Short Palate Research Articles

Clinical and molecular characterization of a Brazilian cohort of campomelic dysplasia patients, and identification of seven new SOX9 mutations.

Campomelic dysplasia (CD) is an autosomal, dominantly inherited, skeletal abnormality belonging to the subgroup of bent bone dysplasias. In addition to bowed lower limbs, CD typically includes the following: disproportionate short stature, flat face, micrognathia, cleft palate, bell-shaped thorax, and club feet. Up to three quarters of 46, XY individuals may be sex-reversed. Radiological signs include scapular and pubic hypoplasia, narrow iliac wings, spaced ischia, and bowed femora and tibiae. Lethal CD is usually due to heterozygous mutations in SOX9, a major regulator of chondrocytic development. We present a detailed clinical and molecular characterization of nine Brazilian CD patients. Infants were either stillborn (n = 2) or died shortly after birth and presented similar phenotypes. Sex-reversal was observed in one of three chromosomally male patients. Sequencing of SOX9 revealed new heterozygous mutations in seven individuals. Six patients had mutations that resulted in premature transcriptional termination, while one infant had a single-nucleotide substitution at the conserved splice-site acceptor of intron 1. No clear genotype-phenotype correlations were observed. This study highlights the diversity of SOX9 mutations leading to lethal CD, and expands the group of known genetic alterations associated with this skeletal dysplasia.

Mammalian short palate lung and nasal epithelial clone 1 (SPLUNC1) in pH-dependent airway hydration

Mammalian short palate lung and nasal epithelial clone 1 (SPLUNC1) in pH-dependent airway hydration

The antimicrobial protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is differentially modulated in eosinophilic and noneosinophilic chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous disease with aberrant host defense responses. However, whether innate immunity is similarly impaired in patients with eosinophilic and those with noneosinophilic CRSwNP remains unclear. We sought to evaluate the expression and possible modulation of short palate, lung, and nasal epithelium clone 1 (SPLUNC1), an innate immune molecule, in the 2 CRSwNP subsets. Polyp tissue and uncinate processes were collected from 40 patients with CRSwNP, 27 patients with chronic rhinosinusitis without nasal polyps (CRSsNP), and 22 control subjects. Expression of SPLUNC1; Toll-like receptor (TLR) 2, TLR3, and TLR4; and the proinflammatory cytokines IL-1α, IL-4, IL-13, IL-17A, and IFN-γ was examined in nasal tissues. Additionally, SPLUNC1 expression in response to specific inflammatory stimulation was measured in cultured polyp epithelial cells and A549 cells. Polyp tissues exhibited significantly decreased expression of SPLUNC1 and other innate immune molecules compared with uncinate process tissues from patients with CRSwNP (P < .05), patients with CRSsNP, and healthy control subjects. Moreover, the eosinophilic CRSwNP subset exhibited significantly decreased SPLUNC1 expression and numbers of submucosal glands, as well as significantly increased IL-4 and IL-13 mRNA levels, compared with the noneosinophilic subset (P < .05). Accordingly, SPLUNC1 expression in polyp epithelial cells was significantly inhibited by IL-4 and IL-13 stimulation in vitro but was significantly upregulated after stimulation with TLR agonists and glucocorticoids (P < .05). Differential SPLUNC1 suppression between the eosinophilic and noneosinophilic CRSwNP subsets suggests that they possess distinct pathogenic mechanisms. This finding might benefit the design of appropriate therapeutic interventions targeted to each subset.

α1-antitrypsin promotes SPLUNC1-mediated lung defense against Pseudomonas aeruginosa infection in mice

