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How to cite this article Bau ALW, Lavinsky L, Bonorino C, Munari L, Lavinsky-wolff M, Wolff FH. Objectives: To establish the frequency of auto-antibodies anti-HSP 70 using ELISA and Western Blot (WB) methods and to compare the results of each method among patients with the Meniere's Disease (MD) and internal ear diseases (IED) who do not fulfill criteria for MD. In 1861, during a medical conference, Prosper Meniere described for the first time a set of symptoms that up to then were attributed to a cerebral condition defined as cerebral apoplexy1. The histopathology of the endolymphatic hydrops was described for the first time in articles of Hallpike and Cairns in England and of Yamakawa in Japan, both published in 1938, reporting characteristic alterations in the labyrinth and temporal bones associated to the Meniere Disease (MD). Historically, MD is one of the most studied otorhinolaryngological pathology, it remained for several years a disease with scarce treatment possibilities, of difficult management and many times frustrating9. The IE was considered to be segregated from the immune system as it is isolated inside the optical capsule and as it does not have lymphatic drainage14. The immunomediated etiopathogeny was described in researches conducted by some authors suggesting this is the cause for part of MD's cases previously classified as of idiopathic etiology7,12,16-18.
The clinical course of the MD presenting hearing fluctuations, asymptomatic periods and oscillations in the illness' severity, which results in slow and progressive deterioration of the affected organ, is similar to the clinical course observed in several autoimmune diseases.
This study aim was to evaluate the relation between anti-HSP 70 antibody and clinical factors of MD, if this relation varies during the course of the disease, and if the presence of antibodies is useful to distinguish a subgroup of MD. We conducted a prospective case-control study21 to evaluate the frequency of anti-HSP 70 antibodies with the ELISA and the WB methods. We invited all patients from Meniere Disease Clinics of the Otorhinolaryngology Department at the Clinics Hospital of Porto Alegre to participate in the study, through invitation letters sent by mail. Patients were included in the study group if they had the syndromic diagnosis of MD according to the American Academy of Otorhinolaryngology and Head Neck Surgery criteria2,22.
Patients with history of treatment with immune suppressor or anti-inflammatory corticosteroids within the last three months, burns, diabetes mellitus, Alzheimer disease, ulcerative colitis, cancer, fever or recent infection by influenza, were excluded from the study as these conditions alter the values of the HSP7023. The control group encompassed patients who were seen at the Otology Clinics of the Otorhinolaryngology Department at the Clinics Hospital of Porto Alegre with diagnosis of other IED, without criteria to MD. Clinical investigation in both groups included anamneses, otorhinolaryngological examination, otoneurological examination, etiological search examination, auto-antibodies' research via ELISA and WB methods, imaging examinations in instances of unilateral hearing loss. During testing with immunological methods we counted on the cooperation of a nurse technician, who drew the 5 ml blood samples of each patient from both groups, following duly guidance provided by the researcher regarding the completion of the informed consent form. In addition to sex and age, the clinical symptoms, such as characteristics of the vertigo, hearing loss, presence of aural plenitude, tinnitus, family history, duration of the disease, laterality and activity of the disease were evaluated. The time elapsed from the most recent vertigo episode or the hearing fluctuation was evaluated and the disease was considered to be active whenever there had been an episode of vertigo or hearing fluctuation within the 30 day period prior to the drawing of blood samples24.
The otorhinolaryngological examination consisted of a review of the clinical history to detect the presence, previous or current, of relevant clinical manifestations. Physical examination was characterized by inspection and handling of the head and neck, otoscopy, anterior rhinoscopy and acumetry.
Imaging examinations, such as computed tomography with contrast and, if necessary, the magnetic resonance imaging (MRI) were performed to exclude lesions on the VIII cranial nerve in patients who presented unilateral SNHL or involvement of the central nervous system and, also, in those who either presented results of the audiometric tests of the brain stem or presented clinical data suggesting retro-cochlear involvement.
The audiologic characteristics were established according to the conventional tonal and vocal audiometry with the determination of the aerial-osseous thresholds via an acoustic cabin and an AD 17 model audiometer of the Interacoustics brand. Still, the immitance audiometry was conducted, including the study of tympanometry, acoustic reflexes and of the static complacence, using an AZ 26 immitance audiometry of the Interacoustics brand. The study group patients underwent a glycerol test, which was considered to be positive whenever there was either a 10dB improvement in three frequencies (500, 1000 e 2000 Hz) or an increase of 8 % of the hearing discrimination (data not shown). ELISA and WB tests results were analyzed considering time of the onset of the symptoms, presence of unilateral or bilateral disease, and age and disease activity.
Five ml of blood were drawn from each patient, and from these 2ml were placed in a non heparinazed tube for coagulation and to secure serum; the remaining blood was transferred to heparinazed tubes.
The serum was initially kept at 4oC for auto-antibodies' analyzes, azida at 0.01% being added to prevent the growth of microorganisms. The HSP70 of human origin was produced at the Laboratory of Immunorheumathology of the Biomedical Research Institute of PUCRS in a recombinant fashion. As there may be a difference in the reactivity of patients to the protein made in human cells as opposed to a recombinant synthesized in bacteria, we tested a second strategy using cellular extracts of mammal lineage bacteria, such as, for example, the K562, a lineage of human myelocytic leukemia before and after a heat-shock at 42oC followed by two hours of recuperation at 37oC. On the membrane that was incubated with the patients' serum and with the positive control of WB, was used a monoclonal antibody of Sigma, that recognizes the HSP as well as the HSC70. We tested the patients, also, with the method of ELISA that we developed, using a HSP70 protein.
