Suture fistula revisited for long gap esophageal atresia.

BACKGROUND: Long gap esophageal atresia presents a challenge to pediatric surgeons due to the variability in surgical management when primary anastomosis is not feasible. Mechanical traction procedures enhance tissue growth that allows early anastomosis, before delayed primary closure (DPC) would be attempted to allow for rapid oral feeding, or when operative discoveries require flexibility of the surgical plan. The Suture Fistula procedure, first published by Alan Shafer and Tirone David in 1974, is a simple, effective, but underutilized single-stage procedure which results in spontaneous fistulization of approximated, non-anastomosed esophageal segments using tension sutures. METHODS: A retrospective chart review was performed of patients who underwent the Suture Fistula procedure at a single institution since 1992. A literature review of all published case series of patients who underwent this procedure was also performed. RESULTS: There were 5 case series found with a total of 24 patients, and three new cases presented. Patients were noted to have spontaneous fistulization with gastrostomy tube feed reflux noted in the upper esophagus or mouth on average of post-operative day 14, which occurred in over 85% of patients. While nearly all patients required esophageal dilation and anti-reflux procedures, the morbidity of the procedure, including esophageal leak, is very low, and similar to the Foker or Kimura procedures, which have been more popular despite their surgical complexity. CONCLUSION: We propose the Suture Fistula technique to be a simpler, more effective, and safe alternative to other mechanical traction suture procedures in cases where primary anastomosis is not feasible. LEVEL OF EVIDENCE: Level IV.

Effect of Systemic Iron Overload and a Chelation Therapy in a Mouse Model of the Neurodegenerative Disease Hereditary Ferritinopathy.

Mutations in the ferritin light chain (FTL) gene cause the neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic iron-containing ferritin inclusion bodies (IBs) in glia and neurons throughout the central nervous system (CNS) and in tissues of multiple organ systems. Herein, using primary mouse embryonic fibroblasts from a mouse model of HF, we show significant intracellular accumulation of ferritin and an increase in susceptibility to oxidative damage when cells are exposed to iron. Treatment of the cells with the iron chelator deferiprone (DFP) led to a significant improvement in cell viability and a decrease in iron content. In vivo, iron overload and DFP treatment of the mouse model had remarkable effects on systemic iron homeostasis and ferritin deposition, without significantly affecting CNS pathology. Our study highlights the role of iron in modulating ferritin aggregation in vivo in the disease HF. It also puts emphasis on the potential usefulness of a therapy based on chelators that can target the CNS to remove and redistribute iron and to resolubilize or prevent ferritin aggregation while maintaining normal systemic iron stores.

Role of p85α in neutrophil extra- and intracellular reactive oxygen species generation.

Drug resistance is a growing problem that necessitates new strategies to combat pathogens. Neutrophil phagocytosis and production of intracellular ROS, in particular, has been shown to cooperate with antibiotics in the killing of microbes. This study tested the hypothesis that p85α, the regulatory subunit of PI3K, regulates production of intracellular ROS. Genetic knockout of p85α in mice caused decreased expression of catalytic subunits p110α, p110ß, and p110δ, but did not change expression levels of the NADPH oxidase complex subunits p67phox, p47phox, and p40phox. When p85α, p55α, and p50α (all encoded by Pik3r1) were deleted, there was an increase in intracellular ROS with no change in phagocytosis in response to both Fcγ receptor and complement receptor stimulation. Furthermore, the increased intracellular ROS correlated with significantly improved neutrophil killing of both methicillin-susceptible and methicillin-resistant S. aureus. Our findings suggest inhibition of p85α as novel approach to improving the clearance of resistant pathogens.

Interprofessional education through service-learning: lessons from a student-led free clinic.

