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Keywords:

  • Hearing loss;
  • Immune-mediated cochleovestibular disease;
  • Positron emission tomography;
  • Etanercept

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Objective

Immune-mediated cochleovestibular disorders (IMCVDs) continue to present a diagnostic and therapeutic challenge. Antirheumatic agents, commonly employed for IMCVDs, are associated with variable efficacy and sometimes with serious side effects. The objective of the current study was to preliminarily evaluate the efficacy of etanercept therapy for IMCVD.

Methods

In this open-label prospective pilot study, 23 patients with bilateral IMCVDs or symptoms of bilateral Meniere's disease were treated with etanercept (25 mg twice weekly, by subcutaneous injection) for 24 weeks. All participants showed progressive hearing loss within 3 months prior to the study and responded to prednisone therapy. Hearing improvement was defined as an improvement of sensorineural hearing from baseline, in at least one ear, of 15 dB or more in the pure-tone air conduction thresholds, or an increase of more than 12% in word identification score. When present, vertigo and tinnitus were assessed by frequency and severity of attack and a functional level scale. Limited serial positron emission tomography (PET) of the inner ear region was performed in 5 patients to assess disease activity.

Results

There were 12 female (52%) and 11 male patients with a mean age of 48 years. Hearing improved in 7 (30%) patients, was unchanged in 13 (57%), and worsened in 3 (13%). Of 21 patients with tinnitus, this symptom improved in 7 (33%), was unchanged in 10 (48%), and worsened in 3 (13%). Of 16 patients with vertigo, 8 (50%) were improved, 7 (47%) unchanged, and 1 (3%) worse at the end of the study. Etanercept was generally well tolerated. PET was positive on one ear of 2 of 5 patients, remained positive with treatment on 1 of these, and was initially positive in 1 deaf ear, becoming negative at followup.

Conclusion

These prospective pilot data suggest that etanercept therapy is safe among patients with IMCVDs. However, these data do not suggest substantial efficacy of etanercept among patients with IMCVDs in improving hearing loss. There appeared to be stabilization or improvement of hearing in 87% in this group of patients with pretreatment intractable progressive hearing loss. However, the study endpoint of improvement in 70% of patients was not attained. This short-term effect of possible stabilization requires further study. PET scanning was not useful as a tool to evaluate hearing loss in a limited subset of patients.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Immune-mediated cochleovestibular disorders (IMCVDs; often referred to as autoimmune inner ear disease) are clinical syndromes of uncertain pathogenesis. They are often associated with rapidly progressive hearing loss that, especially early in the disease course, may be unilateral but is most commonly bilateral, presenting as sudden deafness or rapidly progressive sensorineural hearing loss (1, 2). The hearing loss may be associated with vestibular symptoms.

A rapid response to corticosteroid therapy has suggested that autoimmune processes cause hearing loss and vestibular symptoms in IMCVD, although the mechanism of disease is poorly understood. Clinically, sudden sensorineural hearing loss has been observed in association with other autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and polyarteritis nodosa (1, 3–5). Cogan's syndrome is accepted as an autoimmune disease, and it is likely that at least some cases of Meniere's disease, especially when bilateral, are autoimmune in nature (6–9).

IMCVDs continue to present a management challenge to specialists caring for these patients. Management involves immunosuppressive agents that are used for rheumatic diseases (10–12). Although administration of corticosteroids and/or conventional second-line immunosuppressive agents can improve or slow the disease process, a substantial number of patients do not achieve durable significant efficacy. Such agents usually include prednisone and methotrexate, although other immunosuppressive agents such as cyclophosphamide are sometimes used. Even with use of these agents, normal hearing is rarely, if ever, restored, and hearing is stabilized in no more than ∼60–65% of patients (13, 14).

Despite initial optimistic reports suggestive of a therapeutic effect of methotrexate, a recent double-blinded, randomized trial failed to demonstrate efficacy of this drug for management of IMCVDs (15). Ultimately, many patients become deaf because of this disorder. Furthermore, these agents have been associated with serious and occasionally life-threatening adverse effects (16). Management of IMCVDs clearly calls for more efficacious and less toxic treatment options.