Polystyrene plaques of ELISA, with 96 wells were sensitized with human HSP70, blocked with PBS 5% of milk and incubated with different serum dilutions of the tested subjects, allowing specific antibodies to link to the antigen. From the 64 MD patients invited to participate in the study, 15 did not show up for the medical evaluation. Anti-HSP70 by ELISA had a sensibility of 12,9% (CI 95% 4,2 - 30,8%), specificity of 75,6% (CI 95% 64,4 - 84,3%), positive predictive value of 17,4% (CI 95% 5,7 - 39,5%) and the negative predictive value of 68,6% (CI 95% 57,6 - 77,9%) for diagnosis of MD. Clinical diagnosis of MD was considered the golden- standard and based on it we compared the results of the tests ELISA and Western Blot. Etiological diagnosis of MD was metabolic (n=24), otosclerosis (n=1), otitis media (n=3), chronic otitis media associated with carbohydrate metabolism disorder (n=2), and undefined etiology (n=5). In the control group, etiological diagnosis of inner ear disease were considered unknown in 32 (41%), sudden hearing loss in 14 (18%), presbycusis in 11 (14%), otosclerosis in 12 (15%), and other causes in 9 (12%) patients. The 5 patients with MD and undefined etiology presented negative serology in both WB and ELISA tests. Among MD patients, Globular Sedimentation Rate (GSR) and specific immunity tests (ANA, RF, C3, C4) were normal, except in 1 (3%) patient who presented an elevated C3. One patient with a positive ELISA presented active disease, while 3 patients with positive results presented inactive disease. The 4 patients of the study group with ELISA positive results and the 8 WB positive from study group do not present alterations in the inflammatory activity examinations. Considering the choice of the control group, Soliman suggested that upon conducting a study to evaluate the validity of auto-immunity tests of IE, controls with otological pathologies would be more appropriated than healthy patients; not only hearing-loss patients, but also patients with idiopathic hearing loss not suggestive of immunomediated etiology.
Regarding clinical characteristics of the evaluated groups, we did not observe statistical significant association between the higher positive results in the tests of anti-HSP70 and younger patients, such as Garcia Berrocal et al.31 found in a prior study. In the control, group the bilateral hearing loss was predominant, differently from the study group, in which the unilateral vestibulocochlear disease was more frequent. The most prevalent etiology in patients with MD was the carbohydrate metabolism disorder, data in accordance with some studies that were published32-34. Our study did not find association between MD with unknown etiology and the presence of auto-antibodies. Among MD patients there was no association between the results of inflammatory activity examinations and the anti-HSP70. Hirose and cols35, 36 evaluated the usefulness of laboratorial tests in the IED of probable immunomediated etiology. Our study demonstrated that the request for specific examinations upon the routine of the etiologic investigation without symptoms of auto-immune disease was not useful. In our MD group there was no association between the presence of auto-antibodies (ELISA and WB), disease activity and bilateral disease. The test of the anti-HSP 70 by WB was conducted at the Immunorheumathology laboratory of PUC-RS and some issues were observed. The major issue of the WB conducted in the tracking of the IED is that it generally uses a whole cellular lysate potentially leading to false positive results that recognize other proteins of 70 KD, which are not actually the HSP70. A third, and even more important issue, is the antigen's lack of purity associated to the fact that the serum of both patient and control presents antibodies reactive to additional antigen present in the raw extract, such as bovine albumin that has a molecular weigh slightly lower than 68 kDa, frequently distorting this region of the Blot38,39. The ELISA29 test is semi-quantitative and therefore more appropriate to identify positive and negative results.
In a study published by Harris and cols, anti-HSP70 by WB presented a sensibility of 42%, a specificity of 90% and positive predictive value of 91% among patients with positive response to corticoids, as they fulfilled the criteria for immunomediated IED and improved their condition using the immune suppressor treatment.
Rahman et al.18 report that about one third of the patients with MD presented positive anti-HSP-70 autoantibodies, fact also reported by other authors.
In our study, in the MD patients, the sensibility and positive predictive value of the tests were low, while the specificity and negative predicted value were high.
The role of the tests is not clear as there was small or no link between the activities of the human HSP in serum of patients with MD. The presence of anti-HSP 70 by ELISA and WB methods did not show clinical usefulness in the diagnosis of the MD.
Luiz Lavinsky, MD, PhD Otorhinolaryngology Service, Hospital de Clinicas de Porto Alegre Rua Ramiro Barcelos, 2350 Porto Alegre, RS, Brazil. How to cite this article Mahmoudian S, Shahmiri E, Rouzbahani M, Jafari Z, Keyhani MR, Rahimi F, Mahmoudian G, et al. Tinnitus is a debilitating condition that is widespread yet difficult to successfully diagnose and treat.
Regarding to McFadden (1982), Tinnitus is the "conscious experience of a sound that originates in an involuntary manner in the head of its owner or may appear to him to be so"1.
Some underlying causes were defined for tinnitus, including otological diseases, tumors, ototoxicity, acoustic trauma, noise-induced and psychological disturbances. Since almost the majority of tinnitus questionnaires for assessing individual's reaction to tinnitus are in English language and there is still no standardized and valid questionnaire in Persian, the provision of a Persian version of these questionnaires according to the culture of Iranian society is inevitable.
The first aim of the current study was to investigate the linguistic validity of the Persian Version of the THI as a measure of tinnitus-related reactions in three dimensions 95 International Tinnitus Journal, Vol. The present study consists of translation and standardization of the original version of THI [25] into Persian version (Linguistic validation) then the reliability of the Persian version of THI has been identified. In the first step according to the IQOLA protocol26, THI questionnaire was translated by two native speakers of Persian, bilingual in English and Persian, separately translated the original instrument into Farsi (Persian) language (translator 1 and 2). At the second step of linguistic validation, we used two other translators (5 and 6) with high level of proficiency in Persian and English languages.
Before the THI-P can be administered to assess the effects of tinnitus, its test-retest stability must be determined.
The degree of hearing loss is defined as the severity of the hearing loss measured by audiometry. Patients were asked to complete the THI-P questionnaire as part of the initial assessment at the tinnitus center. All analyses were performed using statistical package for social science (SPSS) version 16. Student t-test was used to test for differences between THI scores and its subscales and gender, hearing impairments and tinnitus laterality. From the 25 items of THI-US version translated to Persian version of THI, thirteen items (52%) including 2, 3, 7, 8, 9, 10, 11, 15, 16, 20, 21 and 22 as an easy translation, six items (24%) including 6, 12, 14, 17, 18 and 19 as a relatively easy translation and finally other six items including 4, 5, 13, 23, 24 and 25 as a difficult translation were evaluated by translators.
