Annexin A2 regulates ß1 integrin internalization and intestinal epithelial cell migration.

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from the underlying tissue compartment and is vital in maintaining mucosal homeostasis. Mucosal wounds in inflammatory disorders compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) rapidly migrate to reseal wounds. We have previously observed that a membrane-associated, actin binding protein, annexin A2 (AnxA2), is up-regulated in migrating IECs and plays an important role in promoting wound closure. To identify the mechanisms by which AnxA2 promotes IEC movement and wound closure, we used a loss of function approach. AnxA2-specific shRNA was utilized to generate IECs with stable down-regulation of AnxA2. Loss of AnxA2 inhibited IEC migration while promoting enhanced cell-matrix adhesion. These functional effects were associated with increased levels of ß1 integrin protein, which is reported to play an important role in mediating the cell-matrix adhesive properties of epithelial cells. Because cell migration requires dynamic turnover of integrin-based adhesions, we tested whether AnxA2 modulates internalization of cell surface ß1 integrin required for forward cell movement. Indeed, pulse-chase biotinylation experiments in IECs lacking AnxA2 demonstrated a significant increase in cell surface ß1 integrin that was accompanied by decreased ß1 integrin internalization and degradation. These findings support an important role of AnxA2 in controlling dynamics of ß1 integrin at the cell surface that in turn is required for the active turnover of cell-matrix associations, cell migration, and wound closure.

Tumor suppressor scribble regulates assembly of tight junctions in the intestinal epithelium.

Formation of the epithelial barrier and apico-basal cell polarity represent two characteristics and mutually dependent features of differentiated epithelial monolayers. They are controlled by special adhesive structures, tight junctions (TJs), and polarity protein complexes that define the apical and the basolateral plasma membrane. The functional interplay between TJs and polarity complexes remains poorly understood. We investigated the role of Scribble, a basolateral polarity protein and known tumor suppressor, in regulating TJs in human intestinal epithelium. Scribble was enriched at TJs in T84 and SK-CO15 intestinal epithelial cell monolayers and sections of normal human colonic mucosa. siRNA-mediated knockdown of Scribble in SK-CO15 cells attenuated development of epithelial barrier and inhibited TJ reassembly independently of other basolateral polarity proteins Lgl-1 and Dlg-1. Scribble selectively co-imunoprecipitated with TJ protein ZO-1, and ZO-1 was important for Scribble recruitment to intercellular junctions and TJ reassembly. Lastly, Scribble was mislocalized from TJs and its expression down-regulated in interferon-gamma-treated T84 cell monolayers and inflamed human intestinal mucosa in vivo. We conclude that Scribble is an important regulator of TJ functions and plasticity in the intestinal epithelium. Down-regulation of Scribble may mediate mucosal barrier breakdown during intestinal inflammation.

Technique for Ensuring Type I Bubble Formation for Pre-Descemet Endothelial Keratoplasty Preparation.

PURPOSE: To describe a technique that ensures the production of a type 1 bubble when preparing pre-Descemet endothelial keratoplasty (PDEK) grafts with a high rate of predictability. METHODS: Donor corneas were placed on a support disc, and a blunt instrument was used to score 360 degrees of the peripheral Descemet membrane and endothelium just inside the trabecular meshwork. Air was injected in several short bursts and several stages with a 30-gauge needle on a 3-mL syringe 2.0 mm away from the limbus to create a type 1 big bubble. The technique was tested by 2 operators (M.S. and A.S.-J.) in 26 human donor corneas, including 12 for possible transplantation, over a 9-month period. Anterior segment optical coherence tomograph (AS-OCT) was performed in 1 case proving a type 1 bubble. RESULTS: A type 1 big bubble was successfully created in 24 of 26 attempted cases (92.3%). The technique was used successfully to obtain PDEK tissue for transplant in 9 eyes. One case was not technically acceptable because of diffuse cell loss (>10%); however, the bubble preparation itself was successful. One case had a mixed bubble because of incomplete scoring, resulting in a Descemet membrane endothelial keratoplasty graft used for transplant. One case failed to form any bubble likely because the scoring was too central. Of a total of 26 cases, 14 cases were for practice. CONCLUSIONS: The Soper technique significantly improved the success rate of creating a type 1 bubble for PDEK preparation.

Trabeculotomy ab interno with Trabectome as surgical management for systemic fluoroquinolone-induced pigmentary glaucoma: A case report.

