CH-π Interactions Are Required for Human Galectin-3 Function.

Glycan-binding proteins, or lectins, recognize distinct structural elements of polysaccharides, to mediate myriad biological functions. Targeting glycan-binding proteins involved in human disease has been challenging due to an incomplete understanding of the molecular mechanisms that govern protein-glycan interactions. Bioinformatics and structural studies of glycan-binding proteins indicate that aromatic residues with the potential for CH-π interactions are prevalent in glycan-binding sites. However, the contributions of these CH-π interactions to glycan binding and their relevance in downstream function remain unclear. An emblematic lectin, human galectin-3, recognizes lactose and N-acetyllactosamine-containing glycans by positioning the electropositive face of a galactose residue over the tryptophan 181 (W181) indole forming a CH-π interaction. We generated a suite of galectin-3 W181 variants to assess the importance of these CH-π interactions to glycan binding and function. As determined experimentally and further validated with computational modeling, variants with smaller or less electron-rich aromatic side chains (W181Y, W181F, W181H) or sterically similar but nonaromatic residues (W181M, W181R) showed poor or undetectable binding to lactose and attenuated ability to bind mucins or agglutinate red blood cells. The latter functions depend on multivalent binding, highlighting that weakened CH-π interactions cannot be overcome by avidity. Two galectin-3 variants with disrupted hydrogen bonding interactions (H158A and E184A) showed similarly impaired lactose binding. Molecular simulations demonstrate that all variants have decreased binding orientation stability relative to native galectin-3. Thus, W181 collaborates with the endogenous hydrogen bonding network to enhance binding affinity for lactose, and abrogation of these CH-π interactions is as deleterious as eliminating key hydrogen bonding interactions. These findings underscore the critical roles of CH-π interactions in carbohydrate binding and lectin function and will aid the development of novel lectin inhibitors.

Cluster analysis of nasal cytokines during rhinovirus infection identifies different immunophenotypes in both children and adults with allergic asthma.

BACKGROUND: Infection with rhinovirus (RV) is a major risk factor for disease exacerbations in patients with allergic asthma. This study analysed a broad set of cytokines in the noses of children and adults with asthma during RV infection in order to identify immunophenotypes that may link to virus-induced episodes. METHODS: Nasal wash specimens were analysed in children (n = 279 [healthy, n = 125; stable asthma, n = 64; wheeze, n = 90], ages 2-12) who presented to a hospital emergency department, and in adults (n = 44 [healthy, n = 13; asthma, n = 31], ages 18-38) who were experimentally infected with RV, including a subset who received anti-IgE. Cytokines were measured by multiplex bead assay and data analysed by univariate and multivariate methods to test relationships to viral load, allergic status, airway inflammation, and clinical outcomes. RESULTS: Analysis of a core set of 7 cytokines (IL-6, CXCL8/IL-8, IL-15, EGF, G-CSF, CXCL10/IP-10 and CCL22/MDC) revealed higher levels in children with acute wheeze versus those with stable asthma or controls. Multivariate analysis identified two clusters that were enriched for acutely wheezing children; one displaying high viral load ("RV-high") with robust secretion of CXCL10, and the other displaying high IgE with elevated EGF, CXCL8 and both eosinophil- and neutrophil-derived mediators. Broader assessment of 39 cytokines confirmed that children with acute wheeze were not deficient in type 1 anti-viral responses. Analysis of 18 nasal cytokines in adults with asthma who received RV challenge identified two clusters; one that was "RV-high" and linked to robust induction of anti-viral cytokines and anti-IgE; and the other associated with more severe symptoms and a higher inflammatory state featuring eosinophil and neutrophil factors. CONCLUSIONS: The results confirm the presence of different immunophenotypes linked to parameters of airway disease in both children and adults with asthma who are infected with RV. Such discrepancies may reflect the ability to regulate anti-viral responses.

Chronic Laryngotracheal Granulomatous Disease Secondary to Sporothrix schenckii in an Immunocompromised Patient.

