[Skip to Content]
Access to paid content on this site is currently suspended due to excessive activity being detected from your IP address 54.205.87.3. Please contact the publisher to request reinstatement.
[Skip to Content Landing]
Download PDF
Table 1. 
Characteristics of 20 Families With rAOM*
Characteristics of 20 Families With rAOM*
Table 2. 
Polymorphism of TNF-α-308, IL-1α-889, IL-1β-511, and IL-1β+3953 in 63 Patients With rAOM vs 400 Controls*
Polymorphism of TNF-α-308, IL-1α-889, IL-1β-511, and IL-1β+3953 in 63 Patients With rAOM vs 400 Controls*
Table 3. 
Polymorphism of IL-1Ra in 63 Patients With rAOM vs 400 Controls*
Polymorphism of IL-1Ra in 63 Patients With rAOM vs 400 Controls*
1.
Alho  OP How common is recurrent acute otitis media? Acta Otolaryngol Suppl.1997;529:8-10.
2.
Maw  RWilks  JHarvey  IPeters  TJGolding  J Early surgery compared with watchful waiting for glue ear and effect on language development in preschool children: a randomised trial. Lancet.1999;353:960-963.
3.
Uhari  MMäntysaari  KNiemelä  M A meta-analytic review of the risk factors for acute otitis media. Clin Infect Dis.1996;22:1079-1083.
4.
Casselbrant  MLMandel  EMFall  PA  et al The heritability of otitis media: a twin and triplet study. JAMA.1999;282:2125-2130.
5.
Kvaerner  KJTambs  KHarris  JRMagnus  P Distribution and heritability of recurrent ear infections. Ann Otol Rhinol Laryngol.1997;106:624-632.
6.
Wiet  RJDeBlanc  GBStewart  JWeider  DJ Natural history of otitis media in the American native. Ann Otol Rhinol Laryngol Suppl.1980;89:14-19.
7.
Prellner  KHallberg  TKalm  OMansson  B Recurrent otitis media: genetic immunoglobulin markers in children and their parents. Int J Pediatr Otorhinolaryngol.1985;9:219-225.
8.
Kelly  KM Recurrent otitis media: genetic immunoglobulin markers in children and their parents [letter]. Int J Pediatr Otorhinolaryngol.1993;25:279-280.
9.
Kalm  OJohnson  UPrellner  KNinn  K HLA frequency in patients with recurrent acute otitis media. Arch Otolaryngol Head Neck Surg.1991;117:1296-1299.
10.
Kalm  OJohnson  UPrellner  K HLA frequency in patients with chronic secretory otitis media. Int J Pediatr Otorhinolaryngol.1994;30:151-157.
11.
Nicod  LP Cytokines, 1: overview. Thorax.1993;48:660-667.
12.
Bidwell  JKeen  LGallagher  G  et al Cytokine gene polymorphism in human disease: on-line databases. Genes Immun.1999;1:3-9.
13.
Hurme  MLahdenpohja  NSanttila  S Gene polymorphisms of interleukins 1 and 10 in infectious and autoimmune diseases. Ann Med.1998;30:469-473.
14.
McGuire  WHill  AVAllsopp  CEGreenwood  BMKwiatkowski  D Variation in the TNF-α promoter region associated with susceptibility to cerebral malaria. Nature.1994;371:508-510.
15.
Westendorp  RGLangermans  JAHuizinga  TW  et al Genetic influence on cytokine production and fatal meningococcal disease. Lancet.1997;349:170-173.
16.
