Measles Virus Prevalence in Otosclerotic Foci 105 10 McKenna MJ, Mills BG: Immunohistochemical evidence of measles virus antigens in active oto- sclerosis. Otolaryngol Head Neck Surg 1989;101:415–421. 11 Lolov SR, Encheva VI, Kyurkchiev SD, Edrev GE, Kehayov IR: Antimeasles immunoglobulin G in sera of patients with otosclerosis is lower than that in healthy people. Otol Neurotol 2001;22: 766–770. 12 Niedermeyer HP, Arnold W, Neubert WJ, Höfler H: Evidence of measles virus RNA in otosclerotic tissue. ORL J Otorhinolaryngol Relat Spec 1994;56:130–132. 13 Arnold W, Friedmann I: Otosclerosis: an inflammatory disease of the otic capsule of viral aetiol- ogy? J Laryngol Otol 1988;102:865–871. 14 McKenna MJ, Kristiansen AG, Haines J: Polymerase chain reaction amplification of measles virus sequence from human temporal bone sections with active otosclerosis. Am J Otol 1996;17: 827–830. 15 Karosi T, Kónya J, Szabó LZ, Sziklai I: Measles virus prevalence in otosclerotic stapes footplate samples. Otol Neurotol 2004;25:451–456. 16 Grayeli AB, Palmer P, Tran Ba Huy P, Soudant J, Sterkers O, Lebon P, Ferrary E: No evidence of measles virus in stapes samples from patients with otosclerosis. J Clin Microbiol 2000;38:2655–2660. 17 Rima BK: Paramyxoviruses and chronic human diseases. Bone 1999;24(suppl 5):23S–26S. 18 Wiet RJ, Causse JB, Shambaugh G, Causse JR: Otosclerosis. Alexandria, American Academy of Otolaryngology-Head and Neck Surgery Foundation, Inc, 1991. 19 Chole RA, McKenna M: Pathophysiology of otosclerosis. Otol Neurotol 2001;22:249–257. 20 Niedermeyer HP, Arnold W: Etiopathogenesis of otosclerosis. ORL J Otorhinolaryngol Relat Spec 2002;64:114–119. 21 Niedermeyer HP, Arnold W, Neubert WJ, Sedlmeier R: Persistent measles virus infection as a pos- sible cause of otosclerosis: state of the art. Ear Nose Throat J 2000;79:552–558. 22 Niedermeyer HP, Arnold W, Schuster M, Baumann C, Kramer J, Neubert WJ, Sedlmeier R: Persistent measles virus infection and otosclerosis. Ann Otol Rhinol Laryngol 2001;110:897–903. 23 McKenna MJ, Kristiansen AG, Tropitzsch AS: Similar COL1A1 expression in fibroblasts from some patients with clinical otosclerosis and those with type I osteogenesis imperfecta. Ann Otol Rhinol Laryngol 2002;111:184–189. 24 Van Den Bogaert K, Govaerts PJ, De Leenheer EM, Schatteman I, Verstreken M, Chen W, Declau F, Cremers CW, Van De Heyning PH, Offeciers FE, Somers T, Smith RJ, Van Camp G: Otosclerosis: a genetically heterogeneous disease involving at least three different genes. Bone 2002;30: 624–630. 25 Dahlqvist A, Diamant H, Dahlqvist SR, Cedergren B: HLA antigens in patients with otosclerosis. Acta Otolaryngol 1985;100:33–35. 26 Chadwick N, Bruce I, Davies M, van Gemen B, Schukkink R, Khan K, Pounder R, Wakefield A: A sensitive and robust method for measles RNA detection. J Virol Methods 1998;70:59–70. 27 Friedmann I: Pathology of the Ear. Oxford, Blackwell Scientific Publications, 1974. 28 Thiers FA, Valvassori GE, Nadol JB: Otosclerosis of the cochlear capsule: correlation of comput- erized tomography and histopathology. Am J Otol 1999;20:93–95. 29 Gloddek B, Ryan AF, Harris JP: Homing of lymphocytes to the inner ear. Acta Otolaryngol 1991;111:1051–1059. 30 Tomiyama S, Harris JP: The endolymphatic sac: its importance in inner ear immune responses. Laryngoscope 1986;96:685–691. 31 Arnold W, Niedermeyer HP: Herpes simplex virus antibodies in the perilymph of patients with Ménière disease. Arch Otolaryngol Head Neck Surg 1997;123:53–56. 32 Ribári O, Sziklai I, Kiss JG, Sohar I: Cathepsin-B activity in otosclerosis. Arch Otolaryngol 1983;238:123–125. 33 Sziklai I, Grof J, Ribari O, Menyhart J: Possible role of peptides derived from otosclerotic bone in the mechanism of sensorineural hearing loss. Acta Otolaryngol 1985;100:253–259. 34 Sziklai I, Kiss JG, Ribari O: Inhibition of myosin light-chain kinase activity in the organ of Corti by 0.3–5 kilodalton substances of the otosclerotic perilymph. Arch Otorhinolaryngol 1986;243: 229–232. 35 Sziklai I: Human otosclerotic bone-derived peptide decreases the gain of the electromotility in iso- lated outer hair cells. Hear Res 1996;95:100–107. Karosi/Kónya/Szabó/Sziklai 106 36 World Health Organization Regional Office for Europe. Available at: http://www.who.dk/hfadb 37 Gorny G, Shaw A, Oursler MJ: IL-6, LIF, and TNF-alpha regulation of GM-CSF inhibition of osteoclastogenesis in vitro. Exp Cell Res 2004;294:149–158. István Sziklai, MD, DSc University Medical School of Debrecen Department of Otorhinolaryngology Head and Neck Surgery Nagyerdei Krt. 98 HU–4012 Debrecen (Hungary) E-Mail isziklai@jaguar.dote.hu Arnold W, Häusler R (eds): Otosclerosis and Stapes Surgery. Adv Otorhinolaryngol. Basel, Karger, 2007, vol 65, pp 107–113 Antimeasles Immunoglobulin G and Virus-Neutralizing Activity in Sera of Patients with Otosclerosis Stephan Lolov a , George Edrev b , Stanimir Kyurkchiev a a Department of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, b ENT Department, ‘Tzar Boris III’ Hospital, Sofia, Bulgaria Abstract Otosclerosis is considered as an organ-specific measles virus (MV)-induced disease. The majority of people are infected with MV during childhood, and the immune activation is char- acterized by a lifelong persistence. Any kind of depressed systemic antimeasles reaction can lead to induction of a local immune response in the inner ear. MV proteins were resolved by conventional polyacrylamide gel electrophoresis in the presence of a denaturing detergent. In subsequent Western blot, healthy blood donors demonstrated a stronger reaction against most proteins than age- and sex-matched otosclerotic patients. Different serum dilutions were tested for neutralizing activity against constant MV concentration. Nearly complete viral neutraliza- tion was achieved with samples containing inactivated complement and in immunoglobulin-G- containing fractions, and activity in sera from patients with confirmed otosclerosis was significantly weaker than in healthy individuals. Our observations are consistent with viral par- ticipation in otosclerotic pathogenesis, but it is difficult to say if the diminished antimeasles humoral response is a consequence of or the cause for a local measles infection. Copyright © 2007 S. Karger AG, Basel Background The etiopathogenesis of otosclerosis is unexplained. There are some data for measles virus (MV) in cases of otosclerosis (fig. 1). The majority of people are infected with MV during childhood, and the immune activation is characterized by a lifelong persistence. It seems that the average age of patients with otosclerosis has increased since the introduction of the MV vaccination program. Lolov/Edrev/Kyurkchiev 108 Vaccination program and increased average age of patients [11]? Natural or induced MV infection Lifelong persistence of immune activation Depressed systemic antimeasles reaction? MV replication in otosclerotic focus Filamentous structures similar to paramyxoviral nucleocapsid [6] MV RNA [8, 9] MV antigens [1, 7] Induction of a local immune response in the inner ear [2] Induction of local autoimmunity [12]? Induction of local inflammation [1, 3] Reactivation of bone turnover MV persistence [10]? ? ? ? Fig. 1. Suspected routes of MV participation in bone turnover reactivation [1–3, 6–12]. Antimeasles IgG and Virus-Neutralizing Activity in Sera of Patients with Otosclerosis 109 Hypothesis Any kind of depressed systemic antimeasles reaction can lead to induction of a local immune response in the inner ear and possibly to reactivation of bone turnover in this specific region. Antimeasles Immunoglobulin G MV proteins were resolved by conventional preparative SDS-PAGE. After subsequent Western blot, the membranes were air-dried and subjected to densit- ometry. According to statistical analysis, healthy blood donors demonstrated a stronger reaction against most proteins than age- and sex-matched otosclerotic patients (fig. 2) [4]. Plot of means Male Optical density 10 20 30 40 50 60 70 80 90 100 Healthy Otosclerosis Female 10 20 30 40 50 60 70 80 90 100 Healthy Otosclerosis pϭ0.0076 pϭ0.0188 pϭ0.0075 pϭ0.0212 pϭ0.0004 pϭ0.0028 pϭ0.0018 pϭ0.0034 pϭ0.0002 X Y P N M H Fig. 2. Systemic IgG activity (in relative units) in otosclerotic patients and healthy individuals against separated viral proteins and their respective statistical signifi- cance. H ϭ Hemagglutinin; M ϭ matrix; N ϭ nucleocapsid; P ϭ polymerase; X ϭ x-band; Y ϭ y-band. Lolov/Edrev/Kyurkchiev 110 MV-containing mixture: (a) virus only; (b) virus with different dillutions of whole sera; (c) virus with treated/separated sera MTT assay (1) MTT solution was added to each well and the plates were incubated for 4h under cell culturing conditions. (2) Solubilization solution was added to dissolve the formazan crystals. (3) After 24h, the absorption at 600nm was measured on an ELISA reader Cell monolayer in culture well Viral absorption Prolonged culturing Fig. 3. Schematic presentation of the combination between in vitro virus neutralization procedure and quantitative MTT reaction. 1,200 1,000 800 600 400 200 0 28/28 29/30 33/34 37/39 Age (years) 40/40 41/41 Healthy Otosclerosis Fig. 4. Titer for 50% MV-neutralizing activity for several sex-matched pairs of healthy individuals and otosclerotic patients. Antimeasles IgG and Virus-Neutralizing Activity in Sera of Patients with Otosclerosis 111 30 min, 56˚C 1.6 1.4 1.2 1.0 0.8 Optical density (600 nm) Untreated serum 1.5 1.4 1.3 Optical density (600 nm) 1.2 1.1 1.0 Otosclerosis Healthy individual pϭ0.000120 Inactivated complement Effluent lgG fraction 0.4 0.6 0.2 0 Protein G Ϯ1.96 standard error Ϯ1.00 standard error Mean Otosclerosis Healthy individual 1.5 1.4 1.3 Optical density (600 nm) 1.2 1.1 1.0 pϭ0.000027 a b cd Fig. 5. Schematic presentation of sample separation (a) and box-whiskers plots of virus-neutralizing activities (mean Ϯ standard error, see legend in (b) in different fractions: all samples (b), only sera with inactivated complement (c) and IgG fractions (d). Lolov/Edrev/Kyurkchiev 112 Measles Virus Neutralization Assay with Whole Sera The ability of MV to infect and replicate in the cell monolayer was detected by enumeration of living and growing cells with a colored reaction, constructed especially for this study (fig. 3) [5]. Virus-neutralizing activity in sera from patients with confirmed otosclero- sis was significantly weaker than in healthy individuals (p ϭ 0.032). When age- and sex-matched pairs were compared, in 5 cases, the neutralizing activity in the healthy counterpart was higher. The opposite situation was observed in 1 healthy/otosclerosis couple only (fig. 4). Measles Virus Neutralization Activity in Separated Sera Samples from selected sera were incubated at 56ЊC for 30 min to inactivate the complement. Immunoglobulin G (IgG) fractions were isolated using protein G sepharose 4 fast flow affinity columns (fig. 5a). Nearly complete viral neutralization was achieved with samples containing inactivated complement, and in IgG-containing fractions (fig. 5b), and activity in sera from patients with confirmed otosclerosis was significantly weaker than in healthy individuals (fig. 5c, d) [5]. Conclusion Our observations are consistent with viral participation in otosclerotic pathogenesis, but it is difficult to say if the diminished antimeasles humoral response is a consequence of or the cause for a local measles infection. References 1 Arnold W, Friedmann I: Otosclerosis: an inflammatory disease of the otic capsule of viral aetiol- ogy? J Laryngol Otol 1988;102:865–871. 2 Arnold W, Niedermeyer HP, Lehn N, Neubert W, Hofler H: Measles virus in otosclerosis and the specific immune response of the inner ear. Acta Otolaryngol 1996;116:705–709. 3 Lim DJ, Robinson M, Saunders WH: Morphologic and immunohistochemical observation of oto- sclerotic stapes: a preliminary study. Am J Otolaryngol 1987;8:282–295. 4 Lolov SR, Encheva VI, Kyurkchiev SD, Edrev GE, Kehayov IR: Antimeasles immunoglobulin G in sera of patients with otosclerosis is lower than that in healthy people. Otol Neurotol 2001;22:766–770. 5 Lolov S, Zarzalanova P, Edrev G, Kyurchiev S: Decreased measles virus-neutralizing activity in sera from otosclerotic patients. ORL J Otorhinolaryngol Relat Spec 2003;65:279–283. Antimeasles IgG and Virus-Neutralizing Activity in Sera of Patients with Otosclerosis 113 6 McKenna MJ, Mills BG, Galey FR, Linthicum FH Jr: Filamentous structures morphologically similar to viral nucleocapsids in otosclerotic lesions in two patients. Am J Otol 1986;7:25–28. 7 McKenna MJ, Mills BG: Immunohistochemical evidence of measles virus antigens in active oto- sclerosis. Otolaryngol Head Neck Surg 1989;101:415–421. 8 McKenna MJ, Kristiansen AG, Haines J: Polymerase chain reaction amplification of a measles virus sequence from human temporal bone sections with active otosclerosis. Am J Otol 1996;17: 827–830. 9 Niedermeyer H, Arnold W, Neubert WJ, Hofler H: Evidence of measles virus RNA in otosclerotic tissue. ORL J Otorhinolaryngol Relat Spec 1994;56:130–132. 10 Niedermeyer HP, Arnold W, Neubert WJ, Sedlmeier R: Persistent measles virus infection as a pos- sible cause of otosclerosis: state of the art. Ear Nose Throat J 2000;79:552–554. 11 Niedermeyer HP, Arnold W, Schwub D, Busch R, Wiest I, Sedlmeier R: Shift of the distribution of age in patients with otosclerosis. Acta Otolaryngol 2001;121:197–199. 12 Yoo TJ: Etiopathogenesis of otosclerosis: a hypothesis. Ann Otol Rhinol Laryngol 1984;93:28–33. Stephan Lolov 73 Tzarigradsko shousse BG–1113 Sofia (Bulgaria) Tel. ϩ3592 872 09 25, Fax ϩ3592 989 05 71, E-Mail Dr_Lolov@yahoo.com Arnold W, Häusler R (eds): Otosclerosis and Stapes Surgery. Adv Otorhinolaryngol. Basel, Karger, 2007, vol 65, pp 114–118 Phenotype-Genotype Correlations in Otosclerosis: Clinical Features of OTSC2 Frank Declau a , Kris Van den Bogaert b , Paul Van De Heyning a , Erwin Offeciers c , Paul Govaerts d , Guy Van Camp b a Department of Otorhinolaryngology, Head and Neck Surgery and Communication Disorders, and b Department of Genetics, University of Antwerp, Antwerp, c Department of Otorhinolaryngology, Head and Neck Surgery, Sint-Augustinus Hospital, Wilrijk, and d The Eargroup, Deurne, Belgium Abstract As part of the GENDEAF consortium, a European multi-centre otosclerotic database is under construction to collect the clinical data of as many otosclerotic patients as possible. Otosclerosis represents a heterogeneous group of heritable diseases in which different genes may be involved regulating the bone homeostasis of the otic capsule. The purpose of the GENDEAF otosclerosis database is to explore the otosclerotic phenotype more in depth. Subtle phenotypic differences otherwise not visible, may become statistically relevant in a large number of patients. Their identification can lead towards the discovery of new genes involved in the pathway of abnormal bone metabolism in the human labyrinth. As soon as one of the otosclerotic genes is identified, it would allow us to identify genotype-phenotype correlations. From other deafness genes, it is know that different mutations in the same gene may cause similar phenotypes of varying severity. Also the variability in treatment outcomes after surgery or fluoride therapy may result not only from differences in practice or surgical skill among physicians, but also on the nature of the underlying disorder. Screening large numbers of patients would make it possible to undertake clinical trials comparing different treatments. Identifying a genetic susceptibility would allow us to dissect out possible envi- ronmental factors that prevent the expression of clinical otosclerosis in those that carry the mutated gene and yet retain normal hearing. Copyright © 2007 S. Karger AG, Basel Otosclerosis is a disorder in which both genetic and environmental etiolog- ical factors are involved [1]. Although a limited number of large autosomal dominant families have been described, most patients occur in small families [...]... results after stapes surgery in otosclerosis 1 The effect of using different audiologic parameters and criteria on success rates Otolaryngol Head Neck Surg 2001;1 24: 76–83 Prof R Probst HNO-Klinik Universitätsspital Basel CH 40 31 Basel (Switzerland) Tel 41 61 265 41 05, Fax 41 61 265 40 29, E-Mail rprobst@uhbs.ch Probst 126 Arnold W, Häusler R (eds): Otosclerosis and Stapes Surgery Adv Otorhinolaryngol... Seta 1 34 0 B-pre 10 A-pre B-post A-post dB HL 20 30 40 50 60 70 80 250 500 1,000 2,000 4, 000 Hz Fig 1 Mean tonal audiograms of the 15 patients included in the study, collected a day before and 30 days after stapedotomy A ϭ Air conduction threshold; B ϭ bone conduction threshold 10 pre intra peri post 5 0 Ϫ5 Ϫ10 0 08 0 2, 40 0 2, 56 0 3, 04 0 3, 20 0 3, 76 0 4, 32 0 4, 80 0 5, 36 0 5, 84 0 6, 40 0 6,... Offeciers FE, Somers T, Van Camp G: Otosclerosis: A genetically heterogeneous disease involving at least 3 different genes Bone 2002;30:6 24 630 Frank Declau, MD, PhD Department of Otorhinolaryngology, Head and Neck Surgery and Communication Disorders University Hospital of Antwerp, Wilrijkstraat 1 BE–2650 Edegem (Belgium) Tel./Fax ϩ32 3 44 05162, E-Mail otosclerosis@ pandora.be Declau/Van den Bogaert/Van... (eds): Otosclerosis and Stapes Surgery Adv Otorhinolaryngol Basel, Karger, 2007, vol 65, pp 119–126 Audiological Evaluation of Patients with Otosclerosis Rudolf Probst Department of Otorhinolaryngology, University Hospital, Basel, Switzerland Abstract Even though the diagnosis of otosclerosis is confirmed definitively during surgery, preoperative diagnosis and determination of the indication for surgery. .. frequency between 250 and 4, 000 Hz The annual threshold deterioration indicated a progression of 0.37–0.82 dB/year for bone conduction and 0.81–1.32 dB/year for air conduction On the ARAB plots, the maximal air-bone gap was situated at 500 and 4, 000 Hz and the Carhart notch at 1–2 KHz was also clearly visible The air-bone gap deterioration at 500 Hz amounted to 0 .41 dB/year Discussion and Conclusion The... essential part of the audiological assessment is to demonstrate the need for rehabilitative measures, and the need for surgery in the particular case of suspected otosclerosis A small air-bone gap with little overall hearing loss will not be an indication for surgery, even if the diagnosis of otosclerosis can be established Three major components are to be considered when recommending surgery for otosclerosis: ... Brownstein Z, Frydman M, Avraham KB: Identification of a new gene for otosclerosis, OTSC4 ARO Meeting, Guildford, 20 04 Van den Bogaert K, de Leenheer EMR, Chen W, Lee Y, Nürnberg P, Pennings RJE, Vanderstraeten K, Thys M, Cremers CWRJ, Smith RJH, van Camp G: A fifth locus for otosclerosis, OTSC5, maps to chromosome 3q22– 24 J Med Genet 20 04; 4: 145 0– 145 3 Van den Bogaert K, Govaerts PJ, De Leenheer EMR, Schatteman... men; mean age 51 years; range 32–69 years) with advanced otosclerosis All the patients were evaluated with the use of pure-tone audiograms (preoperatively, 5 and 30 days after surgery) , stapedial reflexes (preoperatively), and DPOAE recordings (preoperatively, at the end of surgery, and 5 and 30 days after surgery) Changes in the hearing thresholds and in the DPOAE amplitudes were compared Preoperative... Usually stapes surgery, and particularly stapedotomy, is the first-choice treatment The aim of the study was to investigate changes in middle ear dynamic characteristics caused both by the disease and the surgical steps (platinotomy, prosthesis positioning, ossicular chain maneuver) and to evaluate their influence on DPOAE responses in patients with otosclerosis [3, 4] A group of 15 patients (12 women and. .. the maximal effect of the disease on Phenotype-Genotype Correlations in Otosclerosis 115 Ϫ20 0 Hearing loss (dB) 20 40 60 80 100 120 125 250 500 1,000 2,000 4, 000 8,000 air-bone gap Frequency (Hz) Fig 1 Box -and- whisker plot: air conduction versus air-bone gap at 500 Hz hearing, only the thresholds of the worst ear were used in the statistical analysis Pure-tone hearing thresholds were analyzed in relation . neutralization procedure and quantitative MTT reaction. 1,200 1,000 800 600 40 0 200 0 28/28 29/30 33/ 34 37/39 Age (years) 40 /40 41 /41 Healthy Otosclerosis Fig. 4. Titer for 50% MV-neutralizing activity. 2,000 4, 000 8,000 air-bone gap Hearing loss (dB) Ϫ20 0 20 40 60 80 100 120 Fig. 1. Box -and- whisker plot: air conduction versus air-bone gap at 500 Hz. Phenotype-Genotype Correlations in Otosclerosis. Wilrijkstraat 1 BE–2650 Edegem (Belgium) Tel./Fax ϩ32 3 44 05162, E-Mail otosclerosis@ pandora.be Arnold W, Häusler R (eds): Otosclerosis and Stapes Surgery. Adv Otorhinolaryngol. Basel, Karger, 2007,