Quantitative representation of Eustachian tube component movements during swallowing.

OBJECTIVE: Describes a method of dynamic video-endoscopy of the Eustachian tube (ET) orifice at the nasopharynx to quantitatively represent ET component movements during swallowing using a graphic function and analyze their importance to its opening mechanics. METHODS: This was a pilot study of relational event capture using a polar coordinate system applied to trans-nasal video-endoscopic recordings of the ET during 3 swallows in 5 adults. After topical anesthesia of the nose, a 45° telescope was introduced unilaterally and focused on the ipsilateral ET orifice. For each recording, consecutive still-frame images were analyzed by identifying 4 fixed-point locators; the luminal apex, lateral and medial walls and the torus. A frame-normal, horizontal line was constructed through the apex and, then, the medial angles defined at the intersection of the horizontal line and the lines from apex to each point locator were measured. The magnitudes of these angles were plotted as a function of time (i.e. successive frames) for each swallow. RESULTS: The resulting graphs captured the "in plane" relational movements for the locator points during a swallow. Complex interactions among the ET components were resolvable and the patterns were reproducible across swallows. Individual peculiarities observable on review of the corresponding "movies" such as double-swallows, delayed swallows and ET luminal constriction were easily identified in the graphic representation. CONCLUSION: This methodology is potentially useful for summary presentations of the ET mechanics of individual patients and for quantifying differences in those mechanics between groups defined by their history of middle-ear disease.

Histamine Applied Topically to the Nasal Mucosa Increases the Transmucosal Nitrous Oxide Exchange for the Middle Ear.

OBJECTIVE: Determine if the middle ear transmucosal nitrous oxide (N2O) exchange rate is affected by nasal inflammation caused by topical application of histamine. METHODS: In a randomized, double-blind, crossover study, 20 adults were challenged intranasally with histamine (5 mg) and placebo on separate occasions. At each session, the subjects were fitted with a non-rebreathing mask and breathed room air for 20 minutes, 50% N2O:50% O2 for 20 minutes, and 100% O2 for 10 minutes. Throughout, heart rate, blood pressure, and blood O2 saturation were monitored, and bilateral middle ear pressure was recorded by tympanometry every minute. The primary outcome measure was the slope of the middle ear pressure-time function for the 50% N2O:50% O2 breathing period, which is a measure of the transmucosal N2O exchange-constant. The effects of challenge substance, session, and period on the measured vital signs and of treatment, session, ear disease history, and test ear on the pressure-time slopes were evaluated using repeated measures ANOVAs. RESULTS: The post-challenge total symptom score and the slope of the middle ear pressure-time function were greater after histamine when compared to placebo challenge. Of the signs, only heart rate was affected, responding to challenge substance and study period. CONCLUSION: The transmucosal N2O exchange rate for the middle ear is increased during inflammation caused by nasal histamine exposure.

Cyclooxygenase-2 inhibition for the prevention of subglottic stenosis.

OBJECTIVE: To evaluate the role of targeted cyclooxygenase-2 inhibition in reducing scarring associated with a subglottic airway mucosal injury. DESIGN: Thirty-four New Zealand white rabbits underwent anterior cricothyroidotomy. Subglottic stenosis (SGS) was created by carbon dioxide laser injury. INTERVENTION: Treatment consisted of intraperitoneal injection of celecoxib or vehicle for 4 days. Endoscopies were performed to assess injury and healing. Subglottic mucosal secretions were collected with Gelfoam swabs (Pfizer Inc) before and after injury and at subsequent time points. Animals were humanely killed at 3 or 8 weeks after injury and airways were excised, followed by gross examination and histologic analysis to assess the severity of SGS. Secretions were analyzed for interleukin-1ß, prostaglandin E2 (PGE2), and matrix metalloproteinase-8 by enzyme-linked immunosorbent assays. RESULTS: Endoscopy showed mild to moderate stenosis in the celecoxib group, but mild to severe stenosis in the vehicle group. Histologic assessment confirmed and quantified reduction in stenosis and scarring as well as advanced reepithelialization. In the healing tissue, mucosal thickening (stenosis) was reduced significantly (P = .02) in celecoxib-treated animals compared with those treated with vehicle, at 3 and 8 weeks (decrease in thickness by 32% and 49%, respectively). Collagen density (fibrosis) was also reduced 25% at both 3 and 8 weeks but the difference was not statistically significant (P = .20). Reduced level of PGE2 in the subglottic mucosal secretions was correlated with mucosal thickness at 8 weeks (P = .02). CONCLUSION: Short-duration, anti-inflammatory therapy resulted in reduced stenosis and fibrosis with correlation of PGE2 levels in subglottic mucosal secretions.