Helminen  MLahdenpohja  NHurme  M Polymorphism of the interleukin-10 gene is associated with susceptibility to Epstein-Barr virus infection. J Infect Dis.1999;180:496-499.
17.
Sato  KKawana  MNonomura  NNakano  Y Course of IL-1β, IL-6, IL-8, and TNF-α in the middle ear fluid of the guinea pig otitis media model induced by nonviable Haemophilus influenzaeAnn Otol Rhinol Laryngol.1999;108:559-563.
18.
Lindberg  KRynnel-Dagoo  BSundqvist  KG Cytokines in nasopharyngeal secretions: evidence for defective IL-1β production in children with recurrent episodes of acute otitis media. Clin Exp Immunol.1994;97:396-402.
19.
Yellon  RFLeonard  GMarucha  PT  et al Characterization of cytokines present in middle ear effusions. Laryngoscope.1991;101:165-169.
20.
Wilson  AGdi Giovine  FSBlakemore  AIDuff  GW Single base polymorphism in the human tumour necrosis factor alpha (TNF-α) gene detectable by NcoI restriction of PCR product. Hum Mol Genet.1992;1:353.
21.
McDowell  TLSymons  JAPloski  RForre  ODuff  GW A genetic association between juvenile rheumatoid arthritis and a novel interleukin-1 alpha polymorphism. Arthritis Rheum.1995;38:221-228.
22.
di Giovine  FSTakhsh  EBlakemore  AIDuff  GW Single base polymorphism at -511 in the human interleukin-1 beta gene (IL1 beta). Hum Mol Genet.1992;1:450.
23.
Pociot  FMolvig  JWogensen  LWorsaae  HNerup  J A TaqI polymorphism in the human interleukin-1 beta (IL-1β) gene correlates with IL-1β secretion in vitro. Eur J Clin Invest.1992;22:396-402.
24.
Tarlow  JKBlakemore  AILennard  A  et al Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat. Hum Genet.1993;91:403-404.
25.
Yellon  RFLeonard  GMarucha  P  et al Demonstration of interleukin 6 in middle ear effusions. Arch Otolaryngol Head Neck Surg.1992;118:745-748.
26.
Maxwell  KSFitzgerald  JEBurleson  JA  et al Interleukin-8 expression in otitis media. Laryngoscope.1994;104:989-995.
27.
Juhn  SKGarvis  WJLees  CJLe  CTKim  CS Determining otitis media severity from middle ear fluid analysis. Ann Otol Rhinol Laryngol Suppl.1994;163:43-45.
28.
Bernstein  JMRich  GAOdziemiec  CBallow  M Are thymus-derived lymphocytes (T cells) defective in the nasopharyngeal and palatine tonsils of children? Otolaryngol Head Neck Surg.1993;109:693-700.
29.
Tong  HHFisher  LMKosunick  GMDeMaria  TF Effect of tumor necrosis factor alpha and interleukin 1-α on the adherence of Streptococcus pneumoniae to chinchilla tracheal epithelium. Acta Otolaryngol.1999;119:78-82.
30.
Wilson  AGdi Giovine  FSDuff  GW Genetics of tumour necrosis factor-α in autoimmune, infectious, and neoplastic diseases [review]. J Inflamm.1995;45:1-12.
31.
Dinarello  CA Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int Rev Immunol.1998;16:457-499.
32.
Mackowiak  PA The normal microbial flora. N Engl J Med.1982;307:83-93.
33.
Ruuskanen  OHeikkinen  T Viral-bacterial interaction in acute otitis media. Pediatr Infect Dis J.1994;13:1047-1049.
Original Article
January 2002

