into pregnant rat dams induced EA-TEF formation in rat embryos. Control and Adriamycin-exposed embryos were harvested on the 13th gestational day, and the developing foregut was isolated with microdissection. mRNA was isolated from the developing fistula tract, embryonic lung, and normal embryonic esophagus. Reverse transcription-polymerase chain reaction (RT-PCR) for the IIIb splice variant of the FGF2R receptor was performed. Foregut specimens also were processed for histologic analysis, and immunofluorescence for FGF1 was performed. Results: FGF2R-IIIb is specifically absent from the developing fistula tract in TEF, whereas it is present in the normal developing lung and esophagus. FGF1 also is uniquely absent from the developing fistula tract, but it is present in the normal lung mesenchyme. Conclusions: FGF1, FGF7, and FGF10 are critical mesenchymal factors that mediate proliferation and branching morphogenesis by the developing respiratory epithelium. The absence of FGF2R-IIIb, the obligate common receptor for FGF7 and FGF10, from the fistula tract, and the absence of FGF1 in the fistula tract mesenchyme, collectively imply the absence of a specific FGF signaling pathway in the developing fistula tract. This absence of FGF signaling could explain the lack of branching by the developing fistula tract as it grows caudally in the abnormally developing embryo. Downregulation of these components of the FGF signaling pathways may allow for a patterned compensation by the embryo for the proximal foregut atresia in this anomaly. This compensation may then reestablish gastrointestinal continuity as the fistula tract connects to the developing stomach. J Pediatr Surg 35:1421-1425. Copyright © 2000 by W.B. Saunders Company.

Background. We have recently proposed that the “distal esophagus” in esophageal atresia with tracheo-esophageal fistula (EA/TEF) is actually embryologically derived from the middle branch of a trifurcation of the embryonic lung bud, which subsequently grows caudally in the foregut to connect with the developing stomach. We hypothesized that differential mRNA expression of the lung-specific patterning transcription factor, thyroid transcription factor 1 (TTF-1), in the developing fistula tract in TEF relative to the bronchi (the other branches of the lung bud trifurcation) might explain the unique nonbranching pattern of growth of the fistula tract. Materials and methods. EA/TEF was induced in Sprague–Dawley rat embryos via intraperitoneal injection of 2.2 mg/kg adriamycin into pregnant dams on Days 6–9 of gestation. The foregut from embryos developing EA/TEF and from control embryos (no adriamycin) were isolated on Gestational Days 13.5, 15.5, and 17.5 (term = 21 days). Some were processed for whole-mount in situ hybridization for TTF-1, while others were embedded and sectioned for histologic analysis via in situ hybridization for TTF-1. Results. The expression of the respiratory-specific transcription factor TTF-1 is conserved in the epithelium of the developing fistula tract in TEF. The pattern of expression of TTF-1 in the fistula tract mirrors the expression in the large airways of the developing lungs, despite the fact that the fistula tract does not form secondary branches to give rise to a lung. Conclusions. The fistula tract in TEF is a respiratory-derived structure that expresses the lung-specific transcription factor TTF-1 throughout its development in the foregut. Contrary to the patterning role that it normally plays in the developing lung, TTF-1 does not induce branching morphogenesis in the fistula tract. Thus, the nonbranching pattern of growth of the fistula tract may be attributable to local mesenchymal–epithelial interactions that override TTF-1 patterning activity.

We previously showed that the undifferentiated pancreatic epithelium can differentiate into islets, ducts, or acini depending on its milieu and that laminin is necessary for pancreatic duct formation. Therefore we wanted to study the plasticity of laminin-induced duct differentiation the better to understand mechanisms of pancreatic duct lineage selection induced by basement membrane. Mouse embryonic pancreases were dissected at gestational day 11 (E11.5), and epithelium was isolated from its surrounding mesenchyme. Some epithelia were cultured in a collagen gel devoid of laminin. These epithelia were "rescued" at days 1-7 of culture by transferring them to a laminin-rich matrix (Matrigel) for 7 additional days. Other epithelia were instead first cultured in Matrigel, and then placed into collagen. Immunohistochemistry was performed for insulin, amylase, and carbonic anhydrase II. Pancreatic epithelia rescued from collagen into laminin during days 1-4 after harvest were still able to form ducts, whereas epithelia deprived of laminin for longer than this 4-day window were not. Pancreatic epithelia exposed to laminin for as little as 1 day, and then placed into collagen, still retained the ability to make ducts. Thus there is a clear cut-off in the development of the pancreatic epithelium at E11.5, after which laminin appears necessary to induce duct formation. We believe that such "windows of competence" in embryonic development imply that developmental programs in the embryo allow some flexibility.