Extracellular high mobility group box-1 (HMGB1) inhibits enterocyte migration via activation of Toll-like receptor-4 and increased cell-matrix adhesiveness.

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.

Prostaglandin E2 differentially regulates contraction and structural reorganization of anchored collagen gels by human adult and fetal dermal fibroblasts.

Contraction and remodeling of granulation tissue by fibroblasts is a crucial component of dermal wound healing. Postnatal wounds heal with imperfect repair and scar formation, whereas tissue repair in fetal wounds is regenerative. Prostaglandin E2 (PGE2) modulates the behavior of fibroblasts in the wound bed. This study was designed to investigate the mechanism by which PGE2 regulates an in vitro model of granulation tissue, anchored collagen gels, by human adult and fetal dermal fibroblasts. We hypothesized that PGE2 differentially regulates contraction and remodeling of anchored collagen gels by these fibroblast phenotypes. These results indicate that once tension was generated, fetal fibroblasts exerted lower contractile forces resulting in less collagen contraction. This coincided with less prominent stress fibers, yet fetal fibroblasts were able to substantially remodel the collagen architecture. This mechanism was differentially modulated by PGE2 and was mimicked with a PGE2 receptor agonist, indicating a cyclic adenosine monophosphate (cAMP)-dependent mechanism through the EP2 receptor. However, direct up-regulation of cAMP led to decreases in contraction and remodeling by both fibroblast phenotypes indicating an altered signaling pathway. Therefore, targeting cAMP via the EP2 receptor could potentially decrease adult fibroblast contractile forces to the levels of the fetal fibroblast phenotype in order to decrease dermal scarring.

A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in preterm infants and is characterized by translocation of LPS across the inflamed intestine. We hypothesized that the LPS receptor (TLR4) plays a critical role in NEC development, and we sought to determine the mechanisms involved. We now demonstrate that NEC in mice and humans is associated with increased expression of TLR4 in the intestinal mucosa and that physiological stressors associated with NEC development, namely, exposure to LPS and hypoxia, sensitize the murine intestinal epithelium to LPS through up-regulation of TLR4. In support of a critical role for TLR4 in NEC development, TLR4-mutant C3H/HeJ mice were protected from the development of NEC compared with wild-type C3H/HeOUJ littermates. TLR4 activation in vitro led to increased enterocyte apoptosis and reduced enterocyte migration and proliferation, suggesting a role for TLR4 in intestinal repair. In support of this possibility, increased NEC severity in C3H/HeOUJ mice resulted from increased enterocyte apoptosis and reduced enterocyte restitution and proliferation after mucosal injury compared with mutant mice. TLR4 signaling also led to increased serine phosphorylation of intestinal focal adhesion kinase (FAK). Remarkably, TLR4 coimmunoprecipitated with FAK, and small interfering RNA-mediated FAK inhibition restored enterocyte migration after TLR4 activation, demonstrating that the FAK-TLR4 association regulates intestinal healing. These findings demonstrate a critical role for TLR4 in the development of NEC through effects on enterocyte injury and repair, identify a novel TLR4-FAK association in regulating enterocyte migration, and suggest TLR4/FAK as a therapeutic target in this disease.

One-dimensional elastic continuum model of enterocyte layer migration.