Etanercept (Enbrel, Amgen, Thousand Oaks, CA) is a fusion protein consisting of 2 recombinant p75 tumor necrosis factor (TNF) receptors linked to the Fc portion of human IgG1 (17). The medication is a powerful antagonist of TNF, binding to and inactivating the cytokine. TNF has been shown to play a critical role in several chronic inflammatory conditions (18–23). Results of a preliminary study of etanercept in 12 patients suggested improvement (in 7 of 12 patients) or stabilization (in 4 of 12 patients) of hearing over a period of 5–12 months (24).

There are no established methods for the assessment of active inflammatory disease in IMCVD. Both detection of an acute-phase reactant, heat-shock protein 70 (HsP70), and positron emission tomography (PET) have been posited as a means for following disease activity in patients with this condition (25, 26).

To evaluate the safety and efficacy of etanercept in the management of IMCVD, we conducted this investigator initiated, open-label, multicenter trial of anti-TNF therapy-naive patients with steroid-responsive disease. We also further preliminarily evaluated the utility of PET for assessing disease activity in IMCVD.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Study population.

A total of 23 adult patients were treated. All patients exhibited a typical pattern of fluctuating, generally bilateral sensorineural hearing loss with or without vertigo and/or symptoms of bilateral Meniere's disease. In addition, all patients had a positive response to prednisone (1 mg/kg/day), which was given for 3 weeks prior to enrollment. A positive response was defined as a >10 dB increase in pure-tone average by air conduction pure-tone audiography.

Patients with a history of a systemic inflammatory disease including rheumatoid arthritis and inflammatory bowel diseases; patients with a history of acute, chronic, recurrent serious infections including tuberculosis; or patients with otologic diseases suggesting a non-IMCVD etiology were excluded from participation. Screening chest radiographs were obtained and were unremarkable in all patients, and tuberculosis skin testing by purified protein derivative was negative in all patients prior to receiving the study drug.

The study was approved by the human studies ethics committees of the 3 participating centers.

Treatment plan.

All patients received etanercept, 25 mg subcutaneously twice weekly by self injection for 24 weeks. This is the usual and available dosage of etanercept used in the treatment of rheumatoid arthritis (11). Standard symptomatic treatments of Meniere's disease, e.g., severe vertigo with diuretics (e.g., hydrochlorothiazide) and sedatives (e.g., lorazepam and clonazepam) were allowed. The prednisone administered to assess steroid responsiveness was tapered off over 2–4 weeks on an individualized schedule, as is usual in the management of this condition.

Outcome measures.

The primary outcome measurement in this trial was assessment in hearing change by air conduction pure-tone audiogram comparing the audiogram results at the time of enrollment with results at week 24. Audiograms were performed at the outset of the study and then every 4 weeks for the next 24 weeks. Hearing improvement was defined as an improvement of sensorineural hearing from baseline, in at least one ear, of >15 dB in the pure-tone air conduction thresholds at any of the 8 frequencies (200, 500, 1,000, 2,000, 3,000, 4,000, 6,000, and 8,000 Hz), or of 10 dB at 2 consecutive frequencies; or an increase of >12% in word identification/recognition (WR) score; and no significant additional pure-tone air conduction threshold loss at any frequency and no significant additional loss in word identification score in either ear.

Vertigo and tinnitus were assessed using questionnaires completed every 4 weeks by the patients. Questions regarding vertigo were scored on a scale of 1–6, where 1 = dizziness has no effect on activities, 2 = some effect on activities, 3 = moderate effect on activities, 4 = moderately severe effect on activities, 5 = severe effect on activities, and 6 = disabled for >1 year due to dizziness. Tinnitus was scored on a 5-point scale where 1 = none, 2 = marked relief, 3 = partial relief, 4 = unchanged, and 5 = worsening. Improvement was a change in at least one point toward 1; worsening was defined as a change in at least 1 point in the opposite direction.