Activin, a member of the transforming growth factor-beta superfamily, has been shown to be a critical regulator in exocrine and endocrine pancreas formation. The purpose of our study was to describe the ontogeny of activin B and its inhibitor, follistatin, in developing pancreas and to elucidate potential mechanisms for exocrine and endocrine lineage selection. Mouse embryonic pancreata were dissected at various ages (day 10 [E10.5] to birth [E18.5]), sectioned, and immunostained for activin B (one of two existing isomers, A and B), follistatin, insulin and glucagon. In addition, reverse transcriptase-polymerase chain reaction was employed to determine the messenger RNA expression of follistatin in isolated pancreatic epithelia and mesenchyme of various ages. Activin B was first detected at E12.5 in epithelial cells coexpressing glucagon. At E16.5 these coexpressors appeared as clusters in close proximity to early ducts. By E18.5 activin B was localized to forming islets where cells coexpressed glucagon and were arranged in the mantle formation characteristic of mature alpha cells. Follistatin was found to be ubiquitous in pancreatic mesenchyme at early ages by immunohistochemical analysis, disappearing sometime after E12.5. Follistatin reappeared in E18.5 islets and remains expressed in adult islets. Follistatin messenger RNA was first detected in epithelium at E11.5, preceding its protein expression in islets later in gestation. We propose that mesenchyme-derived follistatin inhibits epithelium-derived activin at early embryonic ages allowing for unopposed exocrine differentiation and relative suppression of endocrine differentiation. At later ages the decrease in the amount of mesenchyme relative to epithelium and the subsequent drop in follistatin levels liberates epithelial activin to allow differentiation of endocrine cells to form mature islets by the time of birth.

Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGFbeta) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGFbeta in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-2. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability ofVEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGFbeta1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGFbeta2 and TGFbeta3 yielded 2- to 3-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGFbeta2 and TGFbeta3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGFbeta, we disrupted TGFbeta signal transduction (using adenovirus encoding a truncated form of TGFbeta receptor II), which attenuated TGFbeta1 induction of VEGF mRNA, but did not impede FGF-2 induction ofVEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGFbeta activity.

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cell growth, differentiation, and function in developing mammalian systems. Altering TGF-beta 1 expression in the developing pancreas has been shown to affect both exocrine and endocrine development, suggesting that it is an important regulator of pancreatic organogenesis. We proposed to examine the ontogeny of TGF-beta 1 mRNA expression in the developing pancreas, as well as characterize the patterns of relative TGF-beta 1 gene expression and activity. We performed in situ hybridization for TGF-beta 1 on pancreas specimens obtained from CD-1 mice on gestational days 12.5 (E12.5), 15.5 (E15.5), and 18.5 (18.5). We also isolated mRNA from the pancreas on each of these days and performed a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to assess relative TGF-beta 1 expression as a function of gestational age. Finally, we performed a TGF-beta 1 ELISA with media conditioned by embryonic pancreas from gestational days 15.5 and 18.5. By in situ hybridization, TGF-beta 1 mRNA is expressed exclusively in the E12.5 pancreatic epithelium, sparing the surrounding mesenchyme. As pancreatic organogenesis progresses, TGF-beta 1 mRNA expression localizes predominantly to the developing acini. TGF-beta 1 gene expression appears modest through E15.5 but is upregulated near the end of gestation, at E18.5. TGF-beta 1 activity, by ELISA, is also upregulated at E18.5. TGF-beta 1 may thus be a modulator of pancreatic organogenesis. Modest TGF-beta 1 expression through E15.5 may be permissive for exocrine lineage selection. TGF-beta 1 expression may then become critical for terminal acinar differentiation. Upregulated TGF-beta 1 expression at the end of gestation may be important for islet formation, and it may be necessary to inhibit continued proliferation and differentiation of pluripotent cells within the pancreatic ductal epithelium.

Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II-cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO 2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing.

Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases.

Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone.

The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scarforming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin ( n = 24) and 1-month-old postnatal adult rat skin ( n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur.

The ability to avoid a subsequent bone graft makes the use of gingivoperiosteoplasty (GPP) at the time of cleft lip repair an attractive technique. The use of GPP, in combination with presurgical orthodontics, has been shown to result in successful bony union in the majority of patients. However, secondary bone grafting is still necessary in 30% to 40% of patients due to persistent alveolar bony defects. The elucidation of methods to improve the success rates of these procedures has been hampered by the lack of reproducible animal models. The purpose of this study was, therefore, to develop a rodent model of GPP that would facilitate the investigation of methods to improve osteogenesis in alveolar defects. We report a surgically produced rat model (9 x 5 x 3-mm alveolar defect) that is reproducible, inexpensive (relative to large-animal models), and simple technically. In addition, healing in this model occurs in a predictable manner during a 12-week period, thus enabling analysis of methods designed to accelerate or facilitate osseous regeneration.