The academic community must replicate and strengthen existing models for interprofessional education (IPE) to meet widespread calls for team-based patient-centered care. One effective but under-explored possibility for IPE is through student-led clinics, which now exist in the majority of medical schools. This short report presents the Indiana University Student Outreach Clinic (IU-SOC), which involves seven different professional programs across three institutions, as a model for how IPE can be delivered formally through service learning. Lessons learned, such as nurturing an intentional interprofessional program, structured orientation and reflection, and resource and knowledge sharing between the clinic and academic institutions, can be applied to all student-led clinics, but also can inform other IPE initiatives in health professional curricula.

Systemic and cerebral iron homeostasis in ferritin knock-out mice.

Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect.

Protein-tyrosine phosphatase Shp2 positively regulates macrophage oxidative burst.

Macrophages are vital to innate immunity and express pattern recognition receptors and integrins for the rapid detection of invading pathogens. Stimulation of Dectin-1 and complement receptor 3 (CR3) activates Erk- and Akt-dependent production of reactive oxygen species (ROS). Shp2, a protein-tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt and is crucial for hematopoietic cell function; however, no studies have examined Shp2 function in particulate-stimulated ROS production. Maximal Dectin-1-stimulated ROS production corresponded kinetically to maximal Shp2 and Erk phosphorylation. Bone marrow-derived macrophages (BMMs) from mice with a conditionally deleted allele of Ptpn11 (Shp2(flox/flox);Mx1Cre+) produced significantly lower ROS levels compared with control BMMs. Although YFP-tagged phosphatase dead Shp2-C463A was strongly recruited to the early phagosome, its expression inhibited Dectin-1- and CR3-stimulated phospho-Erk and ROS levels, placing Shp2 phosphatase function and Erk activation upstream of ROS production. Further, BMMs expressing gain of function Shp2-D61Y or Shp2-E76K and peritoneal exudate macrophages from Shp2D61Y/+;Mx1Cre+ mice produced significantly elevated levels of Dectin-1- and CR3-stimulated ROS, which was reduced by pharmacologic inhibition of Erk. SIRPα (signal regulatory protein α) is a myeloid inhibitory immunoreceptor that requires tyrosine phosphorylation to exert its inhibitory effect. YFP-Shp2C463A-expressing cells have elevated phospho-SIRPα levels and an increased Shp2-SIRPα interaction compared with YFP-WT Shp2-expressing cells. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated ROS production in macrophages by dephosphorylating and thus mitigating the inhibitory function of SIRPα and by promoting Erk activation.

PI3K p110δ uniquely promotes gain-of-function Shp2-induced GM-CSF hypersensitivity in a model of JMML.

Although hyperactivation of the Ras-Erk signaling pathway is known to underlie the pathogenesis of juvenile myelomonocytic leukemia (JMML), a fatal childhood disease, the PI3K-Akt signaling pathway is also dysregulated in this disease. Using genetic models, we demonstrate that inactivation of phosphatidylinositol-3-kinase (PI3K) catalytic subunit p110δ, but not PI3K p110α, corrects gain-of-function (GOF) Shp2-induced granulocyte macrophage-colony-stimulating factor (GM-CSF) hypersensitivity, Akt and Erk hyperactivation, and skewed hematopoietic progenitor distribution. Likewise, potent p110δ-specific inhibitors curtail the proliferation of GOF Shp2-expressing hematopoietic cells and cooperate with mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK) inhibition to reduce proliferation further and maximally block Erk and Akt activation. Furthermore, the PI3K p110δ-specific inhibitor, idelalisib, also demonstrates activity against primary leukemia cells from individuals with JMML. These findings suggest that selective inhibition of the PI3K catalytic subunit p110δ could provide an innovative approach for treatment of JMML, with the potential for limiting toxicity resulting from the hematopoietic-restricted expression of p110δ.

Genetic disruption of the PI3K regulatory subunits, p85α, p55α, and p50α, normalizes mutant PTPN11-induced hypersensitivity to GM-CSF.