The tinnitus characteristics of the 112 subjects attending the tinnitus center to consult about tinnitus management have been summarized in Table 1. The frequency distribution of tinnitus subjects who answered the various items in the THI-P questionnaire has been outlined in Table 3.
The correlation of age and total THI-P score was calculated by Pearson's product and for calculating the effect of sex and total THI-P score we applied independent student t-test.
Pearson product-moment correlations were calculated between the first and second administrations of the THI-P among the tinnitus subjects to assess the strength of the relationship between tests and retest (Figure 2). We assessed the percentages of "Yes", "Sometimes" and "No" answers and then we calculated the reproducibility (internal consistency reliability) of items in THI-P using the Cronbach's-alpha coefficient.
In recent years, the need to assess and consider the consequences of diseases as well as distressing symptoms has been specifically considered in all special facilities of medical and rehabilitation. Hence, as a first step, standardization and validation of appropriate tools such as psychometric questionnaires are very important in order to evaluate disease or monitor treatment outcomes. One of our ultimate goals for validating the Persian version of THI and for conducting current study was to develop a standard self-report tinnitus instrument, appropriate to the Persian's culture so that it could be administered as a criterion measure in clinical and research approaches available to clinicians and other researchers who are involved in different aspects of tinnitus subjects. It is widely agreed that in addition to desirable translation to validate an assessment tool, we should also use the terms adapted for socio-cultural setting of the population which will serve.
The translation quality was assessed by translator 3 and 4 using a VAS criterion (from desirable up to absolute undesirable score). In order to determine the divergent or discriminate validity of THI-P, we used the effects of three factors including hearing impairment, gender and age of the tinnitus subjects on the questionnaire scores. Regarding to other studies, in order to identify validity of THI questionnaires (German, Brazilian, Danish versions), authors administered the correlation between items in translated THI with the other standardized questionnaires (convergent validity structure)33.
The most important specifications of one assessing instrument are always including repeatability, consistency and validity of obtained scores.
The other part of consistency was related to the correlation between test-retest which was evaluated by Pearson correlation coefficient. In this study, with omitting any of the THI-P items, the acquired score of Cronbach's alpha coefficient as calculated by the total score of Cronbach's alpha coefficient was very close (r = 0.943).
In conclusion, this study demonstrated that the Persian version of THI (THI-P) is a valid and reproducible tool to assess the psychological complaints of tinnitus sufferers.
We find that THI-P in structural and practical point of view is compatible with the original English version of THI. In this era, the determination of both objective and subjective effects of clinical intervention is critical, because clinicians using outcome measures could make decision about patient's status and the effective treatment. Objective: The aim was to evaluate the oxidative stress and the angiitis in patients with BPPV.
The pathogenesis of the benign paroxysmal positional vertigo (BPPV) is a vestibular lesion like canalolithiasis or cupulolithiasis. It is known that oxidative stress play some role in some of inner ear disease like sensorineural hearing loss due to aging. We postulated that ischemia of the vestibular organ followed by blood recirculation may provoke oxidative stress.
As a treatment for BPPV, the patients were instructed to perform the Brandt - Daroff exercise11 at home by themselves three times a day. The prognosis of BPPV was evaluated as the time until the disappearance of positional nystagmus. Patients who required longer time for the disappearance of positional nystagmus showed high VCAM level, whereas those who required shorter time for remission showed low VCAM1 level. Patients who required longer time for the disappearance of positional nystagmus showed high d-ROM level, whereas those who required shorter correlation between the value of d-ROM and time until remission (Figure 2).
The ischemia of the vestibular organ followed by blood recirculation may provoke generation of cytotoxic radical and produce damage of phospholipids in cytomembrane and result in elevated oxidative stress. In the previous report, we presented that free oxygen radicals, as measured in peripheral blood samples by the d-ROM test, were relatively enhanced in BPPV patients13.
Based on these results, it is reasonable to assume that the angiitis may contribute to the onset of benign paroxysmal positional vertigo (BPPV). In conclusion, the relation between both the values of d-ROMs and of VCAM-1 and the duration of BPPV was found in patients with BPPV.
In summary, it is reasonable to assume that the severe the angiitis, the longer the time required for the remission of vertigo of BPPV.
Controlled study of the frequency of anti-HSP 70 with the ELISA and the Western Blot methods in patients with Meniere Disease. Sensibility, specificity and predictive values of anti-HSP70 test in diagnosis of MD were calculated. He identified and proved that the disease was a result of alterations in the homeostasis of the inner ear (IE) causing sensorineural hearing loss (SNHL), vertigo episodes from twenty minutes to hours of duration and tinnitus in the absence of neurological symptoms or sequel, the patient remaining oriented and conscious1,2.
It is a histopathologic term employed in the description of an alteration of the IE arising from the progressive distension of the endolymphatic space, especially of the cochlear and saccule duct, though the utricle and the ampulla of the semi-circular channels may also be involved4-6. As well as the MD, some other inner ear diseases (IED), such: sudden hearing loss and progressive hearing loss10, remain frequently with unknown etiology11. The difficulty to establish an assured diagnosis of the immunomediated disease remains, as it is not possible to collect patient's cochlear tissue samples. These are exacerbated by physical and psychological stresses, an observation also valid for the MD19,20.
We compared serological findings of patients with MD with a group of patients with non Meniere's disease IED. The study encompassed the above mentioned analyses in a study group of MD patients, according to the criteria defined by the American Academy of Otorhinolaryngology and Head Neck Surgery2,22, compared to the control group of IED patients without criteria for the MD.
The patients, then, received orientation about the objectives of the study and were free to participate or not in it, with no changes or ties attached to their regular treatment plan at the hospital.