BackgroundPseudomonas aeruginosa (PA) infection is involved in various lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. However, treatment of PA infection is not very effective in part due to antibiotic resistance. α1-antitrypsin (A1AT) has been shown to reduce PA infection in humans and animals, but the underlying mechanisms remain unclear. The goal of our study is to test whether a novel endogenous host defense protein, short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is involved in the therapeutic effect of A1AT during lung PA infection.MethodSPLUNC1 knockout (KO) and littermate wild-type (WT) mice on the C57BL/6 background were intranasally infected with PA to determine the therapeutic effects of A1AT. A1AT was aerosolized to mice 2 hrs after the PA infection, and mice were sacrificed 24 hrs later. PA load and inflammation were quantified in the lung, and SPLUNC1 protein in bronchoalveolar lavage (BAL) fluid was examined by Western blot.ResultsIn WT mice, PA infection significantly increased neutrophil elastase (NE) activity, but reduced SPLUNC1 protein in BAL fluid. Notably, PA-infected mice treated with A1AT versus bovine serum albumin (BSA) demonstrated higher levels of SPLUNC1 protein expression, which are accompanied by lower levels of NE activity, lung bacterial load, and pro-inflammatory cytokine production. To determine whether A1AT therapeutic effects are dependent on SPLUNC1, lung PA load in A1AT- or BSA-treated SPLUNC1 KO mice was examined. Unlike the WT mice, A1AT treatment in SPLUNC1 KO mice had no significant impact on lung PA load and pro-inflammatory cytokine production.ConclusionA1AT reduces lung bacterial infection in mice in part by preventing NE-mediated SPLUNC1 degradation.

Dual Acute Proinflammatory and Antifibrotic Pulmonary Effects of Short Palate, Lung, and Nasal Epithelium Clone–1 after Exposure to Carbon Nanotubes

Carbon nanotubes (CNTs; allotropes of carbon with a cylindrical nanostructure) have emerged as one of the most commonly used types of nanomaterials, with numerous applications in industry and biomedicine. However, the inhalation of CNTs has been shown to elicit pulmonary toxicity, accompanied by a robust inflammatory response with an early-onset fibrotic phase. Epithelial host-defense proteins represent an important component of the pulmonary innate immune response to foreign inhalants such as particles and bacteria. The short palate, lung, and nasal epithelium clone-1 (SPLUNC1) protein, a member of the bactericidal/permeability-increasing-fold (BPIF)-containing protein family, is a 25-kD secretory protein that is expressed in nasal, oropharyngeal, and lung epithelia, and has been shown to have multiple functions, including antimicrobial and chemotactic activities, as well as surfactant properties. This study sought to assess the importance of SPLUNC1-mediated pulmonary responses in airway epithelial secretions, and to explore the biological relevance of SPLUNC1 to inhaled particles in a single-walled carbon nanotube (SWCNT) model. Using Scgb1a1-hSPLUNC1 transgenic mice, we observed that SPLUNC1 significantly modified host inflammatory responses by increasing leukocyte recruitment and enhancing phagocytic activity. Furthermore, we found that transgenic mice were more susceptible to SWCNT exposure at the acute phase, but showed resistance against lung fibrogenesis through pathological changes in the long term. The binding of SPLUNC1 also attenuated SWCNT-induced TNF-α secretion by RAW 264.7 macrophages. Taken together, our data indicate that SPLUNC1 is an important component of mucosal innate immune defense against pulmonary inhaled particles.

Increased Susceptibility to Pulmonary Pseudomonas Infection in Splunc1 Knockout Mice

The airway epithelium is the first line of host defense against pathogens. The short palate, lung, and nasal epithelium clone (SPLUNC)1 protein is secreted in respiratory tracts and is a member of the bacterial/permeability increasing (BPI) fold-containing protein family, which shares structural similarities with BPI-like proteins. On the basis of its homology with BPIs and restricted expression of SPLUNC1 in serous cells of submucosal glands and surface epithelial cells of the upper respiratory tract, SPLUNC1 is thought to possess antimicrobial activity in host defense. SPLUNC1 is also reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to determine the in vivo functions of SPLUNC1 following Pseudomonas aeruginosa infection and to elucidate the underlying mechanism by using a knockout (KO) mouse model with a genetic ablation of Splunc1. Splunc1 KO mice showed accelerated mortality and increased susceptibility to P. aeruginosa infection with significantly decreased survival rates, increased bacterial burdens, exaggerated tissue injuries, and elevated proinflammatory cytokine levels as compared with those of their wild-type littermates. Increased neutrophil infiltration in Splunc1 KO mice was accompanied by elevated chemokine levels, including Cxcl1, Cxcl2, and Ccl20. Furthermore, the expression of several epithelial secretory proteins and antimicrobial molecules was considerably suppressed in the lungs of Splunc1 KO mice. The deficiency of Splunc1 in mouse airway epithelium also results in increased biofilm formation of P. aeruginosa. Taken together, our results support that the ablation of Splunc1 in mouse airways affects the mucociliary clearance, resulting in decreased innate immune response during Pseudomonas-induced respiratory infection.

Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways

The ability to maintain proper airway surface liquid (ASL) volume homeostasis is vital for mucus hydration and clearance, which are essential aspects of the mammalian lung's innate defense system. In cystic fibrosis (CF), one of the most common life-threatening genetic disorders, ASL dehydration leads to mucus accumulation and chronic infection. In normal airways, the secreted protein short palate lung and nasal epithelial clone 1 (SPLUNC1) effectively inhibits epithelial Na(+) channel (ENaC)-dependent Na(+) absorption and preserves ASL volume. In CF airways, it has been hypothesized that increased ENaC-dependent Na(+) absorption contributes to ASL depletion, and hence increased disease. However, this theory is controversial, and the mechanism for abnormal ENaC regulation in CF airways has remained elusive. Here, we show that SPLUNC1 is a pH-sensitive regulator of ENaC and is unable to inhibit ENaC in the acidic CF airway environment. Alkalinization of CF airway cultures prevented CF ASL hyperabsorption, and this effect was abolished when SPLUNC1 was stably knocked down. Accordingly, we resolved the crystal structure of SPLUNC1 to 2.8 Å. Notably, this structure revealed two pH-sensitive salt bridges that, when removed, rendered SPLUNC1 pH-insensitive and able to regulate ASL volume in acidic ASL. Thus, we conclude that ENaC hyperactivity is secondary to reduced CF ASL pH. Together, these data provide molecular insights into the mucosal dehydration associated with a range of pulmonary diseases, including CF, and suggest that future therapy be directed toward alkalinizing the pH of CF airways.

Differences in Growth Generate the Diverse Palate Shapes of New World Leaf-Nosed Bats (Order Chiroptera, Family Phyllostomidae)

New World leaf-nosed bats (Family Phyllostomidae) display incredible craniofacial diversity that is associated with their broad range of dietary preferences. The short and broad palates of highly frugivorous bats are functionally linked to high bite forces, and the long and narrow palates of nectarivorous bats to flower feeding. Although the functional correlates and evolutionary history of shape variation in phyllostomid palates are beginning to be understood, the specific developmental processes that govern palate diversification remain unknown. To begin to resolve this issue, this study quantified palate morphology in seven phyllostomid species from a range of developmental stages and in adults. This sample includes species with short and broad, long and narrow, and intermediate palate shapes, and thereby covers the range of palate shapes displayed by phyllostomids. Results indicate that while initial palate shape (i.e., width vs. length) varies among species, the pattern of this variation does not match that observed in adults. In contrast, the relative growth of palate width and length in developing phyllostomids and the ratio of these axes in adults are significantly correlated. These and other results suggest that evolutionary alterations in patterns of palate growth have governed the diversification of palate shapes in adult phyllostomids. This implies that the diverse palate shapes of phyllostomids are the result of relatively subtle evolutionary changes in later rather than earlier development events.

Short Palate, Lung, and Nasal Epithelial Clone–1 Is a Tightly Regulated Airway Sensor in Innate and Adaptive Immunity

Short palate, lung, and nasal epithelial clone-1 (SPLUNC1) is a protein abundantly expressed by the respiratory epithelium of the proximal lower respiratory tract, a site of great environmental exposure. Previous studies showed that SPLUNC1 exerts antimicrobial effects, regulates airway surface liquid and mucociliary clearance, and suppresses allergic airway inflammation. We studied SPLUNC1 to gain insights into its role in host defense. In the lower respiratory tract, concentrations of SPLUNC1 are high under basal conditions. In models of pneumonia caused by common respiratory pathogens, and in Th1-induced and Th2-induced airway inflammation, SPLUNC1 secretion is markedly reduced. Pathogen-associated molecular patterns and IFN-γ act directly on airway epithelial cells to inhibit SPLUNC1 mRNA expression. Thus, SPLUNC1 is quickly suppressed during infection, in response to an insult on the epithelial surface. These experiments highlight the finely tuned fluctuations of SPLUNC1 in response to exposures in the respiratory tract, and suggest that the loss of SPLUNC1 is a crucial feature of host defense across air-breathing animal species.