OBJECTIVE: To describe a rare presentation of laryngotracheal granulomatous disease secondary to sporotrichosis. METHODS: The authors report a case of laryngeal sporotrichosis in an immunocompromised patient, with accompanying endoscopic images and pathology. RESULTS/CASE: A 72-year-old immunocompromised female with a history of rose-handling presented with a year of hoarseness and breathy voice. Flexible nasolaryngoscopy showed diffuse nodularity; biopsy of the lesions demonstrated granulomatous inflammatory changes, and fungal culture grew Sporothrix schenkii. Long-term itraconazole treatment was initiated, with improvement in dysphonia and few residual granulomas on follow-up examination. CONCLUSION: When evaluating granulomatous disease of the airway, a broad differential including infectious or inflammatory etiologies should be considered, especially in immunocompromised patients. Adequate tissue samples should be collected to facilitate special staining. The current recommendations for laryngeal sporotrichosis include treatment with a prolonged course of itraconazole.

Novel Treatment-Refractory Preschool Wheeze Phenotypes Identified by Cluster Analysis of Lung Lavage Constituents.

BACKGROUND: Preschool children with treatment-refractory wheeze often require unscheduled acute care. Current guidelines advise treatment of persistent wheeze with inhaled corticosteroids. Alternative treatments targeting structural abnormalities and specific inflammatory patterns could be more effective. OBJECTIVE: To apply unsupervised analysis of lung lavage (bronchoalveolar lavage [BAL]) variables to identify clusters of preschool children with treatment-refractory wheeze. METHODS: A total of 155 children 6 years or younger underwent bronchoscopy with BAL for evaluation of airway structure, inflammatory markers, and pathogens. Variables were screened with factor analysis and sorted into clusters by Ward's method, and membership was confirmed by discriminant analysis. RESULTS: The model was repeatable in a 48-case validation sample and accurately classified 86% of cases. Cluster 1 (n = 60) had early-onset wheeze, 85% with structural abnormalities, mostly tracheamalacia, with low total IgE and agranulocytic BAL. Cluster 2 (n = 42) had later-onset wheeze, the highest prevalence of gastroesophageal reflux, little atopy, and two-third had increased BAL lipid-laden macrophages. Cluster 3 (n = 46) had mid-onset wheeze, low total IgE, and two-third had BAL viral transcripts, predominately human rhinovirus, with BAL neutrophilia. Cluster 4 (n = 7) was older, with high total IgE, blood eosinophilia, and mixed BAL eosinophils and neutrophils. CONCLUSIONS: Preschool children with recurrent wheeze refractory to inhaled corticosteroid treatment include 4 clusters: airway malacia, gastroesophageal reflux, indolent human rhinovirus bronchoalveolitis, and type-2high inflammation. The results support the risk and cost of invasive bronchoscopy to diagnose causes of treatment-refractory wheeze and develop novel therapies targeting airway malacia, human rhinovirus infection, and BAL neutrophilia in preschool children.

Chemical Control of Quorum Sensing in E. coli: Identification of Small Molecule Modulators of SdiA and Mechanistic Characterization of a Covalent Inhibitor.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of severe diarrheal disease in humans. Cattle are the natural reservoir of EHEC, and approximately 75% of EHEC infections in humans stem from bovine products. Many common bacterial pathogens, including EHEC, rely on chemical communication systems, such as quorum sensing (QS), to regulate virulence and facilitate host colonization. EHEC uses SdiA from E. coli (SdiAEC), an orphan LuxR-type receptor, to sense N-acyl l-homoserine lactone (AHL) QS signals produced by other members of the bovine enteric microbiome. SdiAEC regulates two phenotypes critical for colonizing cattle: acid resistance and the formation of attaching and effacing lesions. Despite the importance of SdiAEC, there is very little known about its selectivity for different AHL signals, and no chemical inhibitors that act specifically on SdiAEC have been reported. Such compounds would represent valuable tools to study the roles of QS in EHEC virulence. To identify chemical modulators of SdiAEC and delineate the structure-activity relationships (SARs) for AHL activity in this receptor, we report herein the screening of a focused library composed largely of AHLs and AHL analogues in an SdiAEC reporter assay. We describe the identity and SARs of potent modulators of SdiAEC activity, examine the promiscuity of SdiAEC, characterize the mechanism of a covalent inhibitor, and provide phenotypic assay data to support that these compounds can control SdiAEC-dependent acid resistance in E. coli. These SdiAEC modulators could be used to advance the study of LuxR-type receptor/ligand interactions, the biological roles of orphan LuxR-type receptors, and potential QS-based therapeutic approaches.