RATIONALE: Bilateral acute iris transillumination (BAIT) is a poorly-understood ocular syndrome in which patients present with acute iridocyclitis and pigmentary dispersion with or without ocular hypertension. The etiology of the disease remains unknown, though recent reports suggest an antecedent upper respiratory tract infection or systemic antibiotic administration may trigger the clinical syndrome. PATIENT CONCERNS: A 55-year-old female was referred for a second opinion regarding her bilateral ocular pain, photophobia, and ocular hypertension. Her medical history was notable for a diagnosis of pneumonia managed with oral moxifloxacin several weeks prior to her initial presentation. DIAGNOSES: Visual acuity was 20/40 with an intraocular pressure (IOP) of 30 mmHg in the affected eye despite maximal tolerated medical therapy. The patient had severe bilateral iris transillumination defects with posterior synechiae formation and 3+ pigment with rare cell in the anterior chamber. This constellation of findings was consistent with a diagnosis of BAIT. INTERVENTIONS: A peripheral iridotomy was placed, which mildly relieved the iris bowing, but did not affect the IOP or inflammatory reaction. The patient then underwent cataract extraction with posterior synechiolysis and ab interno trabeculotomy of the left eye with the Trabectome. OUTCOMES: The patient's IOP on the first post-operative day was 13 mmHg, and anterior chamber inflammation was noted to be significantly reduced at post-operative week 2. The patient was recently seen at a 1-year post-operative visit and her IOP remains in the low teens on a low-dose combination topical agent. LESSONS: Ophthalmologists should remain aware of the association between systemic fluoroquinolones and acute pigmentary dispersion that can progress to glaucoma. The Trabectome remains a viable option for management of pigmentary and uveitic glaucoma resistant to medical treatment.

Clinical, imaging, pathological, and biochemical characterization of a novel presenilin 1 mutation (N135Y) causing Alzheimer's disease.

We present 2 cases of early-onset Alzheimer's disease due to a novel N135Y mutation in PSEN1. The proband presented with memory and other cognitive symptoms at age 32. Detailed clinical characterization revealed initial deficits in memory with associated dysarthria, progressing to involve executive dysfunction, spastic gait, and episodic confusion with polyspike discharges on long-term electroencephalography. Amyloid- and FDG-PET scans showed typical results of Alzheimer's disease. By history, the proband's father had developed cognitive symptoms at age 42 and died at age 48. Neuropathological evaluation confirmed Alzheimer's disease, with moderate to severe amyloid angiopathy. Skeletal muscle showed type 2 fiber-predominant atrophy with pale central clearing. Genetic testing of the proband revealed an N135Y missense mutation in PSEN1. This mutation was predicted to be pathogenic by in silico analysis. Biochemical analysis confirmed that the mutation caused an increased Aß42/Aß40 ratio, consistent with other PSEN1 mutations and with a loss of presenilin function.

Interleukin-4 and interleukin-13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression.

BACKGROUND: Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. METHODS: Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. RESULTS: IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. CONCLUSION: Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity.

Sinonasal epithelial wound resealing in an in vitro model: inhibition of wound closure with IL-4 exposure.

BACKGROUND: Prolonged healing and persistent inflammation following surgery for rhinosinusitis impacts patient satisfaction and healthcare resources. Cytokines interleukin (IL)-4, IL-5, and IL-13 are important mediators in T-helper 2 (Th2) inflammatory rhinosinusitis. Decreased wound healing has been demonstrated with Th2 cytokine exposure, but this has not been extensively studied in sinonasal epithelium. We hypothesized that in vitro exposure of primary sinonasal epithelial cell cultures to Th2 inflammatory cytokine IL-4 and IL-13 would impair wound resealing and decrease expression of annexin A2 at the wound edge. METHODS: Following 24-hour exposure to IL-4, IL-5, or IL-13 vs controls, sterile linear mechanical wounds were created in primary sinonasal epithelial cultures (n = 12 wounds per condition). Wounds were followed for 36 hours or until complete closure, and residual wound areas were calculated by image analysis. Group differences in annexin A2 were assessed by immunofluorescence labeling, confocal microscopy, and Western blots. RESULTS: Significant wound closure differences were identified across cytokine exposure groups (p < 0.001). Mean percentage wound closure at the completion of the 36-hour time course was 98.41% ± 3.43% for control wounds vs 85.02% ± 18.46% for IL-4 exposed wounds. IL-13 did not significantly impair sinonasal epithelial wound resealing in vitro. Annexin A2 protein levels were decreased in IL-4 treated wounds when compared to control wounds (p < 0.01). CONCLUSION: Th2 cytokine IL-4 decreases sinonasal epithelial wound closure in vitro. Annexin A2 is also diminished with IL-4 exposure. This supports the hypothesis that IL-4 exposure impairs sinonasal epithelial wound healing and may contribute to prolonged healing in Th2 inflammatory rhinosinusitis.