Serum aspartate aminotransferase levels, syphilis serology, serum creatinine levels, chest radiography, levels of glucose, and antineutrophil cytoplasmic antibody (ANCA) were obtained, and computerized tomography (CT) or magnetic resonance imaging (MRI) of the brain was performed at enrollment. Measurements of Westergren's erythrocyte sedimentation rate (ESR) and HsP70 were obtained at weeks 0 and 24.

Five patients underwent PET scanning at baseline and again at month 3 of treatment. Patients with diabetes or elevated fasting blood sugar levels of >150 mg/dl were excluded from the PET portion of the study because this may interfere with the assay. PET images were read and scored as previously described, by a trained radiologist (VJL) who participated in the previous study of this imaging modality in IMCVD (26). PET was performed using a GE Advance whole body PET system capable of 3-dimensional and 2-dimensional imaging, and radiochemistry facility with a 16.5 MeV GE PETtrace cyclotron and a selection of PET drug synthesis units incorporating 18F-fluorodeoxyglucose. Analytical techniques for quality assurance included gas chromatography, high performance liquid chromatography, thin layer chromatography, and molecular chemical analysis. PET scans were interpreted visually and semiquantitatively. The visual interpretation consisted of assessing the PET scans for activity in the inner ear region that is greater than background activity. These observations were then compared among the individual study patients and previously obtained controls to correlate with clinical findings (18). The PET images were also superimposed on MR images (MR fusion) for further anatomic comparison.

The study was designed to have 88% power to detect a therapeutic response in 70% of patients based on intent-to-treat methodology.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Patient characteristics.

A total of 23 patients including 12 women (52%) and 11 men with an average age of 48 years (range 27–76 years) were enrolled in the study. Patients had had hearing loss for an average of 3.4 years (range 3 months to 10 years). IMCVD-related hearing loss was idiopathic in 20 patients (87%), and was attributable to Cogans's syndrome in 3 patients. Five patients (22%) had ever smoked, none were smokers at the time of enrollment, and 2 patients had been smokers at the time of initial disease onset.

Hearing loss was bilateral in 20 of 23 patients, including 5 patients who were functionally deaf at enrollment in one ear but had active disease in the other ear (1 with Cogan's syndrome, 4 with idiopathic IMCVD). Hearing was normal in one ear of 3 patients at enrollment. Prior to enrollment, all patients had ongoing episodic worsening of hearing in one or both ears despite traditional medical therapy, including corticosteroids. At enrollment, disequilibrium/vertigo in 16 of 23 patients was present, and 21 of 23 had tinnitus.

All patients had ongoing episodic worsening of hearing in one or both ears despite receiving traditional medical therapy. All had received prednisone therapy at varying doses at some time during their disease course prior to enrollment, and 10 patients (3 with Cogan's syndrome, 7 with idiopathic IMCVD) were receiving long-term prednisone therapy at enrollment at dosages of 5–20 mg/week for >2 weeks and up to 9 years at the baseline evaluation. In addition, 9 patients had taken methotrexate at dosages of 10–25 mg/week 3 months to 7 years prior to enrollment, and 8 patients were receiving methotrexate at these dosages at enrollment. Methotrexate therapy was continued for the duration of the study without a change in dosage in all patients who were taking it at enrollment. One patient had previously received hydroxychloroquine for management of IMCVD, without benefit, and no patients had received any other adjuvant therapies including chemotherapies such as cyclophosphamide or leflunomide. No patients had previously been treated with any monoclonal antibody therapy or anti-TNF therapy.

At the time of enrollment into the study, 32 patients received a challenge of oral prednisone at an initial dosage of 1 mg/kg/day, with tapering over 3 weeks. All 23 patients enrolled in the study had at least partial improvement of hearing as defined above in at least one ear; the remaining patients with suspected IMCVD had no response and were not enrolled in the therapeutic arm of the trial. Chronic daily prednisone therapy was continued during and following tapering of the prednisone challenge in the 10 patients taking it at enrollment. The baseline prednisone dosage was further adjusted in these patients during the active-treatment period with etanercept, according to clinical symptoms.