In the study group patients presenting evidence of compromise of the central nervous system, such as VIII cranial nerve tumor and evidence of other clinical entities that may mime the clinical manifestation of MD (Meniere-like) were excluded. The duration of the disease was defined as the moment of onset of the patient's symptoms, which fulfilled the criteria for the MD2,22. A standard protocol was used for all patients encompassing: general otorhinolaryngological, audiologic and otoneurological examination. Globular Sedimentation Rate (GSR), an unspecific examination to verify the inflammatory activity, examinations to evaluate the specific immunity, anti-nuclear antibody (ANA), rheumatoid factor (RF), seric complement 3 and 4 dosage (C3 and C4) and research for metabolic alterations via a lipid profile (total cholesterol and fractions, triglycerides) and the glycemic and insulinemic curves after fasting and following 100 g of glucose and in 60, 90, 120, 180, 240 and 300 minutes were evaluated27(data not shown). The study was double-blind: the lab team tested serum without the knowledge of the group of referred patients and the medical team managed the patients according to the clinical diagnosis, without knowledge of the results of the tests conducted in the Laboratory of Immunorheumathology of the Biomedical Research Institute of PUCRS. Following the procedure, the remaining blood was diluted in a RPMI (Sigma) medium and centrifuged over 1077 (Sigma) histopaque to set aside circulating cells, which were frozen at -80 oC. The HSC70 is present at a larger concentration in cells before the heat-shock and the HSP70 I is present in cells that were subjected to the 42oC.


Both purified proteins, commercially secure, and cultivated cells, as described above, were placed in solution in a tampon of samples of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and applied onto a gel of SDS-PAGE 12%. The reaction was revealed with a solution of DAB, H2O2 and PBS (Phosphate Buffered Saline). The method of ELISA detects the presence of antibody in serum with the aid of an enzymatic reaction.
After an incubation period, the plaque was washed to retrieve antibodies that had not linked. To verify the association among categorical variables, Chi-square and Fisher's exact tests were used whenever appropriate.
Among the remaining 49 individuals, 18 were excluded as they did not fulfill the standard criteria for MD. Anti-HSP70 by Western Blot presented the sensibility of 25,8% (CI 95% 12,5 - 44,9%), the specificity of 82,1% (CI 95% 71,4 - 89,5%), positive predictive value of 36,4% (CI 95% 18,0 - 59,2%) and negative predictive value of 73,6% (CI 95% 62,8 - 82,2%) for diagnosis of MD, as presented in Table 2. Patients with the diagnosis of chronic otitis media did not present either suppuration or infection of the middle ear for over three months. Among the 4 patients who presented ELISA positive results, disease was unilateral and carbohydrate metabolism disorder was the etiology. A positive WB result was found in 2 patients with active disease, and in 6 patients with inactive disease. These patients had the following etiologic diagnosis of the 10 patients: otosclerosis (n=1), bilateral SNHL of unknown etiology (n=3), and sudden deafness (n=6). Authors concluded that GSR was able to detect the presence of acute inflammatory activity of immunomediated IED.
Even though the laboratorial tests may aid investigation of the immunomediated IED, the diagnosis still is based upon the improvement of the symptoms as a response to corticosteroids, which is the golden-standard considering the lack of a test with these characteristics37. No significant difference was found when comparing ELISA and WB results, disease activity and duration of disease among study group and control group patients. The first issue was that the test was positive for some of the patients when tested with a whole cellular lysate and negative when the antigen used was purified.
In a previous study from our group, we verified that the amount of HSP70 present is crucial in the WB and that high amounts of HSP70, even in normal patients, may lead to a positive test. The solution for the issue of antigen's impurity would be the conduction of tests with recombinant protein.
Also some auto-immune diseases are associated to auto-antibodies that are specific for organs and tissues, consequently it may be more difficult to establish the significance of the auto-antibodies' positivism in the etiology of the disease.
However, according to the author, patients with negative results in the WB may also benefit from corticoids. These characteristics would be useful for the confirmation of diagnosis in a diagnostic test.
The prognostic and therapeutic value of anti-HSP70 auto-antibodies in the IED, including MD, was not yet established.
We did not find association between idiopathic MD nor unspecific etiology MD and the presence of anti- HSP70 auto-antibodies. Committee on Hearing and Equilibrium guidelines for the diagnosis and evaluation of therapy in Meniere's disease. Otorhinolaryngology Service, Hospital de Clinicas de Porto Alegre (HCPA), Porto Alegre, Brazil.
Immunorheumathology Laboratory, Instituto de Pesquisas Biomedicas, Pontificia Universidade Catolica do Rio Grande do Sul (PUCRS), Porto Alegre, Brazil. Persian language version of the "Tinnitus Handicap Inventory": translation, standardization, validity and reliability. This symptom is a debilitating condition that is widespread yet difficult to successfully diagnose and treat. For some patients with tinnitus this sensation does not interfere with their daily activities and for others it is disabling and restricts their work, sleep, and social activities10,11. Numerous studies using various open-ended or self-report structured questionnaires have been conducted.
For this reason we perform according to the International Quality of Life Assessment protocol (IQOLA) approved by WHO in 1991.
Craig Newman, author of the original questionnaire of THI, has permitted us to develop and use the Tinnitus Handicap Inventory in Persian Language Version. They were asked, if needed, to mention a list of possible translations for some words, sentences and phrases. On the other hand, the words and items on the level of reading ability of 14 years old or less were noticed for linguistic validation of THI-P. All subjects gave written informed consent in accord with the declaration of Helsinki before examinations. So, in retest after one week from filling the first THI-P, the questionnaire was filled again by the same patients. Descriptive statistics, including frequency distributions, means and standard deviation were computed for each item of THI-P. Multiple linear regression analysis was used to study the effect of gender, hearing loss and the tinnitus laterality on the overall THI score as well as the functional, emotional and catastrophic subscales.
In this study, the research method and the role of subjects were explained clearly and completely.
The translation clarity exception in one item (item 17) which was evaluated as relatively desirable, in the other 24 items (96%) of THI-P was evaluated as desirable.
Most of tinnitus subjects (72.7%) perceived one type of tinnitus sound, while others complained of sensation of more than one type of sound on different occasions.