Human Neutrophil Elastase Degrades SPLUNC1 and Impairs Airway Epithelial Defense against Bacteria

BackgroundAcute exacerbations of chronic obstructive pulmonary disease (AECOPD) are a significant cause of mortality of COPD patients, and pose a huge burden on healthcare. One of the major causes of AECOPD is airway bacterial (e.g. nontypeable Haemophilus influenzae [NTHi]) infection. However, the mechanisms underlying bacterial infections during AECOPD remain poorly understood. As neutrophilic inflammation including increased release of human neutrophil elastase (HNE) is a salient feature of AECOPD, we hypothesized that HNE impairs airway epithelial defense against NTHi by degrading airway epithelial host defense proteins such as short palate, lung, and nasal epithelium clone 1 (SPLUNC1).Methodology/Main ResultsRecombinant human SPLUNC1 protein was incubated with HNE to confirm SPLUNC1 degradation by HNE. To determine if HNE-mediated impairment of host defense against NTHi was SPLUNC1-dependent, SPLUNC1 protein was added to HNE-treated primary normal human airway epithelial cells. The in vivo function of SPLUNC1 in NTHi defense was investigated by infecting SPLUNC1 knockout and wild-type mice intranasally with NTHi. We found that: (1) HNE directly increased NTHi load in human airway epithelial cells; (2) HNE degraded human SPLUNC1 protein; (3) Recombinant SPLUNC1 protein reduced NTHi levels in HNE-treated human airway epithelial cells; (4) NTHi levels in lungs of SPLUNC1 knockout mice were increased compared to wild-type mice; and (5) SPLUNC1 was reduced in lungs of COPD patients.ConclusionsOur findings suggest that SPLUNC1 degradation by neutrophil elastase may increase airway susceptibility to bacterial infections. SPLUNC1 therapy likely attenuates bacterial infections during AECOPD.

Submucous Cleft Palate

This study aimed to investigate the age at diagnosis, palatal characteristics, and symptoms of submucous cleft palate (SMCP) and to compare the speech outcomes between 3 operative techniques for primary repair of SMCP.It was a retrospective review of 92 patients diagnosed with SMCP between 1994 and 2008, where patients were treated with 1 of 3 surgical procedures: double opposing z-palatoplasty, radical intravelar veloplasty, or pharyngeal flap.The need for a second procedure was considered a primary outcome measure, with operative failure defined by the need for a secondary operation. Postoperative speech was evaluated perceptually using the Great Ormond Street Speech Assessment.We found that SMCP was diagnosed at a mean age of 3.6 years. At diagnosis, 8% were asymptomatic, 67% had abnormal speech, 49% had recurrent otitis media with effusions, and 47% had hearing loss. Ear, nose, and throat surgery was performed on 37% without the diagnosis of SMCP being made. Subjective impression of a short palate was a predictor for secondary surgery (P < 0.02). Age at repair did not affect velopharyngeal insufficiency outcome; however, repair after 18 months of age led to a higher likelihood of developing articulatory speech errors resulting in the need for more speech therapy.In conclusion, the triad of features of SMCP is well recognized, but our study reveals that a high percentage is seen by physicians who have failed to make the diagnosis despite signs and symptoms being evident. Surgical correction is successful regardless of technique and age, but earlier recognition to prevent speech impairment should be sought.

SPLUNC1/BPIFA1 Contributes to Pulmonary Host Defense against Klebsiella pneumoniae Respiratory Infection