Cytokine Gene Polymorphism in Recurrent Acute Otitis Media

Author Affiliations

From the Departments of Otorhinolaryngology, Head and Neck Surgery (Drs Joki-Erkkilä and Puhakka), and Clinical Microbiology (Dr Hurme), Tampere University Hospital, and the Department of Microbiology and Immunology, University of Tampere Medical School (Dr Hurme), Tampere, Finland.

Arch Otolaryngol Head Neck Surg. 2002;128(1):17-20. doi:10.1001/archotol.128.1.17
Abstract

Background  There is increasing evidence that a strong genetic component is involved in the predisposition to recurrent acute otitis media (rAOM). Cytokines play a key role in the pathogenesis of otitis media. Constitutional polymorphisms in cytokine genes may lead to individual variations in cytokine secretion.

Objective  To elucidate the role of cytokine gene polymorphisms in rAOM.

Setting  University hospital.

Participants and Methods  Blood samples for genetic analysis were obtained from 63 individuals with rAOM from 20 different families and from 400 healthy blood donors. The medical history of the rAOM group was based on medical records and interview data. We studied the polymorphisms of tumor necrosis factor α, interleukin (IL) 1α, IL-1β, and IL-1 receptor antagonist genes.

Results  The distribution of cytokine alleles in the rAOM group did not differ significantly from that of the control group. However, in patients with rAOM without a history of allergic disorders, allele frequencies of IL-1α-889 differed significantly from those of controls (P = .03).

Conclusions  There is no clear association between the polymorphism of studied cytokine genes and rAOM. However, the IL-1α gene polymorphism may be associated with recurrent middle ear infections in a subgroup of patients without allergic disorders.

ACUTE OTITIS media (AOM) is one of the most common childhood illnesses. Although nearly all children experience at least one otitis episode, only about 15% of children experience recurrent attacks.1 Recurrent AOM (rAOM) is a challenging clinical problem consuming many medical resources. Moreover, frequent otitis episodes are also associated with a child's developmental delay.2

Recurrent AOM has a multifactorial background that is still only partly understood. Epidemiologic data have revealed several external factors predisposing to otitis media, including day care outside the home, use of a pacifier, and parental smoking.3 However, recent twin and triplet studies4,5 have suggested that there is a strong genetic component involved in middle ear infections. This is supported by earlier findings6 of incidence differences of AOM in different ethnic groups and epidemiologic analyses3 that have shown that a positive family history of AOM is the most important risk factor for AOM. Genetically determined immunoglobulin markers have also been studied in children with rAOM, and allotype G2m(23) has been linked with otitis proneness.7,8 Moreover, an association between the HLA-A2 antigen and rAOM has been reported but not between HLA-A2 and secretory otitis media.9,10

Cytokines are bioactive proteins that widely mediate host responses to inflammatory stimuli. In the pathogenesis of an inflammation in the respiratory tract, they regulate proliferation, chemotaxis, and the activation of inflammatory cells.11 Constitutional polymorphisms in cytokine genes may lead to individual variations in cytokine secretion.12,13 The outcomes of many infectious diseases, such as malaria,14 meningococcal meningitis,15 and Epstein-Barr virus,16 have been associated with these polymorphisms. Cytokines also play a key role in the pathogenesis of otitis media.1719 The aim of the present work was to study the polymorphisms of tumor necrosis factor (TNF) α, interleukin (IL) 1α, IL-1β, and IL-1 receptor antagonist (IL-1Ra) genes in patients with rAOM.

PARTICIPANTS AND METHODS

This study was conducted at Tampere University Hospital, Tampere, Finland, and included 20 different families with a high occurrence of AOM. In each family, at least 1 parent and all the children had a history of rAOM. The criterion for rAOM was at least 6 AOM episodes within 12 months or at least 10 AOM episodes during a lifetime. The medical history was based on medical records and interview data. A blood sample for genetic analysis was obtained from 63 patients with rAOM.

Control blood samples were obtained from 400 unselected healthy blood donors (The Finnish Red Cross Blood Transfusion Centre, Tampere) living in the same area as the study group members. Donors were aged 18 to 60 years. The history of AOM in the control population was not known.

The study was approved by the ethical committee of Tampere University Hospital, and written informed consent was received from the parents in each family with rAOM.

ANALYSIS OF CYTOKINE GENE POLYMORPHISMS

DNA specimens from citrated whole blood samples were prepared using standard methods.

Tumor Necrosis Factor α-308

A 107–base pair fragment of the TNF-α gene promoter region containing the G→A substitution was amplified using polymerase chain reaction (PCR) primers 5′-AGGCAATAGGTTTTGAGGGCCAT-3′ and 5′-TCCTCCCTGCTCCGATTCCG-3′. The amplified product was digested with NcoI and analyzed using electrophoresis on 9% polyacrylamide gel.20

Interleukin 1α-889

The base exchange at position -889 of the IL-1α gene was analyzed as previously described.21 Oligonucleotides 5′-AAGCTTGTT CTACCACCTGAACTAGGC-3′ and 5′-TTACATATGAGC CTTCCATG-3′ flanking the polymorphic site were used as primers in PCR. The products were digested with NcoI, and the resultant products were analyzed using 9% polyacrylamide gel electrophoresis.

Interleukin 1β-511

The region that contains the AvaI polymorphic site at position -511 of the IL-1-β gene was amplified using PCR.22 The oligonucleotides 5′-TGGCATTGATCTGGTTCATC-3′ and 5′-GTTTAGGAATCTTCCCACTT-3′ were used as primers. Fragments were analyzed using electrophoresis on 9% polyacrylamide gel stained with ethidium bromide.