Scatter plots illustration the relationship between the THI-P score and the age in all subscales.
As shown in Figure 2, the scatter plot indicates the correlation between the first (test) and second (retest) administration of the THI-P and their subscales on tinnitus subjects. Scatter plots illustration the correlation between the first (test) and second (retest) administration of the THI-P and their subscales. Researches in many different countries around the world in this area indicate the global emphasis on the importance of these assessments. This symptom is as a common problem which can affect several aspects of patients' life including: going to sleep, understanding speech, depression, irritation, concentration and worse on awakening.
To acquire this expressed goal, it was necessary to translate and standardize the original version of THI and then to evaluate the test-retest reliability of the THI-P. The measure of difficulty of Persian translation of THI was evaluated by translator 1 and 2 using a visual analogue scale from easy up to difficult (VAS; 100 mm total length).
The measure of this criterion administers three measures including clarity scores, common language and identifies conceptual equivalence. Both convergent and discriminate validity methods were used for identifying validity of original English version of THI19. In current research the reliability of THI-P was evaluated from two 102 International Tinnitus Journal, Vol. We obtained 0.96 in Persian THI version, consistent with significant correlation in retest. The findings showed that none of the items of the Persian version of the questionnaire need to be removed. Also, THI-P could be administered as a standard measuring instrument for patients and clinicians all over Iran.
Henceforth, THI-P could be used for both clinical and research purposes by different professionals like audiologists, otolaryngologists, psychiatrists and could be also used for monitoring, treatment and rehabilitative effects on the life's quality of tinnitus sufferers. ENT and Head & Neck Research Center, Hazrat Rasoul Akram Hospital, Tehran University of Medical Sciences (TUMS), Tehran, Iran. Department of Audiology, School of Rehabilitation Sciences, Tehran University of Medical Sciences (TUMS). Department of Biometry, School of Rehabilitation Sciences, Tehran University of Medical Sciences (TUMS).
The possible contribution of angiitis to the onset of benign paroxysmal positional vertigo (BPPV). Method: Patients with benign paroxysmal positional vertigo (BPPV) within 14 days of onset were analyzed. Physical examination was performed for the evaluation of vertigo including pure tone audiometry and vestibular examination including positional nystagmus.
Patients are asked to visit each week for evaluation (positional nystagmus) by a physician to confirm the cure of BPPV. Patients who required longer time for the disappearance of positional nystagmus showed high d-ROM level, whereas those who required shorter time for remission showed low d-ROM level. Some interfering factors must be considered when the D-ROM test is used because several vascular diseases, the use of oral contraceptives, and a state of fatigue after physical exercises have been shown to increase U.
It may be useful to control dizziness or vertigo by reducing free radicals as a product of oxidative stress. The diagnostic value could be such that enhanced values of oxidative stress would be a marker for BPPV and provide additional information for a diagnosis of BPPV.
It is considered that the angiitis and following oxidative stress can be one of the causes of BPPV. Morimoto K, Morikawa M, Kimura H, Ishii N, Takamata A, Hara Y, Uji M, Yoshida K, Ohta Y, Chiba S, Tada M, Imai Y, Kitagawa A. Cesarone MR, Belcaro G, Carratelli M, Cornelli U, De Sanctis MT, Incandela L, Barsotti A, Terranova R, Nicolaides A.
The disease carrying his name presents variations in its clinical presentation, characterized by remissions and exacerbations, leading to a difficult and many times frustrating disease management3. When the cause of the hydrops can not be identified we name it MD, whereas cases with symptoms that may be related to a known cause are named Meniere's Syndrome7,8. It is possible that a hidden immunological reaction may be the cause of these diseases8,12,13.
A recent study of Yimtae and colleagues has found evidences of an IE's drainage through the medium ear to cervical lymph nodes. Sensibility, specificity and predictive values of anti-HSP 70 test in diagnosis of MD, by both ELISA and Western Blot methods, were calculated. The cells were cultivated in a RPMI medium with 10% of bovine fetal serum at 37oC with 5% CO2. Following the separation of gel proteins, they were transferred to a nitrocellulose membrane.
The visualization of one band allows the determination of the presence of specific antibodies to a protein extract antigen. To detect antibodies linked to antigen a conjugated (antiimoglobulin antibody chemically linked to an enzyme) was applied. Among MD patients, three individuals (10%) presented positive results for both tests and 22 (71%) presented negative results in both tests.
Among the 8 patients with positive WB results, 1 patient was diagnosed as idiopathic MD, and other 7 as carbohydrate metabolism disorder etiology. Among these GSR altered patients, 5 presented positive results in one or both anti-HSP70 methods. It is believed that the bilateral disease could be associated to the systemic processes, thus one could expect a more prevalent presence of auto-antibodies in the bilateral disease.
The 5 patients of the MD group with idiopathic etiology, presented negative serology in the WB and in the ELISA. The longer duration of disease and high prevalence of inactive disease could explain the low level of positive auto-antibodies.
The second relevant issue was that if large quantities of purified HSP70 were used, both the patients and the controls would be positive.
Results suggest that the WB is not a suitable method to identify auto-antibodies for the HSP70 and that ELISA is a more trustworthy alternative for diagnosis25. However, patients in this study were treated less than two weeks from the onset of symptoms, a period during which the disease was in activity11.
However, we are not able to make such statement regarding the test of the anti- HSP70 via ELISA and WB in patients with MD, as we did not find significance in the statistical analysis. Only prospective studies with a larger number of patients of different subgroups of the MD will allow the determination of if such origin of disease's subgroups is based upon different pathogenic mechanisms and if there is really participation of the immune response in this process. The cause (multifactorial inheritance) and pathogenesis (endolymphatic malabsorption) of Meniere's disease and its symptoms (mechanical and chemical). Committee on Hearing and Equilibrium guidelines for reporting treatment results in Meniere's disease.