Juvenile myelomonocytic leukemia is a lethal disease of children characterized by hypersensitivity of hematopoietic progenitors to granulocyte macrophage-colony stimulating factor. Mutations in PTPN11, the gene encoding the protein tyrosine phosphatase Shp2, are common in juvenile myelomonocytic leukemia and induce hyperactivation of the phosphoinositide-3-kinase pathway. We found that genetic disruption of Pik3r1, the gene encoding the Class IA phosphoinositide-3-kinase regulatory subunits p85α, p55α and p50α, significantly reduced hyperproliferation and hyperphosphorylation of Akt in gain-of-function Shp2 E76K-expressing cells. Elevated protein levels of the phosphoinositide-3-kinase catalytic subunit, p110δ, in the Shp2 E76K-expressing Pik3r1-/- cells suggest that p110δ may be a crucial mediator of mutant Shp2-induced phosphoinositide-3-kinase hyperactivation. Consistently, treatment with the p110δ-specific inhibitor, IC87114, or the clinical grade pan-phosphoinositide-3-kinase inhibitor, GDC-0941, reduced granulocyte macrophage-colony stimulating factor hypersensitivity. Treatment with the farnesyltransferase inhibitor, tipifarnib, showed that Shp2 E76K induces hyperactivation of phosphoinositide-3-kinase by both Ras-dependent and Ras-independent mechanisms. Collectively, these findings implicate Class IA phosphoinositide-3-kinase as a relevant molecular target in juvenile myelomonocytic leukemia.

Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice.

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 (176)RXXR(179)/S(180) proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene-environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Rho kinase regulates the survival and transformation of cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL.

We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene-bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient-derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth, and loss of actin polymerization in oncogene-bearing cells leading to significantly prolonged life span of leukemic mice. In summary we describe a pathway involving PI3K/Rho/ROCK/MLC that may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans.

In vivo interaction of steroid receptor coactivator (SRC)-1 and the activation function-2 domain of the thyroid hormone receptor (TR) beta in TRbeta E457A knock-in and SRC-1 knockout mice.

The activation function-2 (AF-2) domain of the thyroid hormone (TH) receptor (TR)-beta is a TH-dependent binding site for nuclear coactivators (NCoA), which modulate TH-dependent gene transcription. In contrast, the putative AF-1 domain is a TH-independent region interacting with NCoA. We determined the specificity of the AF-2 domain and NCoA interaction by evaluating thyroid function in mice with combined disruption of the AF-2 domain in TRbeta, due to a point mutation (E457A), and deletion of one of the NCoAs, steroid receptor coactivator (SRC)-1. The E457A mutation was chosen because it abolishes NCoA recruitment in vitro while preserving normal TH binding and corepressor interactions resulting in resistance to TH. At baseline, disruption of SRC-1 in the homozygous knock-in (TRbeta(E457A/E457A)) mice worsened the degree of resistance to TH, resulting in increased serum T(4) and TSH. During TH deprivation, disruption of AF-2 and SRC-1 resulted in a TSH rise 50% of what was seen when AF-2 alone was removed, suggesting that SRC-1 was interacting outside of the AF-2 domain. Therefore, 1) during TH deprivation, SRC-1 is necessary for activating the hypothalamic-pituitary-thyroid axis; 2) ligand-dependent repression of TSH requires an intact AF-2; and 3) SRC-1 may interact with the another region of the TRbeta or the TRalpha to regulate TH action in the pituitary. This report demonstrates the dual interaction of NCoA in vivo: the TH-independent up-regulation possibly through another domain and TH-dependent down-regulation through the AF-2 domain.

Effects of maternal levels of thyroid hormone (TH) on the hypothalamus-pituitary-thyroid set point: studies in TH receptor beta knockout mice.