Epithelial host defense proteins comprise a critical component of the pulmonary innate immune response to infection. The short palate, lung, nasal epithelium clone (PLUNC) 1 (SPLUNC1) protein is a member of the bactericidal/permeability-increasing (BPI) fold-containing (BPIF) protein family, sharing structural similarities with BPI-like proteins. SPLUNC1 is a 25 kDa secretory protein that is expressed in nasal, oropharyngeal, and lung epithelia, and has been implicated in airway host defense against Pseudomonas aeruginosa and other organisms. SPLUNC1 is reported to have surfactant properties, which may contribute to anti-biofilm defenses. The objective of this study was to assess the importance of SPLUNC1 surfactant activity in airway epithelial secretions and to explore its biological relevance in the context of a bacterial infection model. Using cultured airway epithelia, we confirmed that SPLUNC1 is critically important for maintenance of low surface tension in airway fluids. Furthermore, we demonstrated that recombinant SPLUNC1 (rSPLUNC1) significantly inhibited Klebsiella pneumoniae biofilm formation on airway epithelia. We subsequently found that Splunc1(-/-) mice were significantly more susceptible to infection with K. pneumoniae, confirming the likely in vivo relevance of this anti-biofilm effect. Our data indicate that SPLUNC1 is a crucial component of mucosal innate immune defense against pulmonary infection by a relevant airway pathogen, and provide further support for the novel hypothesis that SPLUNC1 protein prevents bacterial biofilm formation through its ability to modulate surface tension of airway fluids.

SPLUNC1 Regulates Cell Progression and Apoptosis through the miR-141-PTEN/p27 Pathway, but Is Hindered by LMP1

Little is known about the role of the host defensive protein short palate, lung and nasal epithelium clone 1 (SPLUNC1) in the carcinogenesis of nasopharyngeal carcinoma (NPC). Here we report that SPLUNC1 plays a role at a very early stage of NPC carcinogenesis. SPLUNC1 regulates NPC cell proliferation, differentiation and apoptosis through miR-141, which in turn regulates PTEN and p27 expression. This signaling axis is negatively regulated by the EBV-coded gene LMP1. Therefore we propose that SPLUNC1 suppresses NPC tumor formation and its inhibition by LMP1 provides a route for NPC tumorigenesis.

Decreased SPLUNC1 expression is associated with pseudomonas infection in surgically treated chronic rhinosinusitis patients who may require repeated sinus surgery

Chronic rhinosinusitis colonized with Pseudomonas aruginosa is difficult to treat and is related to biofilm formation. Repeated sinus surgery is often required for these patients. Short palate, lung, and nasal epithelial clone 1 (SPLUNC1) is an epithelium-secreted protein that is involved in innate immunity and has anti-Pseudomonas and antibiofilm functions. This study examined if SPLUNC1 expression was related to sinusitis with bacterial culture positive for Pseudomonas and the possibility of using SPLUNC1 to predict treatment outcomes for sinusitis. Nonrandomized retrospective study. This was a retrospective study of patients at a tertiary referral center. Pseudomonas aruginosa infection was compared to clinical variables such as SPLUNC1 mRNA expression levels, immunohistochemical (IHC) stain intensity, Lund-Mackay sinus computed tomography scores, rapid recurrent sinusitis, requirement for repeat sinus surgery, Phadiatop test results, age, gender, nasal polyp(s), and patients' presence/absence of diabetes mellitus. Comparisons between groups were performed using the χ(2) test or Fisher exact test when one confronter was <5. The statistical analyses were carried out with SPSS version 13. P. aeruginosa sinus infections were associated with lower sinus mucosa SPLUNC1 expression (P = .0018), weaker SPLUNC1 IHC staining intensity (P = .011), and poor postoperative outcome (i.e., need repeated sinus surgery) (P < .001). Other factors were not associated with Pseudomonas sinus infection. Sinusitis with positive P. aeruginosa bacterial culture is associated with decreased SPLUNC1 sinus mucosa expression. Repeated sinus surgeries are more frequently needed for these patients.

Airway Epithelial NF-κB Activation Promotes Mycoplasma pneumoniae Clearance in Mice

Background/ObjectiveRespiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp) contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD). Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB). We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1) serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression.Methodology/Main ResultsNon-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB) was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-CAIKKβ) with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+), but not transgene negative (Tg−) mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice.ConclusionBy using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression.