Interleukin 1β+3953

Position +3953 within exon 5 of the IL-1β gene has a single base pair polymorphism.23 The polymorphic region containing the TaqI restriction site was amplified using the following primers: 5′-GTTGTCATCAGACTTTGACC-3′ and 5′-TTCAGTTCATATGGACCAGA-3′. Fragments were analyzed using electrophoresis on 9% polyacrylamide gel stained with ethidium bromide.

Interleukin 1Ra

The IL-1Ra exon 2 polymorphism was analyzed as described previously.24 Oligonucleotides 5′-CTCAGCAACACTCCTAT-3′ and 5′-TCCTGGTCTGCAGGTAA-3′ were used as primers in PCR. The final PCR product was analyzed using electrophoresis on a 2% agarose gel stained with ethidium bromide.

STATISTICAL ANALYSIS

The distribution of the TNF-α-308, IL-1α-889, IL-1β-511, IL-1β+3953, and IL-1Ra allele frequencies in the rAOM families was compared with that of healthy blood donors. Statistical analysis was performed using the χ2 test.

RESULTS

The families included in the study were extremely prone to otitis (Table 1), but none of the patients had been known to have an immunologic deficiency. In every patient, the primary condition was rAOM, but secretory otitis media later developed in 20 (32%). Three patients also experienced subacute or chronic mastoiditis, and mastoidectomy was performed in 2 patients. In the study group, 18 patients were adults and 45 were children; 57% were male (n = 36) and 43% were female (n = 27). Seventeen patients from 7 different families had a history of allergic disorders (atopic dermatitis, asthma, or allergic rhinitis): 10 in 2 families and 7 in 5 families.

The distribution of proinflammatory cytokine alleles (TNF-α-308, IL-1α-889, IL-1β-511, and IL-1β+3953) (Table 2) and anti-inflammatory IL-1Ra alleles (Table 3) did not differ significantly in the rAOM group vs the control group. When calculating the children's data separately, the result remained basically the same. The greatest difference was in the distribution of IL-1α-889 alleles, but this did not reach statistical significance (P = .07). However, in a subgroup of patients with rAOM without a history of allergic disorders (atopy, asthma, or allergic rhinitis), allele frequencies of IL-1α-889 differed significantly from those in the control group (allele 1: 0.78 vs 0.67; P = .03).

COMMENT

Cytokines are of central importance in the pathogenesis of inflammation. Three main proinflammatory cytokines—TNF-α, IL-1, and IL-6—and anti-inflammatory cytokines such as IL-1Ra and IL-10 down-regulate the inflammatory process.12,13 The production of cytokines is regulated by genetic elements. Polymorphisms associated with these genes are thought to lead to individual variations in cytokine secretions and are inherited in mendelian fashion.12,13 Cytokine gene polymorphism is associated with the outcomes of various infectious diseases.1416

In the past few years, several studies have been published regarding cytokines and otitis media, and it has been possible to determine various types of cytokines from middle ear effusions (IL-1β, IL-2, TNF-α, interferon γ,19 IL-6,25 and IL-826) and from nasopharyngeal secretions (IL-1β, IL-6, and TNF-α),18 indicating that cytokines play a key role in the pathogenesis of AOM. High concentrations of TNF-α in middle ear effusions correlate with the persistence of secretory otitis media.27 Furthermore, IL-1β, IL-6, and TNF-α concentrations measured from the nasopharyngeal secretions of children with rAOM are lower compared with healthy children.18 It has also been postulated that children with rAOM may have low IL-2 production, leading to lower serum IgA levels in mucosal secretions.28 On the other hand, inflammatory cytokines can also increase bacterial adherence to and invasion of cells29; hence, if a respiratory tract virus infection, which usually precedes an AOM attack, induces too high or prolonged cytokine production, this might theoretically increase the risk of developing AOM.