Da frequencia nistagmica a prova calorica pendular decrescente, estudo eletronistagmografico em individuos normais. Validity of the Western blot immunoassay for heat shock protein-70 in associated and isolated immunorelated inner ear disease. Autoimmune sensorineural hearing loss as aggravatin factor in Meniere's: a preliminary report. Comparison of antiheat shock protein 70 (anti-hsp70) and anti-68-kDa inner ear protein in the sera of patients with Meniere's disease. Objectives: The aim of current study was to compose and validate a Persian version of the Tinnitus Handicap Inventory (THI-P).
The prevalence of chronic tinnitus in the general population is estimated between 5-15% and causes to develop serious psychosocial complications in 1% to 3%1. It is reported that tinnitus can be severe enough to cause significant discomfort in one's daily life12.
Finding the effects of tinnitus on the quality of life and assessing the individual's reaction to tinnitus are very important. The aim of IQOLA is to translate and provide health questionnaires with high validity and reliability for using in different countries by their formal language26. This version is both simple to administer and interpret and also a powerful psychometric tool. After that, each of the expert translators were asked about the difficulties of the translations and they were attributed a score according to the 100 point visual analogue scale (VAS).
Afterward, the pooled back translation was compared and formed by translators and then an agreement was obtained for one English version of THI questionnaire for finding the coherency with original version.
Therefore the formulated initial Persian version of the instrument was attended by 24 (10 female and 14 male) high school students in Tehran.
The Cronbach's Coefficient Alpha test (where values>0.70 indicate a satisfactory internal consistency) was used to test for the evaluating internal stability of the THI-P29, and Pearson correlation coefficient was calculated between the total score of the THI-P and the scores of its subscales, age and pure tone average (PTA).
The common language score of THI-P was assessed as desirable in all 25 items (100%) and also the conceptual equivalence for three items (4, 10, 16) undesirable response (12%), for two items (23 and 14) in a relatively desirable level (8%), and the other items were desirable response (80%). The type of sound reported by most tinnitus subjects was subjectively described as high-pitch tone, matched by external stimuli (described by 52.3% of subjects).
Table 2 summarized the mean air conduction thresholds across frequencies of 500-4000 Hz for left and right ears. As shown in Figure 1, the total score of THI-P and its subscales were not correlated with the age of tinnitus subjects. Total score of the THI-P and its associated subscales are very close to corresponding values in the original THI-US (Table 6).
In Iran, in order to converge and coordinate with the ongoing research at the international level concerning the evaluation of consequences of diseases, impairments, handicaps, treatments and rehabilitation, we should create a new movement.
Sense of the various problems among the subject sufferer from tinnitus and understanding the impact of tinnitus on their quality of life are of the uttermost importance.
Translation of the original questionnaire of THI in Persian language in overall was easy due to its simple sentences, whereas, the translators in six items acknowledged difficult translation (items of 4, 5, 13, 23, 24 and 25).
The translation clarity in 24 items of THI-P was evaluated desirable and only one item (item 17) evaluated as relatively desirable. Majority of epidemiological tinnitus studies have reported a positive relationship between tinnitus and hearing loss4,34-35. This valid tool allows us to quantify how much tinnitus impacts the life's quality of these individuals.
Functional brain abnormalities localized in 55 chronic tinnitus patients: fusion of SPECT coincidence imaging and MRI.
Prevalence of tinnitus and hearing thresholds of a non-noise-exposed population with and without tinnitus. Assessment of psychopathological aspects and psychiatric comorbidities in patients affected by tinnitus. Psychologic profile of tinnitus patients using the SCL-90-R and tinnitus handicap inventory. Psychological profile and somatic complaints between help-seeking and non-help-seeking tinnitus subjects.
Tinnitus reaction questionnaire: psychometric properties of a measure of distress associated with tinnitus. Developmental and Psychometric Adequacy of the Screening Version of the Tinnitus Handicap Inventory. Psychometric Adequacy of the Tinnitus Handicap Inventory (THI) for Evaluating Treatment Outcome.


Tinnitus with normal hearing sensitivity: extended high-frequency audiometry and auditory nerve brain-stem-evoked responses. Validity of the Italian adaptation of the Tinnitus Handicap Inventory; focus on quality of life and psychological distress in tinnitus-sufferers. Brazilian Portuguese Language version of the "Tinnitus Handicap Inventory": Validity and Reproducibility.
Impact of tinnitus as measured by the Tinnitus Handicap Inventory among tinnitus sufferers in Singapore. The level of diacron reactive oxygen metabolites (d-ROM) and circulating soluble vascular cell adhesion molecule 1 (VCAM-1), were evaluated. The incidence of BPPV increases with each decade of life2, peaking at the sixth to seventh decade of life1. The diagnosis of BPPV was performed by the typical positional nystagmus evoked by Dix-Hallpike procedure for PC-BPPV. ROMs were measured in blood samples by the Diacron reactive oxygen metabolite (D-ROM) test.
The relation between these time periods and either initial VCAM-1 or d-ROM levels were evaluated.
Patients who required longer time for the disappearance of positional nystagmus showed high VCAM level, whereas those who required shorter time for remission showed low VCAM-1 level. Benign positional vertigo: incidence and prognosis in a population-based study in Olmsted County, Minnesota. Reactive oxygen metabolites (ROMs) as an index of oxidative stress in obstructive sleep apnea patients. The relevance of an elevation in the plasma vasopressin levels to the pathogenesis of Meniere's attack. Mental stress induces sustained elevation of blood pressure and lipid peroxidation in postmenopausal women. Development of oxidative stress and cell damage in the liver of rats with water-immersion restraint stress. Soluble intercellular adhesion molecule 1 and soluble vascular cell adhesion molecule 1 in sudden hearing loss. Oxidative stress and Meniere's disease and benign paroxysmal positional vertigo (in Japanese). Evaluation of an automated spectrophotometric assay for reactive oxygen metabolites in serum. Methods: Blood samples were collected from 31 patients with MD and 78 patients with non Meniere IED. There are evidences that the endolymphatic saccule has lymphatic vases that may function as a drainage path to regional lymph nodes15. We purified the HSP70 of the cellular extract of K562, using the same chromatography method of ATP-agarose used to purify the recombinant. This phase was completed via the overlapping of the membrane to the gel and an electric current of 150 volts was applied for 1 hour. Again, after an incubation period the plaque was washed to retrieve the conjugated that were not linked.