The liver is the solid organ most commonly injured as a result of blunt abdominal trauma. Complete avulsion of the common hepatic duct is a rare and devastating type of hepatobiliary trauma. Here the authors report the case of a 7-year-old child who had complete biliary disruption as a result of an abdominal crush injury that was not diagnosed correctly preoperatively. The intraoperative diagnosis and treatment of this injury is discussed.

Purpose : Using an established rat model of esophageal atresia with tracheoesophageal fistula (EA-TEF), the authors have studied the organogenesis of this congenital anomaly. The authors previously have proposed that the "distal esophagus" actually is of respiratory lineage. In this report this hypothesis is tested by examining the expression of two foregut patterning transcription factors, thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3β (HNF-3β), within the developing TEF. Methods: Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 to 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. Immunohistochemistry was performed using polyclonal antibodies to TTF-1 and HNF-3β. Results: TTF-1 is a homeodomain protein that previously has been shown to be expressed in the lung and trachea but not in the gastrointestinal tract, and which, when deleted in a developing lung, results in a mouse with no peripheral lung parenchyma. TTF-1 was expressed strongly in the lung, fistula, and distal esophagus, but not in the proximal esophagus. HNF-3β is a forkhead transcription factor important in foregut patterning that binds and activates the TTF-1 promotor sequence. HNF-3β was expressed globally in the fistula and lung as well as the esophagus. Conclusions: The expression of the lung-specific transcription factor TTF-1 within the TEF strongly implies that the "distal esophagus" is a respiratory-derived structure and thus supports our theory of TEF organogenesis. The conservation of HNF-3β expression both in the TEF as well as the normal developing trachea and esophagus suggests that global foregut patterning is intact in the formation of this anomaly, and the defect lies at the level of the respiratory versus gastrointestinal commitment.

A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound.

The role of transforming growth factor beta (TGF-beta) in the regulation of cranial suture fusion has been studied by various qualitative techniques such as in situ hybridization and immunohistochemistry. Although the relative expression of TGF-beta isoforms has been assessed in these studies, increased expression of TGF-beta has not been demonstrated in a quantitative fashion. Therefore, the purpose of this study was to quantify TGF-beta production by fusing (posterofrontal [PF]) and nonfusing (sagittal) mouse sutures using two different quantitative TGF-beta assays. The PF and sagittal sutures of 25-day-old mice were harvested and cultured separately in vitro. Culture media conditioned for 48 hours were collected after 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 days of culture, and total TGF-beta production was assessed using a TGF-beta bioassay. For a quantitative TGF-beta1 immunoassay, media conditioned for 48 hours were collected after 3, 5, 7, 9, 14, 22, and 28 days of culture. The TGF-beta bioassay revealed large amounts of total TGF-beta activity in both PF and sagittal sutures during the first week of culture, with decreasing amounts thereafter. Absolute TGF-beta activity in conditioned media collected from PF sutures at several early time points was higher than those obtained from sagittal sutures; however, these differences were not statistically significant. The results of the TGF-beta1 immunoassay (enzyme-linked immunosorbent assay) were similar to the bioassay in that the highest TGF-beta1 levels were noted during the first week of culture period and decreased thereafter. Analysis of variance of these samples, however, revealed significantly more TGF-beta1 protein in samples collected from the PF suture compared with the sagittal suture on days 3 and 5 of culture (p0.05). TGF-beta1 levels in the conditioned media obtained from PF sutures remained elevated compared with the sagittal suture on days 7 and 9; however, these differences were not statistically significant. Increased production of TGF-beta in the conditioned media of fusing PF sutures is the first such quantitative demonstration of growth factor upregulation during suture fusion and supports the hypothesis that TGF-beta expression may be important in cranial suture fusion.

Background/Purpose: The mammalian pancreas is thought to develop through a complex interaction between the budding epithelium and the surrounding mesenchyme. The exact nature of this interaction is unclear. Most of what is known to date of these interactions comes from a series of organ culture experiments done in the late 1960s. Nevertheless, these important experiments may have been confounded by less-defined culture media and organ dissection techniques, because the results are not reproducible in our hands. The authors undertook a study to reexplore these basic epithelio-mesenchymal interactions. Methods: Using previously described organ dissection and culture techniques the authors examined the basic interactions between the embryonic pancreatic epithelium and its mesenchyme with histological and immunohistological techniques. Results: The authors found that, contrary to previous reports, the earliest pancreatic anlage did not possess the intrinsic signaling necessary to support normal growth and differentiation in vitro. Intimate contact between the epithelium and the mesenchyme may be necessary until E11.5 for normal growth and differentiation. The age of the mesenchyme seemed to correlate with the degree of acinar differentiation, and proximity of mesenchyme was important for acinar differentiation. Conclusions: Previous investigations into the basic epitheliomesenchymal interactions in the developing mammalian pancreas may have had confounding factors. Extrinsic signals seem necessary for complete pancreatic differentiation, and mesenchymal factors appear important for acinar differentiation.