Slc39a7/zip7 Plays a Critical Role in Development and Zinc Homeostasis in Zebrafish

BackgroundSlc39a7/Zip7, also known as Ke4, is a member of solute carrier family 39 (Slc39a) and plays a critical role in regulating cell growth and death. Because the function of Zip7 in vivo was unclear, the present study investigated the function of zip7 in vertebrate development and zinc metabolism using zebrafish as a model organism.Principal FindingUsing real-time PCR to determine the gene expression pattern of zip7 during zebrafish development, we found that zip7 mRNA is expressed throughout embryonic development and into maturity. Interestingly, whole mount in situ hybridization revealed that while zip7 mRNA is ubiquitously expressed until 12 hours post-fertilization (hpf); at 24 hpf and beyond, zip7 mRNA was specifically detected only in eyes. Morpholino-antisense (MO) gene knockdown assay revealed that downregulation of zip7 expression resulted in several morphological defects in zebrafish including decreased head size, smaller eyes, shorter palates, and shorter and curved spinal cords. Analysis by synchrotron radiation X-ray fluorescence (SR-XRF) showed reduced concentrations of zinc in brain, eyes, and gills of zip7-MO-injected embryos. Furthermore, incubation of the zip7 knockdown embryos in a zinc-supplemented solution was able to rescue the MO-induced morphological defects.SignificanceOur data suggest that zip7 is required for eye, brain, and skeleton formation during early embryonic development in zebrafish. Moreover, zinc supplementation can partially rescue defects resulting from zip7 gene knockdown. Taken together, our data provide critical insight into a novel function of zip7 in development and zinc homeostasis in vivo in zebrafish.

SPLUNC1 Deficiency Enhances Airway Eosinophilic Inflammation in Mice

Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is enriched in normal airway lining fluid, but is significantly reduced in airway epithelium exposed to a Th2 cytokine milieu. The role of SPLUNC1 in modulating airway allergic inflammation (e.g., eosinophils) remains unknown. We used SPLUNC1 knockout (KO) and littermate wild-type (C57BL/6 background) mice and recombinant SPLUNC1 protein to determine the impact of SPLUNC1 on airway allergic/eosinophilic inflammation, and to investigate the underlying mechanisms. An acute ovalbumin (OVA) sensitization and challenge protocol was used to induce murine airway allergic inflammation (e.g., eosinophils, eotaxin-2, and Th2 cytokines). Our results showed that SPLUNC1 in the bronchoalveolar lavage fluid of OVA-challenged wild-type mice was significantly reduced (P < 0.05), which was negatively correlated with levels of lung eosinophilic inflammation. Moreover, SPLUNC1 KO mice demonstrated significantly higher numbers of eosinophils in the lung after OVA challenges than did wild-type mice. Alveolar macrophages isolated from OVA-challenged SPLUNC1 KO versus wild-type mice had higher concentrations of baseline eotaxin-2 that was amplified by LPS (a known risk factor for exacerbating asthma). Human recombinant SPLUNC1 protein was applied to alveolar macrophages to study the regulation of eotaxin-2 in the context of Th2 cytokine and LPS stimulation. Recombinant SPLUNC1 protein attenuated LPS-induced eotaxin-2 production in Th2 cytokine-pretreated murine macrophages. These findings demonstrate that SPLUNC1 inhibits airway eosinophilic inflammation in allergic mice, in part by reducing eotaxin-2 production in alveolar macrophages.

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.

Role of x-ray lateral view nasopharynx in the diagnosis of subtle velopharyngeal incompetence

Introduction: Velopharyngeal dysfunction following adenoidectomy is not uncommon. Incidence range from 1 per 1,500 to 1 per 10,000 patients. Children at risk can be identified including cleft palate, sub mucous cleft, and palatal hypotonia, but if it occurred in the absence of structural abnormalities, increased pharyngeal width or short soft palate has been proposed. Methods: The study included 227 children, 71 females and 156 males. Plain x-ray lateral view nasopharynx was done during pronouncing the vowel Eeee aiming to assess the degree of closure and the point of contact between the contracted soft palate and the adenoid pad. Children with cleft palate, bifid uvula , mythsenia gravis and congenital abnormality of the hard palate were excluded from the study Results: Thirteen patients out of 227 were included with a median age of 5.8 years having short functional length of the soft palate. Only 4 (1.7 %) among the studied patients developed velopharyngeal incompetence. All patients with short functional length of the soft palate demonstrate attachment to the adenoid anterior surface rather to others show posterior pharyngeal wall attachment. Results showed that there was an increased incidence of velopharyngeal incompetence with patients having short functional length of the soft palate. Conclusion: Plain x-ray lateral view nasopharynx during phonation of the vowel Eeee could be cheap and sensitive method in predicting velopharyngeal incompetence in children undergoing adenoidectomy.