Understanding the asthmatic response to an experimental rhinovirus infection: Exploring the effects of blocking IgE.

BACKGROUND: Rhinovirus frequently causes asthma exacerbations among children and young adults who are allergic. The interaction between allergen and rhinovirus-induced symptoms and inflammation over time is unclear. OBJECTIVE: Our aim was to compare the response to an experimental inoculation with rhinovirus-16 in allergic asthmatics with the response in healthy controls and to evaluate the effects of administrating omalizumab before and during the infection. METHODS: Two clinical trials were run in parallel. In one of these trials, the response to an experimental inoculation with rhinovirus-16 among asthmatics with high levels of total IgE was compared to the response in healthy controls. The other trial compared the effects of administering omalizumab versus placebo to asthmatics in a randomized, double-blind placebo-controlled investigation. The primary outcome for both trials compared lower respiratory tract symptoms (LRTSs) between study groups over the first 4 days of infection. RESULTS: Frequent comparisons of symptoms, lung function, and blood eosinophil counts revealed differences that were more pronounced among allergic asthmatics than among controls by days 2 and 3 after virus inoculation. Additionally, an augmentation of upper respiratory tract symptom scores and LRTS scores occurred among the atopic asthmatics versus the controls during the resolution of symptoms (P < .01 for upper respiratory symptom tract scores and P < .001 for LRTS scores). The beneficial effects of administering omalizumab on reducing LRTSs and improving lung function were strongest over the first 4 days. CONCLUSIONS: LRTSs and blood eosinophil counts were augmented and lung function was reduced among allergic asthmatics early after rhinovirus inoculation but increased late in the infection during symptom resolution. The effect of administering omalizumab on the response to rhinovirus was most pronounced during the early/innate phase of the infection.

Lung Lavage Granulocyte Patterns and Clinical Phenotypes in Children with Severe, Therapy-Resistant Asthma.

BACKGROUND: Children with severe asthma have frequent exacerbations despite guidelines-based treatment with high-dose corticosteroids. The importance of refractory lung inflammation and infectious species as factors contributing to poorly controlled asthma in children is poorly understood. OBJECTIVE: To identify prevalent granulocyte patterns and potential pathogens as targets for revised treatment, 126 children with severe asthma underwent clinically indicated bronchoscopy. METHODS: Diagnostic tests included bronchoalveolar lavage (BAL) for cell count and differential, bacterial and viral studies, spirometry, and measurements of blood eosinophils, total IgE, and allergen-specific IgE. Outcomes were compared among 4 BAL granulocyte patterns. RESULTS: Pauci-granulocytic BAL was the most prevalent granulocyte category (52%), and children with pauci-granulocytic BAL had less postbronchodilator airflow limitation, less blood eosinophilia, and less detection of BAL enterovirus compared with children with mixed granulocytic BAL. Children with isolated neutrophilia BAL were differentiated by less blood eosinophilia than those with mixed granulocytic BAL, but greater prevalence of potential bacterial pathogens compared with those with pauci-granulocytic BAL. Children with isolated eosinophilia BAL had features similar to those with mixed granulocytic BAL. Children with mixed granulocytic BAL took more maintenance prednisone, and had greater blood eosinophilia and allergen sensitization compared with those with pauci-granulocytic BAL. CONCLUSIONS: In children with severe, therapy-resistant asthma, BAL granulocyte patterns and infectious species are associated with novel phenotypic features that can inform pathway-specific revisions in treatment. In 32% of children evaluated, BAL revealed corticosteroid-refractory eosinophilic infiltration amenable to anti-TH2 biological therapies, and in 12%, a treatable bacterial pathogen.

16S rRNA survey revealed complex bacterial communities and evidence of bacterial interference on human adenoids.

Adenoid microbiota plays an important role in the development of various infectious and non-infectious diseases of the upper airways, such as otitis media, adenotonsillitis, rhinosinusitis and adenoid hypertrophy. Studies have suggested that adenoids could act as a potential reservoir of opportunistic pathogens. However, previous bacterial surveys of adenoids were mainly culture based and therefore might only provide an incomplete and potentially biased assessment of the microbial diversity. To develop an in-depth and comprehensive understanding of the adenoid microbial communities and test the 'pathogen reservoir hypothesis', we carried out a 16S rRNA based, culture-independent survey of bacterial communities on 67 human adenoids removed by surgery. Our survey revealed highly diverse adenoid bacterial communities distinct from those of other body habitats. Despite large interpersonal variations, adenoid microbiota shared a core set of taxa and can be classified into at least five major types based on its bacterial species composition. Our results support the 'pathogen reservoir hypothesis' as we found common pathogens of otitis media to be both prevalent and abundant. Co-occurrence analyses revealed evidence consistent with the bacterial interference theory in that multiple common pathogens showed 'non-coexistence' relationships with non-pathogenic members of the commensal microflora.

Early outcomes of endoscopic transsphenoidal surgery for adult craniopharyngiomas.

OBJECT: Although the transsphenoidal approach for subdiaphragmatic craniopharyngiomas has been performed for many years, there are few reports describing the role of the endoscopic transsphenoidal technique for suprasellar craniopharyngiomas. The purpose of this study was to report the outcomes of the endoscopic transsphenoidal approach for adults with craniopharyngiomas in whom the goal was gross-total resection. METHODS: Twelve patients were identified who were older than 18 years at the time of their pure endoscopic transsphenoidal surgery. Their medical records and imaging studies were retrospectively reviewed. RESULTS: Gross-total resection was achieved in 42% of cases when assessed by intraoperative impression alone and in 75% when assessed by the first postoperative MR imaging study. However, 83% of patients achieved at least a 95% resection when assessed by both intraoperative impression and the first postoperative MR imaging study. Permanent diabetes insipidus occurred postoperatively in 44% of patients. Six (67%) of 9 patients who had a functioning hypothalamic-pituitary axis preoperatively developed panhypopituitarism after surgery. Visual improvement or normalization occurred in 78% of patients with preoperative visual deficits. Although no patient experienced a postoperative CSF leak, 1 patient was treated for meningitis. CONCLUSIONS: The authors have achieved a high rate of radical resection and symptomatic improvement with the endoscopic transsphenoidal technique for both subdiaphragmatic (sellar/suprasellar) and supradiaphragmatic (suprasellar) craniopharyngiomas. However, this is also associated with a high incidence of new endocrinopathy. Endoscopic assessment of tumor resection may be more sensitive for residual tumor than the first postoperative MR imaging study.

Location of bacterial biofilm in the mucus overlying the adenoid by light microscopy.

OBJECTIVE: To determine the location of bacteria and biofilm in adenoid tissue and in mucus overlying the adenoid. DESIGN: Adenoids removed in 1 piece were oriented to the cephalic and caudal ends. Mucus was fixed by the gradual addition of Carnoy fluid. Consecutive histologic sections were stained with periodic acid-Schiff for visualization of the exopolysaccharide matrix, Giemsa for visualization of bacteria and cells, and fluorescent in situ hybridization with a universal probe for visualization of bacteria. SETTING: Department of Otolaryngology-Head and Neck Surgery, University of Virginia. PARTICIPANTS: We obtained adenoids from children 10 years or younger who had chronic adenotonsillitis or obstructive sleep apnea. Twenty-seven adenoids were used to develop the fixation method. We examined histologic sections from 9 of 10 adenoids fixed using the final fixation protocol. One adenoid that was missing the surface epithelium was excluded from further evaluation. MAIN OUTCOME MEASURE: Identification of bacteria by light microscopy. RESULTS: Bacteria in large numbers were present in the mucus overlying the surface of all 9 adenoids; bacteria were not found in the parenchyma of the adenoids below the epithelial surface. Bacterial biofilms were present on 8 of the 9 adenoids. Sessile (attached) biofilm was present on the caudal end of only 1 adenoid. Multiple planktonic (unattached) biofilms were present on 7 adenoids, always in areas not subject to mucus flow. Biofilms were most common on the caudal portions of adenoids. CONCLUSIONS: Bacteria of the adenoid reside in secretions on the surface and in crypts. Biofilms, predominantly planktonic, were present on 8 of 9 adenoids excised because of hypertrophy. Whether biofilms have a role in the causation of adenoid hypertrophy is not known.

Pharyngocutaneous fistula after anterior cervical spine surgery.

Pharyngocutaneous fistulae are rare complications of anterior spine surgery occurring in less than 0.1% of all anterior surgery cases. We report a case of a 19 year old female who sustained a C6 burst fracture with complete quadriplegia. She was treated urgently with a C6 corpectomy with anterior cage and plating followed by posterior cervical stabilization at another institution. Post operatively she developed a pharyngocutaneous fistula that failed to heal despite several attempts of closure and esophageal exclusion with a Jpeg tube. The patient was eventually successfully treated with a three-stage procedure consisting of firstly a posterior approach to reinforce the posterior stabilization of the cervical spine that was felt to be inadequate, secondly an anterior approach with removal of all the anterior instrumentation followed by iliac crest bone graft and thirdly a superior based sternocleidomastoid flap that was interposed between the esophagus and the anterior cervical spine. The patient's fistula healed successfully. However, yet asymptomatic, the anterior iliac crest bone graft resorbed almost completely at 16 months follow up. In light of this complication, we discuss the surgical options for the treatment of pharyngocutaneous fistulae and the closure of this fistula using a superiorly based sternocleidomastoid muscle flap.

Vagal nerve stimulator implantation: an otolaryngologist's perspective.

OBJECTIVE: This study was conducted to compare an otolaryngologist's experience with a cohort of epilepsy patients implanted with a vagal nerve stimulator (VNS) to previously published data. METHODS: Demographics, preoperative seizure frequency, medications, and complications were retrospectively collected from patients implanted by the senior author. Postoperative medications and seizure frequency were obtained from referring neurologists. RESULTS: Seventeen patients were implanted over a 24-month period. Average age was 28.3 years. Patients presented with petit mal (n = 3), tonic-clonic (n = 6), complex partial (n = 5), and grand mal (n = 8) seizures. Mean follow-up postimplantation was 13.5 months. Most patients had at least a 50% reduction of seizure frequency, with 3 patients being seizure free. There were no postoperative infections. One patient had left vocal cord immobility. The most common side effect was voice disturbance during device activation. CONCLUSION: Otolaryngologists are well equipped to perform VNS implantation and to diagnose and treat possible laryngeal side effects. EBM RATING: C-4.

Surgery of the auricle.

Auricular reconstruction is a unique area of facial plastic surgery where a wide array of reconstructive options often must be considered. The external ear is unique in its aesthetic role where the normal auricle often goes unnoticed; yet even a small irregularity can stand out and become conspicuous. The reconstruction of large or total auricular defects is a combination of science and art. Two forms of auricular reconstruction are discussed: (1) those for a congenitally abnormal shape but no acquired tissue deficiency, that is, otoplasty, and (2) repairs requiring a reconstruction of discrete loss of tissue. A general algorithm is presented that can assist with flap selection and covers the techniques for grafts, framework repair, local and pedicled flaps, temporoparietal facial flaps, and auricular prostheses.