To date, many efforts to engineer cartilage have focused on matrix construction with the goal of producing a durable construct as cartilage replaces the resorbing matrix. However, the importance of matrix construction is at least matched by the challenge of efficient chondrocyte extraction, culture expansion, and prevention of dedifferentiation. This challenge is underscored by the large number of chondrocytes needed for a clinically significant construct such as an ear. Because human rib provides a large, readily available source of hyaline cartilage, the authors evaluated human rib chondrocyte extraction and found that maximum viable cell yield occurred after a 6-hour digestion. They also evaluated human microtic auricular remnant chondrocyte extraction and identified fibroblast contamination as a shortcoming of this potential source of chondrocytes. Initially, rib chondrocytes proliferated in vitro with a doubling time of approximately 1 week. As the cells were passaged, proliferation decreased such that the cells stopped proliferating and adopted a large, spindle-shaped morphology by passage 6. Interestingly, no increase in proliferation was noted when rib chondrocytes were stimulated with transforming growth factor beta 1, bone morphogenetic protein 2, and basic fibroblast growth factor. The major obstacles to the use of autologous rib chondrocytes in matrix construction are the low cell yield from a small piece of rib and the limited proliferation that these cells will undergo in vitro. Further investigation of culture systems and mitogenic cytokines may help resolve these limitations.

The etiology of keloid formation is diverse. They are characterized grossly as thick scar tissue that extends beyond the boundaries of the original wound. Histologically, keloids are composed of excessive collagen with an abnormally large number of partially or totally occluded microvessels. This occlusion of keloid microvessels has been hypothesized to contribute to a hypoxic microenvironment within these pathological scars. Vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen, and proangiogenic cytokine have been implicated in normal and pathological wound healing. The purpose of this study was to evaluate the amount of VEGF protein production by fibroblast cell lines derived from keloids and normal human dermal skin in hypoxic compared with normoxic culture conditions. By enzyme-linked immunosorbent protein assay, VEGF was increased in both keloid and normal human dermal fibroblasts in hypoxia over normoxic controls. There was not, however, a significant difference between upregulation of VEGF protein when comparing the keloid and normal fibroblast groups. As the result of the data, alternative hypotheses for hypoxia-induced keloid formation were explored: (1) downstream modulation or signal transduction of VEGF, (2) VEGF production from cells other than fibroblasts, (3) the importance of matrix accumulation stimulated by hypoxia, or (4) increased migration of cells (other than fibroblasts) specific to keloid biology. These hypotheses may help explain the possible role of hypoxia in the pathogenesis of keloid formation. Future studies involving in situ hybridization or immunohistochemical analysis may offer greater insight into the mechanisms underlying keloid formation. Ultimately, our therapeutic goal is the utilization of biomolecular approaches for the suppression of keloid formation.

Background/Purpose: The transforming growth factor-β (TGF-β) cytokines are important regulators of growth and differentiation in multiple mammalian organ systems. Recent studies suggest that they may play a significant role in the regulation of pancreatic organogenesis. The authors proposed to examine the ontogeny of expression of the TGF-β cytokine isoforms (TGF-β1, β2, and β3), as well as that of the type II TGF-β receptor (TβRII), in the pancreas. We hypothesized that their patterns of expression might help to clarify the manner in which they influence the development of this organ. Methods: Embryos from pregnant CD-1 mice were harvested on gestational days 12.5, 15.5, and 18.5. Microdissection was performed on the embryos to isolate their pancreases. The pancreases were fixed, frozen embedded, and sectioned with a cryostat. Immunohistochemistry was performed using polyclonal antibodies to TGF-β1, β2, and β3, and TβRII. Results: The patterns of expression of TGF-β1, β2, and β3 were similar throughout gestation. They were all present, though weakly, early in the development of the pancreas, in the E12.5 epithelial cells. Their expression persisted and became localized to the acinar cells later in gestation. TβRII staining was present in both the E12.5 epithelial cells and the surrounding mesenchyme. As the pancreas developed, TβRII became strongly expressed in the ductal epithelial cells with only minimal staining in the acinar and endocrine cells. Conclusions: TGF-βs may play a role in regulating pancreatic organogenesis. Our data suggest that they may be required for the normal development of acini. As in other cell systems, TGF-β1 may act as a suppressor of pancreatic cellular growth and differentiation. The localization of TβRII to the mature ductal epithelium may indicate a need for ongoing regulation of growth and differentiation in the pancreatic ducts beyond the fetal period.