According to results of twin and triplet studies,4,5 the weight of genetic factors in middle ear infections is 0.45 to 0.64 among males and 0.74 to 0.79 among females. The genetic foundation of susceptibility to rAOM is unclear. Prellner et al found that the genetically determined immunoglobulin marker G2m(23) was present in 95% of children with rAOM but in only 64% of children with no history of AOM (P = .01).7,8 Kalm et al9 revealed that the HLA-A2 antigen occurred significantly more often in the rAOM group than in controls (80% vs 56%; P<.01) and that the frequency of the HLA-A3 antigen was lower. The TNF-α gene is located on the short arm of chromosome 6 in the major histocompatibility complex between HLA class I and III loci and can be coinherited with certain HLA antigens.30 However, in the present study, we could not find differences in allele frequencies of TNF-α-308 between the study groups.

The IL-1 gene family includes IL-1α, IL-1β, and IL-1Ra, which are situated on the short arm of chromosome 2.31 In the present study, the allele frequencies of the IL-1 gene did not differ significantly in the rAOM group vs controls. The greatest difference was in the allelic distribution of the IL-1β-511 gene, but it did not reach statistical significance (P = .08). However, in a subgroup of patients with rAOM who did not have any allergic disorders, there were significant differences in the IL-1α-889 allele frequencies. Because these differences were not so great and because the number of patients in the subgroup analysis was small, the value of this finding should be considered cautiously. On the other hand, it is possible that the pathogenesis of middle ear inflammation in allergic patients differs from that in nonallergic patients.

In the present study we paid extra attention to selecting families who were extremely prone to otitis; however, the control population was not ideal because the occurrence of AOM in this population was not known. We have no reason to assume that our control population would have had an unusually high occurrence of AOM. Also, the frequency of TNF alleles reported20 in other populations did not differ from that in our control population.

Otitis media has a multifactorial background. The pathogenesis of AOM is regulated by many external and internal factors, including normal flora,32 bacterial-viral interactions,33 and individual immunologic reactions. Recent studies have shown that a strong genetic component is involved in middle ear infections,4,5 but the genetic foundation of susceptibility to rAOM is unclear. In the present work, we studied the polymorphisms of TNF-α-308, IL-1α-889, IL-1β-511, IL-1β+3953, and IL-1Ra genes in patients with rAOM, but we did not find any clear association between the studied cytokine genes and susceptibility to recurrent middle ear infections, except in a subgroup of patients without any allergic disorders. More data are needed to assess the value of this finding.

Back to top
Article Information

Accepted for publication August 16, 2001.

This study was supported by a grant from The Medical Research Fund of Tampere University Hospital.

Corresponding author and reprints: Veli-Pekka Joki-Erkkilä, MD, Department of Otorhinolaryngology, Head and Neck Surgery, Tampere University Hospital, PO Box 2000, FIN-33521 Tampere, Finland (e-mail: vp.jokierkkila@nokianlaakariasema.fi).