Epithelial permeability alterations in an in vitro air-liquid interface model of allergic fungal rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory upper-airway disease with numerous etiologies. Patients with a characteristic subtype of CRS, allergic fungal rhinosinusitis (AFRS), display increased expression of T helper 2 (Th2) cytokines and antigen-specific immunoglobulin E (IgE). Various sinonasal inflammatory conditions are associated with alterations in epithelial barrier function. The aim of this study was to compare epithelial permeability and intercellular junctional protein expression among cultured primary sinonasal cells from AFRS patients vs noninflammatory controls. METHODS: Epithelial cells isolated from paranasal sinus mucosa of AFRS and noninflammatory control patients were grown to confluence on permeable supports and transitioned to air-liquid interface (ALI). Transepithelial resistance (TER) was measured with a horizontal Ussing chamber to characterize the functional permeability of each cell type. After TER recordings were complete, a panel of intercellular junctional proteins was assessed by Western blot and immunofluorescence labeling followed by confocal microscopy. RESULTS: After 12 samples were measured from each group, we observed a 41% mean decrease in TER in AFRS cells (296 ± 89 ohms × cm(2) ) compared to control (503 ± 134 ohms × cm(2) , p = 0.006). TER deficits observed in AFRS were associated with decreased expression of the tight junction proteins occludin and junctional adhesion molecule-A (JAM-A), and increased expression of a leaky tight junction protein claudin-2. CONCLUSION: Cultured sinonasal epithelium from AFRS patients displayed increased epithelial permeability and altered expression of intercellular junctional proteins. Given that these cells were not incubated with inflammatory cytokines in vitro, the cultured AFRS epithelial alterations may represent a retained modification in protein expression from the in vivo phenotype.

House dust mite allergen Der p 1 effects on sinonasal epithelial tight junctions.

BACKGROUND: Epithelial permeability is highly dependent upon the integrity of tight junctions, which are cell-cell adhesion complexes located at the apical aspect of the lateral membrane of polarized epithelial cells. We hypothesize that sinonasal epithelial exposure to Der p 1 house dust mite antigen decreases expression of tight junction proteins (TJPs), representing a potential mechanism for increased permeability and presentation of antigens across the sinonasal epithelial layer. METHODS: Confluent cultured primary human sinonasal epithelial cells were exposed to recombinant Der p 1 antigen vs control, and transepithelial resistance measurements were performed over 24 hours. Antibody staining for a panel of TJPs was examined with immunofluorescence/confocal microscopy and Western blotting. Tissue for these experiments was obtained from 4 patients total. RESULTS: Der p 1 exposed sinonasal cells showed a marked decrease in transepithelial resistance when compared to control cells. In addition, results of Western immunoblot and immunofluorescent labeling demonstrated decreased expression of TJPs claudin-1 and junction adhesion molecule-A (JAM-A) in Der p 1-exposed cultured sinonasal cells vs controls. CONCLUSION: Der p 1 antigen exposure decreases sinonasal epithelium TJP expression, most notably seen in JAM-A and claudin-1 in these preliminary experiments. This decreased TJP expression likely contributes to increased epithelial permeability and represents a potential mechanism for transepithelial antigen exposure in allergic rhinitis.

Epithelial tight junction alterations in nasal polyposis.

OBJECTIVE: To explore alterations in expression of tight junction proteins (TJPs) in nasal polyposis and in respiratory epithelium under inflammatory conditions. Our hypothesis is that exposure of nasal and respiratory epithelium to inflammatory cytokines results in the altered expression of specific TJPs. METHODS: Human sinonasal mucosa (3 nasal polyp specimens and 3 nonpolypoid controls) were stained with immunofluorescent markers specific for TJPs claudin-1 and occludin and examined with confocal scanning laser microscopy. A complementary in vitro experiment involving exposure of cultured human bronchial epithelium to interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) was also performed. Alterations in claudin-1 and occludin were localized by immunofluorescence labeling and confocal microscopy and quantified by western blotting. RESULTS: Nasal polyp epithelium from human tissue specimens had reduced claudin-1 expression along the basal aspect of the mucosal layer, whereas occludin expression was reduced in the apical and basal epithelial zones. In vitro experiments demonstrated stable or increased TJP expression after 24 hours of cytokine exposure (43% increase for claudin-1, 9% increase for occludin). However, a reduction in TJP expression was observed after 72 hours of cytokine exposure (18% reduction for claudin-1, and 43% reduction for occludin). CONCLUSION: Nasal polyposis is associated with epithelial TJP alterations. Further, the expression of TJPs in a model of inflamed respiratory mucosa is reduced in a similar fashion. Research on the histopathology of other epithelial inflammatory disorders suggests TJP alterations contribute to a self-perpetuating inflammatory state. Findings of this preliminary study support a similar process in nasal polyposis.