Background/Purpose : The organogenesis of esophageal atresia with tracheoesophageal fistula (EA-TEF) is unknown. Using an established model for EA-TEF in rats, the authors proposed to study this aberrancy of development in the hope of gaining insight into its mechanism of formation. Methods : Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 through 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. The specimens were analyzed histologically with routine H&E staining. Results : The classic EA-TEF developed in the embryos, with proximal EA and distal TEF. As expected, the atresia formed as a blind-ending pouch, but the distal fistula began as an apparent equal trifurcation of the tracheal anlage into two mainstem bronchi and the fistula tract leading to the stomach. Histological analysis of the fistula tract showed respiratorylike pseudostratified columnar epithelium. Conclusions : TEF develops as the middle branch of a tracheal trifurcation. EA-TEF occurs by a primary atresia of the esophagus. As a secondary phenomenon, the distal foregut anlage is switched toward the pulmonary phenotype. It trifurcates, and its middle branch grows caudally to fistulize into the stomach.

Craniosynostosis is a common disorder with an unknown etiology. Recent genetic mapping studies have demonstrated a strong linkage between several familial craniosynostotic syndromes and mutations in fibroblast growth factor receptor 1 (FGF-R1) and 2 (FGF-R2). The purpose of this experiment was to investigate by immunohistochemistry the protein production of these receptors as well as of their most prevalent ligand, basic fibroblast growth factor (bFGF), before, during, and after sutural fusion in rat cranial sutures. The posterior frontal (normally fuses between postnatal days 12 and 22) and sagittal (remains patent) sutures of embryonic day 20 and neonatal days 6, 12, 17, 22, and 62 (n = 3 per group) were harvested, fixed, and decalcified. Five-micrometer sections were stained with polyclonal antibodies against bFGF, FGF-R1, and FGF-R2, and patterns of immunohistochemical staining were assessed by independent reviewers. Our results indicate that increased bFGF production correlates temporally with suture fusion, with increased staining of the dura underneath the fusing suture prior to fusion followed by increased staining within osteoblasts and sutural cells during fusion. FGF-R1 and, to a lesser extent FGF-R2 immunostaining revealed a different pattern of localization with increased immunostaining within the patent sagittal suture at these time points. These results implicate bFGF in the regulation of sutural fusion and may imply autoregulatory mechanisms in fibroblast growth factor receptor expression.

Growth factors such as TGF-beta, PDGF and FGF are thought to play important roles in wound healing. However, their biological activity and signal transduction during wound repair remain poorly understood. Growth factors are often ligands for receptor tyrosine kinase and receptor serine/threonine kinases. With recent advances in signal transduction by receptor kinases, we are beginning to understand the underlying mechanism of how growth factors may regulate cutaneous wound repair. In this paper, we will describe the pharmacological effects of growth factors on wound healing, and discuss the potential underlying signaling mechanisms. Thus, we hope to provide the basis for designing more specific therapeutics for wound healing in the near future.

Incisional skin wounds in the early gestation fetus heal rapidly and without scarring. If the biological characteristics that make fetal skin resistant to scarring are determined, these properties might be replicated in the adult environment with tremendous clinical benefit. This article provides an overview of the biology of fetal repair.

We report the first generation of human cartilage from fibrin glue using a technique of molding chondrocytes in fibrin glue developed in our laboratory. Human costal chondrocytes were suspended in cryoprecipitate and polymerized into a human nasal shape with bovine thrombin. After culture in vitro for 4 weeks, this construct was implanted subcutaneously into a nude mouse. The final construct harvested after 4 weeks in vivo demonstrated some preservation of its original features. Histological analysis showed features of native cartilage, including matrix synthesis and viable chondrocytes by nuclear staining. Biochemical analysis demonstrated active matrix production. Biomechanical testing was performed. To our knowledge this is the first reported creation of human cartilage from fibrin glue, and the first creation of human cartilage in vitro. This technique may become a promising means of engineering precisely designed autogenous cartilage for human reconstruction.

Determination of signaling pathways that regulate beta-cell replication is critical for beta-cell therapy. Here, we show that blocking pancreatic macrophage infiltration after pancreatic duct ligation (PDL) completely inhibits beta-cell proliferation. The TGFβ superfamily signaling inhibitor SMAD7 was significantly up-regulated in beta cells after PDL. Beta cells failed to proliferate in response to PDL in beta-cell-specific SMAD7 mutant mice. Forced expression of SMAD7 in beta cells by itself was sufficient to promote beta-cell proliferation in vivo. M2, rather than M1 macrophages, seem to be the inducers of SMAD7-mediated beta-cell proliferation. M2 macrophages not only release TGFβ1 to directly induce up-regulation of SMAD7 in beta cells but also release EGF to activate EGF receptor signaling that inhibits TGFβ1-activated SMAD2 nuclear translocation, resulting in TGFβ signaling inhibition. SMAD7 promotes beta-cell proliferation by increasing CyclinD1 and CyclinD2, and by inducing nuclear exclusion of p27. Our study thus reveals a molecular pathway to potentially increase beta-cell mass through enhanced SMAD7 activity induced by extracellular stimuli.