Baseline laboratory assessments (including complete blood count, serum aspartate aminotransferase, and syphilis serology) were negative or normal for all patients. Perinuclear antineutrophil cytoplasmic antibody (pANCA) was present in 2 patients; all patients were negative for cytoplasmic ANCA, and HsP70 was positive in only 4 of 20 patients (20%) who underwent this testing. The ESR was slightly elevated (35 mm/hour) in only 1 patient. CT (4 patients) or MRI (19 patients) of the brain was nondiagnostic in all patients.

Clinical efficacy.

During the 24-week period of followup on etanercept therapy, improvement in hearing as defined in the Patients and Methods section was detected in 7 of 23 patients (30%), hearing was worse in 3 patients (13%), and unchanged in 13 patients (57%). A summary of the findings is presented in Table 1.

Table 1. Summary of therapeutic efficacy of etanercept for autoimmune hearing loss in 23 patients at six months of followup*
 Hearing loss (n = 23)Tinnitus (n = 21)Vertigo (n = 16)
  • *

    Values are the number (percentage) of patients with each symptom. Hearing loss was unilateral or bilateral. Improved = increase in pure tone average (PT) by >10 dB or word recognition/identification (WR) by >12% in at least one ear. Worse = PT threshold falling >10 dB from baseline, WR >12% from baseline; No change/stable = change in PT of ± 10 dB from baseline; change in WR ± 12% from baseline.

Improved7 (30)7 (33)8 (50)
No change/stable13 (57)10 (48)7 (47)
Worse3 (13)4 (19)1 (3)

Improvement in pure-tone audiogram in at least one ear was seen in 7 of 23 patients (30%), and WR was improved in at least one ear in 6 of 23 patients (26%), including 2 of 3 patients with Cogan's syndrome. The pure-tone audiogram improved in both ears in only 2 of 18 patients (5 patients had one non-evaluable ear; see above), and WR improved in both ears in only 1 of 18 patients. Both pure-tone audiogram and WR were improved in at least 1 ear in only 5 of 23 patients (22%), and only 1 of 18 patients had improvement in both pure-tone audiogram and WR in both ears. Evaluation of interval audiograms obtained at weeks 4, 8, 12, 16, and 20 revealed that any improvement noted occurred by week 12 of treatment. Table 2 contains a summary of these results. No relationship between disease duration, previous or current use of disease-modifying antirheumatic drugs, nor degree of improvement from the initial prednisone challenge was noted.

Table 2. Summary of evaluation of interval audiograms in 23 patients at six months of followup*
 Pure toneWord recognition
 UnilateralBilateralUnilateralBilateral
  • *

    Values are the number (percentage) of patients. According to the study criteria, improved = improvement in pure tone (PT) average by >10 dB or word recognition/identification (WR) by >12% in at least one ear. Worse = PT threshold falling >10 dB from baseline; WR >12% from baseline. No change/stable = change in PT of ± 10 dB from baseline; WR ± 12% from baseline. Unilateral denotes at least one ear with active disease (23 patients), 18 patients with bilateral disease (2 patients with deafness of at least one ear; 3 patients with a normal hearing and clinically unaffected ear were excluded). The PT and WR changes could occur in different patients. Unilateral patients had at least 1 ear involved, but could have bilateral disease.

Improved7 (30)2 (11)6 (26)1 (6)
No change/  stable14 (61)15 (83)15 (65)16 (88)
Worse2 (9)1 (6)2 (9)1 (6)

The frequency and severity of disequilibrium/vertigo improved in 8 of 16 patients (50%), and tinnitus improved in only 7 of 21 patients (33%) with this symptom (Table 1).

During the course of the study, prednisone therapy was discontinued in 3 of 10 patients who had received long-term therapy prior to enrollment, without change in hearing status. All of these patients had been receiving prednisone for <6 months, with an average dosage of 7.5 mg/day at enrollment. The daily prednisone dose was decreased by 19% in these 10 patients, and the dosage could be reduced from an average of 12.67 mg/day to 8.1 mg/day in the patients with Cogan's syndrome. None of the patients with Cogan's syndrome had active ocular or systemic disease at enrollment, and none of these patients developed these disease manifestations during the study.