In the instance of the comparison of the evolutions' time variable (ordinal) the non-parametric test of Mann-Whitney was applied. There was no association between unspecific immunity tests and the anti-HSP70 results (Fisher's exact test p = 0.7 for ELISA versus GSR). Among MD patients, the floating progressive hearing loss was present in 71%, data similar to the one found in the literature16. Such was not expected as it was believed that these patients with disease of unknown etiology could present an immunomediated mechanism with a causal factor for the disease. Another issue is the denaturation of the protein, which affects the recognition by the patient's serum29. We do not know if IED is a manifestation of the immune response against the increased production of HSP70 or if the antibodies could be an epi-phenomena of the disease that may be associated to other causal factors such as: prior surgery, viral infection37,39,41 . Materials and Methods: The linguistic validation of the original version of THI into Persian version (THI-P) included translation, back translation and data gathering. Epidemiological studies indicated that in developed countries such as Germany about 1.5 million people, in the United States about 10 million out of 40 million tinnitus sufferers and a similar ratio in the United Kingdom have been reported that were seriously affected by tinnitus2-4. Tinnitus symptoms have been linked to various psychological and psychosomatic disorders13-15.
One of the most important methods in the assessment of the severity of tinnitus and its effects on the lifestyle of a patient is the use of psychometrical evaluations. Finally, we formulated the initial Persian version of the instrument with proper and satisfactory translation.
The final version of THI-P was administered in all tinnitus subjects who were managed in our center between September 2009 and May 2010.
The score of "Yes", "No" and "Occasionally" were 4, 0 and 2, respectively in three subscales: emotional, functional and catastrophic.
All personal information in the questionnaire were confidential and only used in this research.
The other descriptions of tinnitus included cricket, sizzling, hissing, humming and other sounds. Tinnitus is a subjective idiopathic symptom in which it is not detected by blood examination and CT scan or like this. In this regard, translator 1 and 2 were asked to provide a list of possible alternative translations for the replacement in relatively difficult and difficult items of THI-P. The prevalence of tinnitus among normal hearing subjects has been reported between 7%-10%36-37. As a treatment the patients were taught to perform the Brandt-Daroff exercise at home by themselves. BPPV is a mechanical problem of the inner ear caused by abnormal stimulation of a semicircular canal.
Radical scavengers and antioxidants play a role protecting the inner ear from oxidative stress.
The elevated oxidative stress can provoke damage in vestibular organ (especially otolith dysfunction). AS for HC-BPPV characteristic direction alternating horizontal nystagmus during right lateral and left lateral position in lying down were examined.
This test is based on the ability of transition metals to catalyze in the presence of peroxides, the formation of free radicals, which are trapped by an alchilamine.
Inflammation is known to play an important role as cause of various angiitis such as atherosclerosis. It is reported that the D-ROM test is a simple, reliable and inexpensive method for the measurement of endogenous hydroperoxides.
The proteins were transferred from the gel to the membrane of nitrocellulose after 2 hours at 100 volts.
The agreement between the ELISA and WB tests was verified by the McNemar's Kappa and Chi-square tests.
Data regarding aural plenitude, tinnitus and vertigo's characteristics were also similar to the ones found in literature16.
It is shown that 48% to 60% of patients with chronic or disabling tinnitus have major depressive disorder (MDD)16. Such self-report questionnaires include the Tinnitus Questionnaire (TQ)18, Tinnitus Handicap Inventory (THI)19, Tinnitus Severity Scale (TSS)20, Tinnitus Handicap Questionnaire (THQ)21, Subjective Tinnitus Severity Scale22 and Tinnitus Reaction Questionnaire (TRQ)23. The THI is a 25-item self-report instrument which is divided into three subscales including functional, emotional and catastrophic reactions to tinnitus.
On the basis of these two translations and their VAS scores to each THI items, we formed the pooled version that was then reviewed by presence of translators for considering suggested replacing words and difficulty in translation.
This questionnaire was used as an initial instrument for gathering data from tinnitus subjects at next steps. Each single item was read loudly and the participants were asked to follow the items on the given sheet. Age, gender, medical history and tinnitus characteristics were recorded as baseline information. In total, more than 92% of tinnitus subjects who attended the current study revealed high pitch tinnitus, matched by external stimuli (>3000 Hz). Independent Student t-tests did not demonstrate statistically difference between total THI-P score and those with or without hearing impairment, neither did those with unilateral or bilateral tinnitus (Table 4).
The main problem with tinnitus is its subjective nature so that evaluation of this complaint always depends upon patient's words. These alternative items were reviewed again by translator 3 and 4 in order to optimize quality of translation and finally revised for appropriate items. In conceptual equivalence of the translation, three items had undesirable response (4, 10 and 16) and two items had relatively desirable response. The prognosis of BPPV, which is measured as the time until the disappearance of positional nystagmus by a physician during the outpatient visit each week, the relation among the level of oxygen metabolites, vascular molecule and the duration until remission were analyzed.
Most BPPVs are thought to be caused by otoconia detaching from the otolithic membrane and moving to the lowest point of the inner ear, usually the posterior canal (PC).
There are some basic data to indicate the relationship between oxidative stress and endolymphatic hydrops.
The elevated oxidative stress can cause canalolithiasis, which is one of pathogenesis of BPPV as a result of otolith aberration to the canal organ. We postulated that ischemia of the vestibular organ followed by blood recirculation may provoke generation of cytotoxic radicals and produce damage of phospholipids in cytomembrane and result in elevated oxidative stress (Figure 3). Cesarone and colleagues have found that in normal subjects the mean (±SD) levels of free radicals were 312 (±49) U.