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Type 2 Diabetes (T2D) is a global epidemic that affects hundreds of millions of individuals. The pancreatic β-cell has long been the focus of studies pertaining to T2D, but the therapies that exist to date are inadequate. Devising new therapies will require a more detailed understanding of the pathogenesis of T2D and of the normal function of the β-cell. In particular, there is a great need to understand the signaling pathways that govern normal β-cell function and that become dysfunctional during the progression to diabetes. The Transforming Growth Factor β (TGF-β) signaling pathway is implicated in nearly all tissue types in the body, and has been shown to play a role in pancreas development and homeostasis, including β-cell regeneration after pancreatic insult. TGF-β exerts its cellular effects through transcriptional activity of downstream SMAD molecules, as well as through cross-talk with other signaling pathways. Accumulating evidence suggests that β-cell failure in T2D is a multifaceted process that may include islet inflammation, increased β-cell apoptosis, reduced β-cell proliferation, and/or β-cell dedifferentiation to a progenitorlike state. This review details the known roles of TGF-β signaling in dedifferentiation-induced and inflammationinduced β-cell failure, and draws on the apparent coordinated regulation of β-cell proliferation and the β-cell differentiation state to offer new hypotheses about β-cell failure in T2D.

Gastroesophageal reflux (GER) often develops in children who have undergone prior repair of esophageal atresia/tracheoesophageal fistula (EA/TEF). Fundoplication is necessary in many of these children. The complete wrap (Nissen) fundoplication is often used in this setting. However, poor results have been noted, with a mean failure rate of 30% reported in four recent studies. A partial wrap fundoplication for GER associated with EA/TEF is theoretically attractive, because the poor esophageal motility and diminished acid clearance (already physiologically present) is exacerbated by a complete wrap fundoplication. The authors reviewed their extensive experience with partial wrap (Thal) fundoplication in EA/TEF to determine if the failure rate was better than that reported for the Nissen fundoplication. In the past 18 years, the authors performed 1,467 fundoplication procedures. During the same period, 143 children underwent repair of EA/TEF. Fifty-nine children underwent fundoplication after a previous EA/TEF repair. Most of the fundoplications (58 of 59, 98%) were Thal procedures. Defining failure strictly as a need for reoperation, the failure rate in our series was 15% (9 of 59 children). Compared with the failure rate in the 1,408 non-EA/TEF patients (61 of 1408, 4.3%), results were significantly worse for the EA/TEF group (P > .001). The failure rate of Thal fundoplication performed for GER in the EA/TEF population is substantially higher than the non-EA/TEF patients. The same factors responsible for the development of reflux in these children (poor acid clearance, altered motility, esophageal shortening) may contribute to the higher failure rate. Although partial wrap fundoplication frequently failed (15%), the results were still substantially better than those reported for Nissen fundoplication in these children (30% failure rate).

Purpose: Children with a wet, draining, or infected umbilicus are often referred to pediatric surgeons. Unfortunately, uniform guidelines regarding diagnostic imaging are lacking. Historically, the persistence of the urachus was attributed to intrauterine distal urinary obstruction. Today, many surgeons continue to advocate preoperative voiding cystourethrogram (VCUG). Methods: Records of children with urachal abnormalities over the past 10 years were reviewed. Demographics, presentation, imaging, genitourinary anomalies, operations, length of stay, and complications were recorded. Statistical evaluation was by descriptive analysis. Results: Fifty-six children were diagnosed with urachal anomalies. Age at operation was 2.5 years (1 day-13 years). Fifty percent of patients were less than 1 year. Ultrasound was used in 88% of cases. Voiding cystourethrogram (34%) and computed tomography (14%) were also used. Average hospitalization was 1.9 (0-13) days. Thirty-two percent underwent operations as outpatients. Seven percent developed wound infections. Eight children (14%) had genitourinary anomalies. However, no VCUG examination (n = 19) documented an obstructive process. Conclusions: The current study represents the largest reported series of symptomatic urachal anomalies in children. Disorders of the urachus are variable in presentation with the diagnosis reliably made by history and ultrasound alone. Further testing, including VCUG, is not warranted, adding additional cost, an invasive procedure, and inconvenience to the child.