A level of thyroid hormone (TH) in agreement with the tissue requirements is essential for vertebrate embryogenesis and fetal maturation. In this study we evaluate the immediate and long-term effects of incongruent intrauterine TH levels between mother and fetus using the TH receptor (TR) beta(-/-) knockout mouse as a model. We took advantage of the fact that the TRbeta(-/-) females have elevated serum TH but are not thyrotoxic due to resistance to TH. We used crosses between heterozygotes with wild-type phenotype (TRbeta(+/-)) males and TRbeta(-/-) females, with a hyperiodothyroninemic (high T(4) and T(3) levels) intrauterine environment (TH congruent with the TRbeta(-/-) fetus and excessive for the TRbeta(+/-) fetus), and reciprocal crosses between TRbeta(-/-) males and TRbeta(+/-) females, providing a euiodothyroninemic intrauterine environment. We found that TRbeta(-/-) dams had reduced litter sizes and pups with lower birth weight but preserved the mendelian TRbeta(-/-) to TRbeta(+/-) ratio at birth, indicating that the incongruous TH levels did not decrease intrauterine survival of a specific genotype. The results of studies in newborns demonstrate that TRbeta(+/-) pups born to TRbeta(-/-) dams have persistent suppression of serum TSH without a peak. On the other hand, TRbeta(-/-) pups born to TRbeta(+/-) dams have lower serum TSH at birth and a tendency to peak higher, compared with TRbeta(-/-) pups born to TRbeta(-/-) dams. The studies in the adult progeny demonstrate that TRbeta(+/-) mice born to TRbeta(-/-) dams and, thus, exposed to higher intrauterine TH levels, have greater resistance to TH at the level of the pituitary when stimulated with TRH. On the other hand, TRbeta(-/-) mice born to TRbeta(+/-) dams and, thus, deprived of TH in uterine life, were more sensitive to TH when similarly stimulated with TRH. Thus, TH exposure in utero has an effect on the regulatory set point of the hypothalamus-pituitary-thyroid axis, which can be seen early in life and persists into adulthood.

Pituitary-thyroid setpoint and thyrotropin receptor expression in consomic rats.

The genetic basis for differences in TSH sensitivity between two rat strains was examined using consomic rats generated from original strains salt-sensitive Dahl (SS) (TSH 1.8 +/- 0.1 ng/ml; free T(4) index 4.9 +/- 0.4) and Brown Norwegian (BN) (TSH 5.5 +/- 0.6 ng/ml, P < 0.05; free T(4) index 4.3 +/- 0.1, P not significant). Consomic rats SSBN6 [BN chromosome (CH) 6 placed in SS rat] and SSBN2 (BN CH 2 placed in SS rat) have TSH concentrations intermediate between pure SS and BN strains (2.9 +/- 0.3 and 3.1 +/- 0.3 ng/ml, respectively; P < 0.05). Candidate genes on rat CH 2 included TSH beta-subunit and on CH 6 the TSH receptor (TSHR). TSH from sera of BN, SS, SSBN6, and SSBN2 strains had similar in vitro bioactivity suggesting that the cause for the variable TSH concentrations was not due to an altered TSH. Physiological response to TSH was measured by changes in serum T(4) concentrations upon administration of bovine TSH (bTSH). Rat strain SS had a greater T(4) response to bTSH than BN (change in T(4), 1.3 +/- 0.1 vs. 0.4 +/- 0.1 microg/dl, P < 0.005), suggesting reduced thyrocyte sensitivity to TSH in BN. Sequencing of the TSHR coding region revealed an amino acid difference in BN (Q46R). This substitution is unlikely to contribute to the strain difference in serum TSH because both TSHR variants were equally expressed at the cell surface of transfected cells and responsive to bTSH. Given similar TSH activity and similar TSHR structure, TSHR mRNA expression in thyroid tissue was quantitated by real-time PCR. BN had 54 +/- 5% the total TSHR expression compared to SS (100 +/- 7%, P < 0.0001), when corrected for GAPDH expression, a difference confirmed at the protein level. Therefore, the higher TSH level in the BN strain appears to reflect an adjustment of the feedback loop to reduced thyrocyte sensitivity to TSH secondary to reduced TSHR expression. These strains of rat provide a model to study the cis- and trans-acting factors underlying the difference in TSHR expression.