There is evidence that the D-ROM assay has both an acceptable stability and an acceptable margin of error14. Results: ELISA tests results were positive in 4(13%) patients and results of WB were positive in 8(26%) patients. The revelation was made with a human anti-IgG conjugated to peroxidase, followed by OPD and the reading was made in a reader of ELISA at 490 nm. In addition, tinnitus is a common symptom of the auditory system disorders that is usually associated with sensoryneural hearing loss6,7. Asplund reported that among subjects with tinnitus poor sleep and frequent waking were more common in men and women17. In this regard the client was questioned in order to find out which activity or performance of the patient is affected by tinnitus. Analysis of the THI items suggested that this self-report instrument had excellent internal consistency reliability and adequate convergent and constructive validity28. It is worth to mention that in this step, for differentiation of the level of difficulty in translation, average scores less than 25 were considered as easy, more than 30 as difficult and between 25 and 30 as relatively easy.
Thus, items with undesirable translation were revised by translator 5 and 6 and revised by suggested replacing proper words, sentences and phrases. As reported in several studies for the original version (US), Italian, Turkish, Danish, Portuguese and Singapore translations of THI19,28,38-41, the Persian version of THI was not significantly different in the handicap scores between those with normal hearing and those with hearing impairment with regard to the degree of handicap severity. Eventually it became clear that internal consistency was high and the response of every item to the response of total items was matched. Results: The patients who required longer time for the disappearance of positional nystagmus showed high d-ROM and VCAM levels, whereas those who required shorter time for remission showed lower d-ROM and VCAM levels. Free oxygen radicals are highly reactive molecules playing pivotal roles in the pathophysiology of such different diseases as neurodegenerative disorders, chronic inflammatory disease, and sleep apnea syndrome4,5. The automatic assay may be regarded as a fast and reproducible method for the quantitative evaluation of oxidative stress. Among patients with positive ELISA results, 1 patient presented active disease and in the remaining 3 patients the disease was inactive. The intensity of the color developed by the substrate was proportional to the specific antibodies for the antigen present in the serum. Calculation of the sensibility, specificity, positive and negative predictive value as well as its respective confidence intervals (95%) were made via Epi Info 6.04d.
All participants complained of chronic unilateral or bilateral subjective idiopathic tinnitus lasting for at least 6 months before consulting about their tinnitus. An epidemiological study in Italy based on a tinnitus questionnaire demonstrated that the prevalence of tinnitus was 8% in normal hearing subjects while this amount rose up to 30.5% in the presence of auditory dysfunctions8. This agreed version of Persian THI translation was given to the two other translators who were expert to Persian philology (translator 3 and 4). Finally, the results of the present investigation suggest that the THI-P was simple and clear, without losing its equivalence towards cultural issues. All values of item-total correlation coefficient are greater than 0.3 which also indicated high internal consistency in THI-P except item 19.
Conclusion: There is an increased expression of VCAM-1 and d-ROM confirming the existence of an angiitis and supporting the vascular involvement in BPPV.
The vestibule connects to the 3 semicircular canals (posterior canal [PC], horizontal canal [HC], and anterior canal). In the previous study diacron reactive oxygen metabolites (d-ROM) was measured by the Free Radical Analytical system 4(FRAS4) from the blood of subjects with BPPV. It is reported that psychological stress is closely related to the onset and course of Meniere's disease6 and BPPV7 Oxidative stress and psychological stress are closely related8, 9.
The increased expression of circulating adhesion molecules confirmed the existence of an endothelial dysfunction and supports the vascular involvement of the disease.
Since it is easily performed, the method is suitable for routine use in clinical laboratories and may provide an estimation of oxidative stress in vivo. Results: There was no significant difference between gender, age, hearing impairment and total score and subscales of THI-P. Therefore, the valid psychometrical evaluations such as THI can be administered as a robust instrument to objectify tinnitus distress, but additional measures might be needed in order to catch the dimensions of tinnitus-related problems.
Inoverall the translation quality of THI-P was evaluated as desirable from both aspects of a careful translation and adapted to the socio-cultural setting. The identification of the high levels of d-ROM and VCAM-1 can open the way to selective pharmacological treatments able to correct the oxidative stress and activation of endothelial cells.
Although there are no statistically significant differences among three groups, the d-ROM value of BPPV and Meniere's disease are higher with respect to control. Based on these considerations, it is hypothesized that BPPV may be linked to increased oxidative stress. Statistical analyses did not establish any association between serologic findings and clinical factors of MD. Pearson product-moment correlations revealed adequate test-retest reliability for the THI-P (r = 0.96). Also the valid questionnaires are valuable in the evaluation of results from before to after special treatment and its effect on the individual's quality of life. The diagnosis of BPPV is based on the patient's history and the findings on positional testing. The purpose of this study was to evaluate free oxygen radicals by a method providing values of reactive oxygen metabolite (d-ROM) concentration and VCAM-1 in blood samples from patients with BPPV. Conclusion: The presence of anti-HSP70 using the ELISA and the WB methods did not demonstrate clinical value for the diagnosis of MD. Therefore, the symptom of tinnitus has forced clinicians to attempt to establish protocols for accuracy of tinnitus diagnosis and treatment9. The Tinnitus Handicap Inventory (THI)25 has gained widespread acceptance as a self-report measure of tinnitus around the world. Therefore, these translators (3 and 4) for each question determined 3 scores on the basis of 100 point criteria. Therefore, the Persian translation of THI was not affected by hearing impairment, gender and age factors.
We did not find association between idiopathic MD nor unspecific etiology MD and the presence of anti-HSP70 auto-antibodies.
In this VAS, zero point means absolutely improper quality and 100 means absolutely proper and satisfactory quality.
After that, the subsequent processing was attended by audiologists from department and research center of otolaryngology of Tehran University of Medical Sciences (TUMS). Once the canal is identified, the patients is treated effectively with an appropriate particle repositioning procedure which is now widely accepted. Criteria for decision making in improper quality of each translation items was the average score less than 90 (determined by translators 3 and 4). Accordingly, in this step, Persian version of the THI was obtained, which regarding to all translators 1 to 4, had satisfactory quality. We should understand the basic pathology why either canalolithiasis or cupulolithiasis occur to invent new pharmacological treatment.



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