Background & Aims Although the cells that contribute to pancreatic regeneration have been widely studied, little is known about the mediators of this process. During tissue regeneration, infiltrating macrophages debride the site of injury and coordinate the repair response. We investigated the role of macrophages in pancreatic regeneration in mice. Methods We used a saporin-conjugated antibody against CD11b to reduce the number of macrophages in mice following diphtheria toxin receptor–mediated cell ablation of pancreatic cells, and evaluated the effects on pancreatic regeneration. We analyzed expression patterns of infiltrating macrophages after cell-specific injury or from the pancreas of nonobese diabetic mice. We developed an in vitro culture system to study the ability of macrophages to induce cell-specific regeneration. Results Depletion of macrophages impaired pancreatic regeneration. Macrophage polarization, as assessed by expression of tumor necrosis factor−α, interleukin 6, interleukin 10, and CD206, depended on the type of injury. The signals provided by polarized macrophages promoted lineage-specific generation of acinar or endocrine cells. Macrophage from nonobese diabetic mice failed to provide signals necessary for β-cell generation. Conclusions Macrophages produce cell type–specific signals required for pancreatic regeneration in mice. Additional study of these processes and signals might lead to new approaches for treating type 1 diabetes or pancreatitis.

Glucagon-producing α-cells are the second-most abundant cell type in the islet. Whereas α-cells make up less than 20% of the cells in a mature mouse islet, they occupy a much larger proportion of the pancreatic endocrine cell population during the early postnatal period, the time when morphological and functional maturation occurs to form adult islets. To determine whether α-cells have a role in postnatal islet development, a diphtheria toxin-mediated α-cell ablation mouse model was established. Rapid and persistant depletion of α-cells was achieved by daily injection of the toxin for 2 wk starting at postnatal day 1 (P1). Total pancreatic glucagon content in the α-cell-ablated mice was undetectable at P14 and still less than 0.3% of that of the control mice at 4 mo of age. Histological analyses revealed that formation of spherical islets occurred normally, and the islet size distribution was not changed despite the near-total lack of α-cells. Furthermore, there were no differences in expression of β-cell maturation marker proteins, including urocortin 3 and glucose transporter 2, in the α-cell-ablated islets at P14. Mice lacking α-cells grew normally and appeared healthy. Both glucose and insulin tolerance tests demonstrated that the α-cell-ablated mice had normal glucose homeostasis. These results indicate that α-cells do not play a critical role in postnatal islet morphogenesis or functional maturation of β-cells.

It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.

Hypoxia promotes angiogenesis, proliferation, invasion, and metastasis of pancreatic cancer. Essentially, all studies of the hypoxia pathway in pancreatic cancer research to date have focused on fully malignant tumors or cancer cell lines, but the potential role of hypoxia inducible factors (HIF) in the progression of premalignant lesions has not been critically examined. Here, we show that HIF2α is expressed early in pancreatic lesions both in human and in a mouse model of pancreatic cancer. HIF2α is a potent oncogenic stimulus, but its role in Kras-induced pancreatic neoplasia has not been discerned. We used the Ptf1aCre transgene to activate KrasG12D and delete Hif2α solely within the pancreas. Surprisingly, loss of Hif2α in this model led to markedly higher, rather than reduced, number of low-grade pancreatic intraepithelial neoplasia (mPanIN) lesions. These lesions, however, failed to progress to high-grade mPanINs, and displayed exclusive loss of β-catenin and SMAD4. The relationship among HIF2α, β-catenin, and Smad4 was further confirmed in vitro, where silencing of Hif2α resulted in reduced β-catenin and Smad4 transcript levels. Thus, with oncogenic Ras expressed in the pancreas, HIF2α modulates Wnt-signaling during mPanIN progression by maintaining appropriate levels of both Smad4 and β-catenin. Cancer Res; 73(15); 4781–90. ©2013 AACR.

Children with neurological impairment (NI) frequently require feeding gastrostomy, and this often aggravates or produces gastroesophageal reflux (GER). From 1976 to 1994, 141 children with severe NI underwent Thal fundoplication and gastrostomy (GT). GER was evident in 80%; in the rest, fundoplication was an adjunct to GT. Ph results were positive in 38 cases, and 57 children had reflux according to the barium studies. There were no major intraoperative complications. Disruption of the repair and/or recurrent GER was noted in 14 cases (10%); 8 were redone as Thals, and 6 were converted to Nissen procedures. Pyloroplasty was done later in 9 children (6%). Bowel obstruction was seen in 4 patients (3%). Clinical follow-up (mean, 54 months) showed improvement in 96%; only 5 of the 141 (3.2%) have residual symptoms. Of the patients with an intact Thal, 67% could burp or vomit. The ability to vomit may protect the Thal fundoplication and avoid disruption of the repair.