Methotrexate therapy was continued without dose alteration per protocol in all 9 patients taking it at the time of study entry. Because of worsening hearing, one patient underwent plasmapheresis without benefit, and one patient received plasmapheresis, methotrexate therapy, and intratympanic corticosteroid injection during the study, with improvement in 1 ear and no improvement in the other. Although this result was regarded as “improvement,” neither including nor excluding this patient as a responder had any significant effect on the study results. One patient with progressive disease received a burst of prednisone (1 mg/kg, with tapering over 3 weeks) without improvement in disease activity parameters.

HsP70 was positive at enrollment in only 4 of 20 patients (20%) for whom data were available, and positive in 2 of 20 patients (10%) at completion of the study. It became negative in 3 of 4 patients who initially were positive, although one of these patients was assessed to have active disease at week 24, with ongoing fluctuating hearing and worsening compared with baseline. One patient in whom HsP70 had been negative at baseline and who had active disease at week 24 was newly positive for HsP70 at week 24. Westergren's ESR was slightly elevated in only one patient at both baseline and week 24, but the finding was of uncertain significance.

PET scanning was positive in one ear of 2 of 5 patients (2 of 10 ears) at enrollment. This included one deaf ear, which did not improve during the study. The other positive scan was in an ear with clinically and audiometrically active disease (fluctuating hearing loss); however, in none of the other 7 ears with active disease was the scan positive (2 ears were normal and one was deaf, as described above). The PET scan was repeated at week 12 and was positive in only one patient, who was considered to have active disease. HsP70 was incidentally positive in this patient at week 24, although it was negative both at enrollment and at week 24 in all 4 other patients who underwent PET scanning. By week 12, the PET scan was negative in 3 patients with inactive disease but was negative in 1 patient with active disease.

Etanercept therapy was generally well tolerated. One patient developed lower extremity edema and cellulitis at week 5, requiring antibiotic therapy for 3 weeks. This patient was also taking methotrexate and prednisone, making adverse event attribution difficult. The methotrexate and etanercept were temporarily discontinued from week 5 to week 13; there was no change in his hearing during this time. Three other patients each missed one week of therapy; no patients discontinued etanercept prematurely.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

IMCVD is an uncommon condition of uncertain etiology. The syndrome is partly defined by observations regarding response to steroid therapy, but the pathogenesis of hearing loss and vestibular symptoms in IMCVD is poorly understood. Response to treatment, usually with corticosteroids and/or immunosuppressive drugs, is uncertain, and many if not most patients eventually develop some degree of permanent hearing loss.

The present study examined the 24-week outcome of patients with IMCVD treated with etanercept at doses commonly used in the treatment of another immune-mediated disorder, rheumatoid arthritis. To ascertain whether the therapy could be beneficial in the studied patients, we first administered a short course of prednisone, enrolling patients who had improvement following the corticosteroid challenge. At the outset of the study, success was defined as improvement in audiometric parameters of pure-tone average by >10 dB or improvement in speech recognition/identification by >12% in at least 1 ear. These parameters were chosen because they represent true differences in hearing based on test–retest audiometrics (27, 28).

According to the audiometric criteria, improvement was seen in 7 of 23 treated patients. Pure tone improved in 7 of 23 patients in at least one ear, while word recognition improved in 6 of 23 patients. However, improvement in both pure-tone and WR in at least one ear occurred in only 5 of 23 patients. It could not be expected that both pure-tone and word recognition would improve in both ears in all patients because 2 patients were functionally deaf in 1 ear at study outset; nevertheless, only 1 patient had improvement in both of these parameters in both ears.

By the end of the 24-week followup, disequilibrium/vertigo was improved in 50% of patients who entered the study with this symptom, and tinnitus was improved in 33% of patients. The assessment of these symptoms was entirely patient derived in this study, and formal testing was not included as a parameter of outcome. Whether this improvement represents a true treatment effect, represents the natural course of the symptom(s) and the accommodation of the patient to it, or represents a placebo effect remains unknown.