Necrotizing enterocolitis is the leading cause of death from gastrointestinal disease in preterm infants. It results from an injury to the mucosal lining of the intestine, leading to translocation of bacteria and endotoxin into the circulation. Intestinal mucosal defects are repaired by the process of intestinal restitution, during which enterocytes migrate from healthy areas to sites of injury. In this article, we develop a mathematical model of migration of enterocytes during experimental necrotizing enterocolitis. The model is based on a novel assumption of elastic deformation of the cell layer and incorporates the following effects: i), mobility promoting force due to lamellipod formation, ii), mobility impeding adhesion to the cell matrix, and iii), enterocyte proliferation. Our model successfully reproduces the behavior observed for enterocyte migration on glass coverslips, namely the dependence of migration speed on the distance from the wound edge, and the finite propagation distance in the absence of proliferation that results in an occasional failure to close the wound. It also qualitatively reproduces the dependence of migration speed on integrin concentration. The model is applicable to the closure of a wound with a linear edge and, after calibration with experimental data, could be used to predict the effect of chemical agents on mobility, adhesion, and proliferation of enterocytes.

Interferon-gamma inhibits intestinal restitution by preventing gap junction communication between enterocytes.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is characterized by interferon-gamma (IFN-gamma) release and inadequate intestinal restitution. Because enterocytes migrate together, mucosal healing may require interenterocyte communication via connexin 43-mediated gap junctions. We hypothesize that enterocyte migration requires interenterocyte communication, that IFN impairs migration by impairing connexin 43, and that impaired healing during NEC is associated with reduced gap junctions. METHODS: NEC was induced in Swiss-Webster or IFN(-/-) mice, and restitution was determined in the presence of the gap junction inhibitor oleamide, or via time-lapse microscopy of IEC-6 cells. Connexin 43 expression, trafficking, and localization were detected in cultured or primary enterocytes or mouse or human intestine by confocal microscopy and (35)S-labeling, and gap junction communication was assessed using live microscopy with oleamide or connexin 43 siRNA. RESULTS: Enterocytes expressed connexin 43 in vitro and in vivo, and exchanged fluorescent dye via gap junctions. Gap junction inhibition significantly reduced enterocyte migration in vitro and in vivo. NEC was associated with IFN release and loss of enterocyte connexin 43 expression. IFN inhibited enterocyte migration by reducing gap junction communication through the dephosphorylation and internalization of connexin 43. Gap junction inhibition significantly increased NEC severity, whereas reversal of the inhibitory effects of IFN on gap junction communication restored enterocyte migration after IFN exposure. Strikingly, IFN(-/-) mice were protected from the development of NEC, and showed restored connexin 43 expression and intestinal restitution. CONCLUSIONS: IFN inhibits enterocyte migration by preventing interenterocyte gap junction communication. Connexin 43 loss may provide insights into the development of NEC, in which restitution is impaired.

Nitric oxide inhibits enterocyte migration through activation of RhoA-GTPase in a SHP-2-dependent manner.

Diseases of intestinal inflammation like necrotizing enterocolitis (NEC) are associated with impaired epithelial barrier integrity and the sustained release of intestinal nitric oxide (NO). NO modifies the cytoskeletal regulator RhoA-GTPase, suggesting that NO could affect barrier healing by inhibiting intestinal restitution. We now hypothesize that NO inhibits enterocyte migration through RhoA-GTPase and sought to determine the pathways involved. The induction of NEC was associated with increased enterocyte NO release and impaired migration of bromodeoxyuridine-labeled enterocytes from terminal ileal crypts to villus tips. In IEC-6 enterocytes, NO significantly inhibited enterocyte migration and activated RhoA-GTPase while increasing the formation of stress fibers. In parallel, exposure of IEC-6 cells to NO increased the phosphorylation of focal adhesion kinase (pFAK) and caused a striking increase in cell-matrix adhesiveness, suggesting a mechanism by which NO could impair enterocyte migration. NEC was associated with increased expression of pFAK in the terminal ileal mucosa of wild-type mice and a corresponding increase in disease severity compared with inducible NO synthase knockout mice, confirming the dependence of NO for FAK phosphorylation in vivo and its role in the pathogenesis of NEC. Strikingly, inhibition of the protein tyrosine phosphatase SHP-2 in IEC-6 cells prevented the activation of RhoA by NO, restored focal adhesions, and reversed the inhibitory effects of NO on enterocyte migration. These data indicate that NO impairs mucosal healing by inhibiting enterocyte migration through activation of RhoA in a SHP-2-dependent manner and support a possible role for SHP-2 as a therapeutic target in diseases of intestinal inflammation like NEC.

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier.

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.

An approach to the writing of a scientific manuscript.

OBJECTIVE: Writing a scientific manuscript is one of the most important tasks facing the academician, and may also be one of the most daunting. MATERIALS: The essentials of any paper include a description of what is known, an assessment of what is unknown, a clear statement regarding the question and hypothesis being addressed by the current study, and a discussion and summary of new information that has been learned as a result of the study. CONCLUSIONS: A formulaic approach is provided to guide the author through this process.

Increased expression and function of integrins in enterocytes by endotoxin impairs epithelial restitution.

BACKGROUND & AIMS: Experimental necrotizing enterocolitis (NEC) is characterized by circulating endotoxin (lipopolysaccharide [LPS]) and impaired enterocyte migration. We hypothesized that LPS increases integrin function and cell-matrix adhesion, leading to impaired enterocyte migration in the pathogenesis of NEC. METHODS: NEC-like intestinal injury was induced in newborn rats by hypoxia/gavage feedings, and restitution was determined by assessing bromodeoxyuridine-labeled enterocytes along the crypt-villus axis. Newborn mice were injected with 5 mg/kg LPS. IEC-6 cells were treated with LPS +/- LY294002 or wortmannin, and beta 1- and alpha 3-integrins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunofluorescence. Beta 1-integrin function was determined by adherence of fibronectin beads to IEC-6 monolayers. Migration of IEC-6 cells into a scraped wound was measured by time-lapse microscopy. RESULTS: Newborn intestinal injury was associated with decreased intestinal restitution and increased alpha 3- and beta 1-integrin expression in the ileal mucosa, which also was observed after LPS injection. In IEC-6 cells, LPS caused an increase in the expression of alpha 3- and beta 1-integrins, a shift of beta 1-integrins from the cytoplasm to the plasma membrane and an increase in fibronectin bead adhesion during which beta 1-integrins accumulated underneath attached beads. These effects could be reversed with LY294002 or wortmannin, suggesting phosphatidylinositol-3-phosphate kinase (PI3K) dependence. The increased integrin-matrix adhesion by LPS led to an inhibition of enterocyte migration, which could be reversed by anti-beta 1-antibodies. CONCLUSIONS: Enterocyte migration is inhibited by LPS through increased expression and function of alpha 3- and beta 1-integrins. Modulation of enterocyte migration via integrins may provide novel insights into the pathogenesis of NEC, in which intestinal restitution is impaired.

Plasma D-lactic acid level: a useful marker to distinguish perforated from acute simple appendicitis.

Early diagnosis of perforated appendicitis is important for reducing morbidity rates. The aim of this study was to determine the value and utility of plasma D-lactic acid levels in identifying the type of appendicitis. In this clinical study, plasma D-lactic acid levels were assessed in 44 consecutive paediatric patients (23 with acute appendicitis, 21 with perforated appendicitis) before laparotomy. D-lactic acid levels were determined by an enzymatic spectrophotometric technique using a D-lactic acid dehydrogenase kit. Patients with perforated appendicitis had higher D-lactic acid levels (3.970 +/- 0.687 mg/dL) than patients in the control group (0.478 +/- 0.149 mg/dL) and patients with acute appendicitis (1.409 +/- 0.324 mg/dL; p < 0.05). For a plasma D-lactic acid level greater than 2.5 mg/dL, the sensitivity and specificity of the D-lactic acid assay were 96% and 87%, respectively. The positive predictive value was 87%, the negative predictive value was 96%, and the diagnostic value was 91%. These results suggest that the measurement of plasma D-lactic acid levels may be a useful adjunct to clinical and radiological findings in distinguishing perforated from acute non-perforated appendicitis in children.

Endotoxin differentially modulates the basolateral and apical sodium/proton exchangers (NHE) in enterocytes.

BACKGROUND: Maintenance of enterocyte activity during extracellular acidosis requires functional sodium/proton exchangers (NHE), which are present at both basolateral and apical surfaces. Necrotizing enterocolitis is characterized by systemic hypoperfusion, metabolic acidosis, and the apical to basolateral translocation of endotoxin (lipopolysaccharide [LPS]). We hypothesized that LPS differentially impairs NHE activity at the basolateral or apical domains of enterocytes, leading to cellular acidification, and explored the mechanisms involved. METHODS: Experimental necrotizing enterocolitis (NEC) was induced in newborn rats using a combination of gavage feeds and hypoxia. NHE isoforms were assessed in primary and cultured enterocytes by Western blot analysis and by confocal microscopy in the presence or absence of LPS. NHE activity was detected by single-cell fluorescent ratiometric imaging with the use of the pH-sensitive dye 2', 7'-bis-(2carboxyethyl) -5-(and-6)-carboxyfluorescein. RESULTS: In both NEC and control rats, NHE1 was basolateral and NHE3 was apical. A similar distribution was observed in polarized IEC-6 cells. LPS caused a dose-dependent reduction in basolateral NHE1 activity in IEC-6 cells, but had no effect on apical NHE3 activity. This effect could not be accounted for by reduced expression or impaired plasma membrane localization of NHE isoforms. Strikingly, LPS-mediated NHE1 impairment caused marked cytoplasmic acidification under conditions of extracellular acidosis, whereas functional NHE1 maintained cytoplasmic pH homeostasis in control cells. CONCLUSIONS: LPS selectively impairs basolateral NHE1 but not apical NHE3, leading to cytoplasmic acidification during extracellular acidosis. This effect could mediate impaired enterocyte function after LPS translocation and suggests a mechanism leading to barrier disruption in NEC.

Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions.

Diseases of gut inflammation such as neonatal necrotizing enterocolitis (NEC) result after an injury to the mucosal lining of the intestine, leading to translocation of bacteria and endotoxin (lipopolysaccharide). Intestinal mucosal defects are repaired by the process of intestinal restitution, during which enterocytes migrate from healthy areas to sites of injury. In an animal model of NEC, we determined that intestinal restitution was significantly impaired compared with control animals. We therefore sought to determine the mechanisms governing enterocyte migration under basal conditions and after an endotoxin challenge. Here we show that the cytoskeletal reorganization and stress fiber formation required for migration in IEC-6 enterocytes requires RhoA. Enterocytes were found to express the endotoxin receptor Toll-like receptor 4, which served to bind and internalize lipopolysaccharide. Strikingly, endotoxin treatment significantly inhibited intestinal restitution, as measured by impaired IEC-6 cell migration across a scraped wound. Lipopolysaccharide was found to increase RhoA activity in a phosphatidylinositol 3-kinase-dependent manner, leading to an increase in phosphorylation of focal adhesion kinase and an enhanced number of focal adhesions. Importantly, endotoxin caused a progressive, RhoA-dependent increase in cell matrix tension/contractility, which correlated with the observed impairment in enterocyte migration. We therefore conclude that endotoxin inhibits enterocyte migration through a RhoA-dependent increase in focal adhesions and enhanced cell adhesiveness, which may participate in the impaired restitution observed in experimental NEC.

Bardet-Biedl syndrome associated with vaginal atresia: a case report.

This is a case report of Bardet-Biedl syndrome associated with vaginal atresia diagnosed in a 15-year-old girl. She had mild mental retardation; obesity; nistagmus, retinitis pigmentosa and optic atrophy in both eyes; accessory digit on the left hand; polydactyly in lower extremities; a mobile, painful, nonfixed mass of 6 cm in diameter in the pelvic region; a palpable cystic mass in front of the rectal wall; and no vaginal opening. Secondary sex characteristics were determined. The vaginal atresia was distinguished from vaginal agenesis by the presence of proximal vagina in radiological examination.