Pancreatic organogenesis has been a classic example of epitheliomesenchymal interactions. The nature of this interaction, and the way in which endocrine, acinar and ductal cell lineages are generated from the embryonic foregut has not been determined. It has generally been thought that mesenchyme is necessary for all aspects of pancreatic development. In addition islets have been thought to derive, at least in part, from ducts. We microdissected 11-day embryonic mouse pancreas and developed several culture systems for assays of differentiation: (i) on transparent filters; (ii) suspended in a collagen I gel; (iii) suspended in a basement membrane rich gel; (iv) under the renal capsule of an adult mouse. Epithelia were grown either with or without mesenchyme, and then assayed histologically and immunohistochemically. Epithelium with its mesenchyme (growth systems i-iv) always grew into fully differentiated pancreas (acinar, endocrine, and ductal elements). In the basement membrane-rich gel, epithelium without mesenchyme formed ductal structures. Under the renal capsule of the adult mouse the epithelium without mesenchyme exclusively formed clusters of mature islets. These latter results represent the first demonstration of pure islets grown from early pancreatic precursor cells. In addition, these islets seemed not to have originated from ducts. We propose that the default path for growth of embryonic pancreatic epithelium is to form islets. In the presence of basement membrane constituents, however, the pancreatic anlage epithelium appears to be programmed to form ducts. Mesenchyme seems not to be required for all aspects of pancreatic development, but rather only for the formation of acinar structures. In addition, the islets seem to form from early embryonic epithelium (which only express non-acinar genes). This formation occurs without any specific embryonic signals, and without any clear duct or acinus formation.

Although the pathogenesis of esophageal atresia with tracheoesophageal fistula (EA/TEF) remains unknown, it has been shown that despite its esophageal appearance, the fistula tract originates from respiratory epithelium. The authors now hypothesize that defects in fibroblast growth factor (FGF) signaling contribute to the esophaguslike phenotype of the fistula tract. FGF2R is critical to normal lung morphogenesis and occurs in 2 isoforms (FGF2RIIIb and FGF2RIIIc), each with different ligand-binding specificity. To characterize FGF signaling in the developing EA/TEF, the authors analyzed levels of FGF2R splice variants in experimental EA/TEF.The standard Adriamycin-induced EA/TEF model in rats was used. Individual foregut components from Adriamycin-treated and control embryos were processed for real-time, fluorescence-activated semiquantitative reverse transcriptase polymerase chain reaction on gestational days 12.5 and 13.5.Both fistula tract and Adriamycin-treated or normal esophagus showed significantly lower levels of FGF2RIIIb than either Adriamycin-treated lung buds (E12.5, P =.02; E13.5, P.005) or normal lung buds (E12.5, P.005; E13.5, P.01). At E13.5, the fistula tract had lower levels of FGF2RIIIc than either treated (P.01) or normal lung (P.05).Levels of FGF2R in the developing fistula tract resemble that of distal esophagus rather than developing lung. This defect in FGF2RIIIb signaling may account for the nonbranching, esophaguslike phenotype of the fistula, despite its respiratory origin.

Adeno-Associated Virus (AAV) has been widely used for in vivo study and preclinical therapy due to its ability to mediate long-term transgene expression, its lack of pathogenicity and low immunogenicity. It has been found that AAV has more than ten serotypes, which each transfect certain types of cells in the viral infected organ. Current methods for purification of different AAV serotypes utilize CsCl or Iodixanol ultrahigh speed density gradient centrifugation, which is expensive and time consuming. We recently developed a simplified method, PEG/(NH(4))(2)SO(4) aqueous two phase partitioning, for purification of AAV serotype 2 and 8. The method does not require ultrahigh speed gradient centrifugation or chromatography. Here we further explore the simplified method for purification of other serotypes of AAV, serotype 6 and 9. This simplified method not only can be used to purify serotype 2 and 8, but also serotype 6 and 9, indicating that a variety of AAV serotypes can be purified by this method.

VEGF-A expression in beta cells is critical for pancreatic development, formation of islet-specific vasculature, and Insulin secretion. However, two key questions remain. First, is VEGF-A release from beta cells coupled to VEGF-A production in beta cells? Second, how is the VEGF-A response by beta cells affected by metabolic signals? Here, we show that VEGF-A secretion, but not gene transcription, in either cultured islets or purified pancreatic beta cells, was significantly reduced early on during low glucose conditions. In vivo, a sustained hypoglycemia in mice was induced with Insulin pellets, resulting in a significant reduction in beta cell mass. This loss of beta cell mass could be significantly rescued with continuous delivery of exogenous VEGF-A, which had no effect on beta cell mass in normoglycemic mice. In addition, an increase in apoptotic endothelial cells during hypoglycemia preceded an increase in apoptotic beta cells. Both endothelial and beta cell apoptosis were prevented by exogenous VEGF-A, suggesting a possible causative relationship between reduced VEGF-A and the loss of islet vasculature and beta cells. Furthermore, in none of these experimental groups did beta cell proliferation and islet vessel density change, suggesting a tightly regulated balance between these two cellular compartments. The average islet size decreased in hypoglycemia, which was also prevented by exogenous VEGF-A. Taken together, our data suggest that VEGF-A release in beta cells is independent of VEGF-A synthesis. Beta cell mass can be regulated through modulated release of VEGF-A from beta cells based on physiological need.