This study was of an exploratory nature and has all of the shortcomings of an open-label trial. The outcome may simply reflect the usual course of autoimmune hearing loss rather than the result of treatment. Although 30% of patients were improved by audiometric criteria, most had no change in findings, but only 13% were worse. It is uncertain what the effect of the initial burst of prednisone on the final audiometric, laboratory, and PET scan outcomes was. However, all patients had active disease at treatment initiation despite frequent use of adjuvant therapies such as methotrexate, which were continued throughout the study. It cannot be excluded that use of etanercept led to stabilization of symptoms, which could be considered a measure of success, albeit an uncertain one in the context of this study. The goal of the study was improvement in the audiometric assessment in 70% of patients. This result was not attained by the primary or secondary parameters examined. This outcome goal was more stringent than that of a preliminary study of etanercept for this indication in 12 other patients, although the result was similar (improvement [58%] or stabilization [33%] of hearing) (24). Patients in that study had a mean age of 47 years, which is virtually identical to the mean age of patients in the current study (48 years), but longer disease duration (average 13 years, compared with 3.4 years in the current study). The fact that a combined 87% of patients in the current study achieved hearing stabilization (57%) or improvement (30%) leaves open the question of whether etanercept may have a role in disease stabilization when steroid treatment is inadequate. Because both of these effects may or may not be treatment related, addressing this question will require longer-term controlled trials.

Interestingly, 2 of 3 patients with Cogan's syndrome appeared to do well. However, the number of patients was too small to permit meaningful conclusions about the ultimate role of etanercept as adjuvant therapy for Cogan's syndrome, but this and other studies suggest that it may be superior to corticosteroid therapy alone.

The hope that detection of HsP70 and use of PET scanning could be useful for identifying and following patients with active disease, as suggested in the literature, was not sustained in our study (15, 26).

Whether the improvement or stabilized symptoms are attributable to the treatment or to the etanercept therapy remains uncertain. Although a randomized, controlled trial would be required to establish the role of etanercept in the treatment of this disease, based on our findings it is unlikely that this therapy will be determined to be effective in achieving improvement of the clinically significant magnitude this study was designed to detect. Future studies may need to require an even larger degree of improvement in audiometric parameters than that employed in this study, including perhaps improvement in both ears, and perhaps a different method for patient-derived assessment. Future studies could also explore higher dosages of etanercept, for example 50 mg twice weekly, as used in treatment of psoriasis, which may be more efficacious than the dosage used in this study (29).

Treatment of IMCVD remains unsatisfactory. Although many patients have a short-term response to steroids, the response is generally not sustained. Understanding of the efficacy of long-term benefits of brief courses of corticosteroids or long-term therapies with cocorticosteroids, chemotherapies, and/or other agents including monoclonal antibody and biologic response-modifier therapy for autoimmune hearing loss is compromised by the limitations of our current classification schemes and lack of knowledge about the natural history of these entities.

Even with treatment, hearing loss may progress in most patients, even to deafness in a substantial number, and can be associated with significant drug-related toxicity, including hypercortisolism (6, 10). Open-label studies have shown that methotrexate may be beneficial in some patients, especially those with mild disease, but controlled randomized trials do not support its use (14, 15). More toxic cytotoxic regimens, such as with cyclophosphamide, are generally not justified for management of this condition when the poor benefit-to-toxicity relationship is taken into account. A source of good news has been the excellent outcome of cochlear implantation in our patients who have undergone this procedure for profound hearing loss due to IMCVD.

IMCVD continues to present a nosologic, diagnostic, and therapeutic challenge. Future research into the pathobiology of the condition should contribute to an improved understanding of it.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors would like to thank Jane Jaquith as study coordinator, and our colleagues Charles W. Beatty, MD, Colin L. W. Driscoll, MD, and Christopher D. Bauch, PhD of the Department of Otorhinolaryngology, Mayo Clinic, Rochester, Minnesota, without whom this study would not have been possible.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES