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11:00: Biofilm Formation on Ossicular Reconstruction Prostheses
P138
Otolaryngology-Head and Neck Surgery, Vol 137, No 2S, August 2007
mesothelial cells in both the homozygous and the heterozygous mutant mice. CONCLUSION: The injection technique for hearing preservation should be considered. SIGNIFICANCE: The data suggest that the adenovirus mediated gene transduction may be successful in the inner ear of GJB2 knockout mice.
10:40 Molecular Diagnostic Strategies of EVAS in China and U.S. Pu Dai, MD (presenter); Dong-Yi Han, MD, PhD; Bai-lin Wu, M.Med., PhD PROBLEM: Mutations in the Pendrin gene (SLC26A4) are associated with congenital autosomal recessive hearing loss (DFNB4), often with accompanying temporal bone and/or thyroid abnormalities (Pendred Syndrome, PS). Evidence to date indicates that different populations exhibit different patterns of mutations, suggesting that local clinical testing strategies should reflect these differences. METHODS: The authors used DHPLC–based mutation analysis and bidirectional DNA sequencing to detect all possible mutations in the open reading frame and flanking sequences of SLC26A4 in a population of patients referred for genetic testing in China, and a comparable group of patients in the United States. RESULTS: The relatively higher proportion of Chinese patients with temporal bone abnormalities and deafness who had SLC26A4 mutations (47 of 50 families) and the different spectrum of SLC26A4 mutations in these two populations were found, and 11 previously unreported SLC26A4 mutations were discovered: 5 in the Chinese population (E303Q, G316X, X329, X467, X573), and 6 in the U.S. population (V250A, D266N, F354S, D697A, K715N, E737D), in addition to 14 reported mutations. CONCLUSION: Since this approach worked well in both clinical laboratory settings, the authors propose a practical diagnostic strategy: In China, to screen the two prevalent mutations IVS7-2A⬎G and H723R first as an effective and affordable alternative to CT examination; in the U.S., information on temporal bone and thyroid abnormalities to be included in the clinical information submitted with clinical samples from deaf patients who are undergoing genetic testing and use two-tier analyses for detection of SLC26A4 mutations. SIGNIFICANCE: This study provides appropriate, efficient, and cost effective molecular diagnostic strategies for EVAS in deaf populations from both China and the U.S. SUPPORT: This work was supported by the Chinese National Nature Science Foundation Research Grant 30572015, Beijing Nature Science Foundation Research Grant 7062062 to Pu Dai and Chinese Capital Medical Development Scientific Funding 2005-1032 to Dongyi Han.
10:50 Axon Guidance Cues in the Developing Inner Ear Alyssa M Hackett, MD (presenter); Wei Gao; Ashish R Shah; Sabrina Kadri; Audra M Webber, MD; Yael Raz, MD PROBLEM: Auditory neurons connect the cochlear sensory epithelium with the CNS while conserving the tonotopic organization established in the organ of Corti. Establishment of this innervation pattern requires restricted expression of guidance molecules. The slit proteins and their robo receptors play a significant role in axon guidance in the CNS. The slit/robo signaling pathway may play a similar role in guiding cochlear afferent fibers. METHODS: The spatiotemporal expression of both slits and robos was determined in the developing mouse inner ear using immunohistochemistry and in situ hybridization techniques. Spiral ganglion explant cultures were then utilized to determine the functional role of this pathway in the auditory system. RESULTS: Robo 1 and robo 2 are expressed in the cochleovestibular ganglion during embryonic development. Robo 1 is down-regulated in the early postnatal period. Slit mRNA expression is localized to a subpopulation of cells in the cochleovestibular ganglion at E13. At E16, slit 1 is expressed in the spiral ganglion, slit 2 is expressed in the mesenchymal cells located between the ganglion and the cochlear epithelium, and slit 3 is expressed in the inner sulcus as well as the stria vascularis. In the early postnatal period, slit 2 is also expressed in the inner sulcus where slit 3 expression is maintained. CONCLUSION: The slit and robo genes are expressed in a restricted fashion during cochlear development. The spatiotemporal pattern of expression suggests a model in which slit 2 and slit 3 define a boundary which excludes robo-expressing neurites directing them towards the organ of Corti. SIGNIFICANCE: An increased understanding of axon guidance in the developing inner ear may prove useful in developing an enhanced interface between cochlear implants and the auditory nerve. SUPPORT: NIDCD, AAO core grants (AHRF, Triological Society), UPMC CMRF grant, the UPMC EEI Foundation, Pennsylvania Lion’s Foundation.
11:00 Biofilm Formation on Ossicular Reconstruction Prostheses Eric Michael Jaryszak, MD, PhD (presenter); Edith Sampson; Patrick J Antonelli, MD PROBLEM: Ossicular chain reconstruction may be complicated by prosthesis extrusion. As prostheses are commonly placed in middle ears contaminated with biofilm-forming bacteria, such as Pseudomonas aeruginosa (PA), extrusion may be
caused by development of a biofilm on the prosthesis and the host response to this biofilm. METHODS: Prostheses made of titanium, hydroxylapatite (HA), and plastic (23 each) were cultured with PA in broth for 96 hours. Biofilm formation was assessed by electron microscopy and quantitative microbiology. RESULTS: Titanium prostheses formed less biofilm than plastic (p ⫽ 0.0003) and HA (p ⫽ 0.003), but there was no difference between HA and plastic. Correction for surface area did not alter these significant differences. CONCLUSION: PA forms biofilm on ossicular reconstruction prostheses, particularly those made of plastic and HA. These differences could, in part, explain the extrusion propensity of certain biomaterials. SIGNIFICANCE: The results of this study could alter which biomaterials are chosen for ossicular chain reconstruction in the setting of different middle ear pathology. SUPPORT: This project was supported by the resident research fund allocated by the Department of Otolaryngology– Head and Neck Surgery of the University of Florida. Ossicular chain reconstruction prostheses were graciously contributed by Medtronic ENT.
11:10 Biofilms in Humans with Chronic Suppurative Otitis Media Amber Uyen Luong, MD, PhD (presenter); Karen S Pawlowski, PhD; Peter S Roland, MD PROBLEM: The recalcitrant nature of chronic suppurative otitis media (CSOM) to antibiotics suggests a presence of biofilms in CSOM. METHODS: Middle ear mucosa specimens were collected
P139 from 7 human adults with CSOM and 7 human adults with a nondraining tympanic membrane perforation. These specimens were analyzed by scanning electron microscopy (SEM) and live/dead bacteria staining for presence of biofilm and live bacteria. Specimens from the CSOM patients were also sent for culture. Mastoid mucosa samples from patients with CSOM were also analyzed in a manner similar to the middle ear tissue. RESULTS: A blind analysis for live/dead bacteria and by SEM was performed on each specimen. Biofilms were clearly identified in 5 of the 7 (71%) samples with suspected CSOM. In 1 of the CSOM samples, there were questionable areas that resembled biofilms on both SEM and live/dead staining. Another CSOM specimen revealed unusual lipid-like structures on SEM without clear evidence of biofilms and no evidence of live bacteria. In comparison, 6 of the 7 (88%) specimens from patients with a nondraining tympanic membrane perforation showed no biofilm. Culture results from the CSOM samples showed no growth, confirming that biofilm bacteria are difficult to culture. Comparison of mucosa from the middle ear and from the mastoid revealed that biofilm when present was present in both locations. CONCLUSION: Direct visualization of biofilms in human adults with CSOM and not in patients with a dry tympanic membrane perforation strongly suggests a role of biofilms in the pathophysiology of CSOM. SIGNIFICANCE: First study to report the direct visualization of biofilms in human adults with CSOM. This data may explain the persistence of drainage and mucosal inflammation despite treatment with topical antibiotics. SUPPORT: UT Southwestern Medical Center Department of Otolaryngology-HNS.
10:30 AM to 12:00 PM MINISEMINARS WCC 207AB Miniseminar: Allergy Vial Mixing and Testing: New Standards in Safety Sandra Y Lin, MD (moderator); Richard C Haydon, III, MD; Steven M Houser, MD; Hector P Rodriguez, MD Recently there has been a trend toward further regulation of sterile medication compounding that may affect allergy vial mixing. The objectives of this miniseminar include introducing otolaryngologists to proposed standards regarding allergen vial mixing, identifying clinical implications, and recommending efforts to address
the issue of vial mixing standards. The topics to be covered include current state and federal regulations in allergy vial mixing and testing, including JCAHO guidelines. The United States Pharmacopeia on sterile compounding, USP ⬍797⬎, and it’s potential effects on vial mixing will be discussed. The impact of these guidelines on the otolaryngology office practicing allergy will be discussed. New allergy vial mixing standards proposed by the AAOA and JCAI will be introduced. Finally, suggestions for creating and implementing an acceptable safety standard for the otolaryngic allergy office will be presented.
WEDNESDAY
Miniseminars—Wednesday
Otolaryngology-Head and Neck Surgery, Vol 137, No 2S, August 2007
mesothelial cells in both the homozygous and the heterozygous mutant mice. CONCLUSION: The injection technique for hearing preservation should be considered. SIGNIFICANCE: The data suggest that the adenovirus mediated gene transduction may be successful in the inner ear of GJB2 knockout mice.
10:40 Molecular Diagnostic Strategies of EVAS in China and U.S. Pu Dai, MD (presenter); Dong-Yi Han, MD, PhD; Bai-lin Wu, M.Med., PhD PROBLEM: Mutations in the Pendrin gene (SLC26A4) are associated with congenital autosomal recessive hearing loss (DFNB4), often with accompanying temporal bone and/or thyroid abnormalities (Pendred Syndrome, PS). Evidence to date indicates that different populations exhibit different patterns of mutations, suggesting that local clinical testing strategies should reflect these differences. METHODS: The authors used DHPLC–based mutation analysis and bidirectional DNA sequencing to detect all possible mutations in the open reading frame and flanking sequences of SLC26A4 in a population of patients referred for genetic testing in China, and a comparable group of patients in the United States. RESULTS: The relatively higher proportion of Chinese patients with temporal bone abnormalities and deafness who had SLC26A4 mutations (47 of 50 families) and the different spectrum of SLC26A4 mutations in these two populations were found, and 11 previously unreported SLC26A4 mutations were discovered: 5 in the Chinese population (E303Q, G316X, X329, X467, X573), and 6 in the U.S. population (V250A, D266N, F354S, D697A, K715N, E737D), in addition to 14 reported mutations. CONCLUSION: Since this approach worked well in both clinical laboratory settings, the authors propose a practical diagnostic strategy: In China, to screen the two prevalent mutations IVS7-2A⬎G and H723R first as an effective and affordable alternative to CT examination; in the U.S., information on temporal bone and thyroid abnormalities to be included in the clinical information submitted with clinical samples from deaf patients who are undergoing genetic testing and use two-tier analyses for detection of SLC26A4 mutations. SIGNIFICANCE: This study provides appropriate, efficient, and cost effective molecular diagnostic strategies for EVAS in deaf populations from both China and the U.S. SUPPORT: This work was supported by the Chinese National Nature Science Foundation Research Grant 30572015, Beijing Nature Science Foundation Research Grant 7062062 to Pu Dai and Chinese Capital Medical Development Scientific Funding 2005-1032 to Dongyi Han.
10:50 Axon Guidance Cues in the Developing Inner Ear Alyssa M Hackett, MD (presenter); Wei Gao; Ashish R Shah; Sabrina Kadri; Audra M Webber, MD; Yael Raz, MD PROBLEM: Auditory neurons connect the cochlear sensory epithelium with the CNS while conserving the tonotopic organization established in the organ of Corti. Establishment of this innervation pattern requires restricted expression of guidance molecules. The slit proteins and their robo receptors play a significant role in axon guidance in the CNS. The slit/robo signaling pathway may play a similar role in guiding cochlear afferent fibers. METHODS: The spatiotemporal expression of both slits and robos was determined in the developing mouse inner ear using immunohistochemistry and in situ hybridization techniques. Spiral ganglion explant cultures were then utilized to determine the functional role of this pathway in the auditory system. RESULTS: Robo 1 and robo 2 are expressed in the cochleovestibular ganglion during embryonic development. Robo 1 is down-regulated in the early postnatal period. Slit mRNA expression is localized to a subpopulation of cells in the cochleovestibular ganglion at E13. At E16, slit 1 is expressed in the spiral ganglion, slit 2 is expressed in the mesenchymal cells located between the ganglion and the cochlear epithelium, and slit 3 is expressed in the inner sulcus as well as the stria vascularis. In the early postnatal period, slit 2 is also expressed in the inner sulcus where slit 3 expression is maintained. CONCLUSION: The slit and robo genes are expressed in a restricted fashion during cochlear development. The spatiotemporal pattern of expression suggests a model in which slit 2 and slit 3 define a boundary which excludes robo-expressing neurites directing them towards the organ of Corti. SIGNIFICANCE: An increased understanding of axon guidance in the developing inner ear may prove useful in developing an enhanced interface between cochlear implants and the auditory nerve. SUPPORT: NIDCD, AAO core grants (AHRF, Triological Society), UPMC CMRF grant, the UPMC EEI Foundation, Pennsylvania Lion’s Foundation.
11:00 Biofilm Formation on Ossicular Reconstruction Prostheses Eric Michael Jaryszak, MD, PhD (presenter); Edith Sampson; Patrick J Antonelli, MD PROBLEM: Ossicular chain reconstruction may be complicated by prosthesis extrusion. As prostheses are commonly placed in middle ears contaminated with biofilm-forming bacteria, such as Pseudomonas aeruginosa (PA), extrusion may be
caused by development of a biofilm on the prosthesis and the host response to this biofilm. METHODS: Prostheses made of titanium, hydroxylapatite (HA), and plastic (23 each) were cultured with PA in broth for 96 hours. Biofilm formation was assessed by electron microscopy and quantitative microbiology. RESULTS: Titanium prostheses formed less biofilm than plastic (p ⫽ 0.0003) and HA (p ⫽ 0.003), but there was no difference between HA and plastic. Correction for surface area did not alter these significant differences. CONCLUSION: PA forms biofilm on ossicular reconstruction prostheses, particularly those made of plastic and HA. These differences could, in part, explain the extrusion propensity of certain biomaterials. SIGNIFICANCE: The results of this study could alter which biomaterials are chosen for ossicular chain reconstruction in the setting of different middle ear pathology. SUPPORT: This project was supported by the resident research fund allocated by the Department of Otolaryngology– Head and Neck Surgery of the University of Florida. Ossicular chain reconstruction prostheses were graciously contributed by Medtronic ENT.
11:10 Biofilms in Humans with Chronic Suppurative Otitis Media Amber Uyen Luong, MD, PhD (presenter); Karen S Pawlowski, PhD; Peter S Roland, MD PROBLEM: The recalcitrant nature of chronic suppurative otitis media (CSOM) to antibiotics suggests a presence of biofilms in CSOM. METHODS: Middle ear mucosa specimens were collected
P139 from 7 human adults with CSOM and 7 human adults with a nondraining tympanic membrane perforation. These specimens were analyzed by scanning electron microscopy (SEM) and live/dead bacteria staining for presence of biofilm and live bacteria. Specimens from the CSOM patients were also sent for culture. Mastoid mucosa samples from patients with CSOM were also analyzed in a manner similar to the middle ear tissue. RESULTS: A blind analysis for live/dead bacteria and by SEM was performed on each specimen. Biofilms were clearly identified in 5 of the 7 (71%) samples with suspected CSOM. In 1 of the CSOM samples, there were questionable areas that resembled biofilms on both SEM and live/dead staining. Another CSOM specimen revealed unusual lipid-like structures on SEM without clear evidence of biofilms and no evidence of live bacteria. In comparison, 6 of the 7 (88%) specimens from patients with a nondraining tympanic membrane perforation showed no biofilm. Culture results from the CSOM samples showed no growth, confirming that biofilm bacteria are difficult to culture. Comparison of mucosa from the middle ear and from the mastoid revealed that biofilm when present was present in both locations. CONCLUSION: Direct visualization of biofilms in human adults with CSOM and not in patients with a dry tympanic membrane perforation strongly suggests a role of biofilms in the pathophysiology of CSOM. SIGNIFICANCE: First study to report the direct visualization of biofilms in human adults with CSOM. This data may explain the persistence of drainage and mucosal inflammation despite treatment with topical antibiotics. SUPPORT: UT Southwestern Medical Center Department of Otolaryngology-HNS.
10:30 AM to 12:00 PM MINISEMINARS WCC 207AB Miniseminar: Allergy Vial Mixing and Testing: New Standards in Safety Sandra Y Lin, MD (moderator); Richard C Haydon, III, MD; Steven M Houser, MD; Hector P Rodriguez, MD Recently there has been a trend toward further regulation of sterile medication compounding that may affect allergy vial mixing. The objectives of this miniseminar include introducing otolaryngologists to proposed standards regarding allergen vial mixing, identifying clinical implications, and recommending efforts to address
the issue of vial mixing standards. The topics to be covered include current state and federal regulations in allergy vial mixing and testing, including JCAHO guidelines. The United States Pharmacopeia on sterile compounding, USP ⬍797⬎, and it’s potential effects on vial mixing will be discussed. The impact of these guidelines on the otolaryngology office practicing allergy will be discussed. New allergy vial mixing standards proposed by the AAOA and JCAI will be introduced. Finally, suggestions for creating and implementing an acceptable safety standard for the otolaryngic allergy office will be presented.
WEDNESDAY
Miniseminars—Wednesday