References
1.
Alho  OP How common is recurrent acute otitis media? Acta Otolaryngol Suppl.1997;529:8-10.
2.
Maw  RWilks  JHarvey  IPeters  TJGolding  J Early surgery compared with watchful waiting for glue ear and effect on language development in preschool children: a randomised trial. Lancet.1999;353:960-963.
3.
Uhari  MMäntysaari  KNiemelä  M A meta-analytic review of the risk factors for acute otitis media. Clin Infect Dis.1996;22:1079-1083.
4.
Casselbrant  MLMandel  EMFall  PA  et al The heritability of otitis media: a twin and triplet study. JAMA.1999;282:2125-2130.
5.
Kvaerner  KJTambs  KHarris  JRMagnus  P Distribution and heritability of recurrent ear infections. Ann Otol Rhinol Laryngol.1997;106:624-632.
6.
Wiet  RJDeBlanc  GBStewart  JWeider  DJ Natural history of otitis media in the American native. Ann Otol Rhinol Laryngol Suppl.1980;89:14-19.
7.
Prellner  KHallberg  TKalm  OMansson  B Recurrent otitis media: genetic immunoglobulin markers in children and their parents. Int J Pediatr Otorhinolaryngol.1985;9:219-225.
8.
Kelly  KM Recurrent otitis media: genetic immunoglobulin markers in children and their parents [letter]. Int J Pediatr Otorhinolaryngol.1993;25:279-280.
9.
Kalm  OJohnson  UPrellner  KNinn  K HLA frequency in patients with recurrent acute otitis media. Arch Otolaryngol Head Neck Surg.1991;117:1296-1299.
10.
Kalm  OJohnson  UPrellner  K HLA frequency in patients with chronic secretory otitis media. Int J Pediatr Otorhinolaryngol.1994;30:151-157.
11.
Nicod  LP Cytokines, 1: overview. Thorax.1993;48:660-667.
12.
Bidwell  JKeen  LGallagher  G  et al Cytokine gene polymorphism in human disease: on-line databases. Genes Immun.1999;1:3-9.
13.
Hurme  MLahdenpohja  NSanttila  S Gene polymorphisms of interleukins 1 and 10 in infectious and autoimmune diseases. Ann Med.1998;30:469-473.
14.
McGuire  WHill  AVAllsopp  CEGreenwood  BMKwiatkowski  D Variation in the TNF-α promoter region associated with susceptibility to cerebral malaria. Nature.1994;371:508-510.
15.
Westendorp  RGLangermans  JAHuizinga  TW  et al Genetic influence on cytokine production and fatal meningococcal disease. Lancet.1997;349:170-173.
16.
Helminen  MLahdenpohja  NHurme  M Polymorphism of the interleukin-10 gene is associated with susceptibility to Epstein-Barr virus infection. J Infect Dis.1999;180:496-499.
17.
Sato  KKawana  MNonomura  NNakano  Y Course of IL-1β, IL-6, IL-8, and TNF-α in the middle ear fluid of the guinea pig otitis media model induced by nonviable Haemophilus influenzaeAnn Otol Rhinol Laryngol.1999;108:559-563.
18.
Lindberg  KRynnel-Dagoo  BSundqvist  KG Cytokines in nasopharyngeal secretions: evidence for defective IL-1β production in children with recurrent episodes of acute otitis media. Clin Exp Immunol.1994;97:396-402.
19.
Yellon  RFLeonard  GMarucha  PT  et al Characterization of cytokines present in middle ear effusions. Laryngoscope.1991;101:165-169.
20.
Wilson  AGdi Giovine  FSBlakemore  AIDuff  GW Single base polymorphism in the human tumour necrosis factor alpha (TNF-α) gene detectable by NcoI restriction of PCR product. Hum Mol Genet.1992;1:353.
21.
McDowell  TLSymons  JAPloski  RForre  ODuff  GW A genetic association between juvenile rheumatoid arthritis and a novel interleukin-1 alpha polymorphism. Arthritis Rheum.1995;38:221-228.
22.
di Giovine  FSTakhsh  EBlakemore  AIDuff  GW Single base polymorphism at -511 in the human interleukin-1 beta gene (IL1 beta). Hum Mol Genet.1992;1:450.
23.
Pociot  FMolvig  JWogensen  LWorsaae  HNerup  J A TaqI polymorphism in the human interleukin-1 beta (IL-1β) gene correlates with IL-1β secretion in vitro. Eur J Clin Invest.1992;22:396-402.
24.
Tarlow  JKBlakemore  AILennard  A  et al Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat. Hum Genet.1993;91:403-404.
25.
Yellon  RFLeonard  GMarucha  P  et al Demonstration of interleukin 6 in middle ear effusions. Arch Otolaryngol Head Neck Surg.1992;118:745-748.
26.
Maxwell  KSFitzgerald  JEBurleson  JA  et al Interleukin-8 expression in otitis media. Laryngoscope.1994;104:989-995.
27.
Juhn  SKGarvis  WJLees  CJLe  CTKim  CS Determining otitis media severity from middle ear fluid analysis. Ann Otol Rhinol Laryngol Suppl.1994;163:43-45.
28.
Bernstein  JMRich  GAOdziemiec  CBallow  M Are thymus-derived lymphocytes (T cells) defective in the nasopharyngeal and palatine tonsils of children? Otolaryngol Head Neck Surg.1993;109:693-700.
29.
Tong  HHFisher  LMKosunick  GMDeMaria  TF Effect of tumor necrosis factor alpha and interleukin 1-α on the adherence of Streptococcus pneumoniae to chinchilla tracheal epithelium. Acta Otolaryngol.1999;119:78-82.
30.
Wilson  AGdi Giovine  FSDuff  GW Genetics of tumour necrosis factor-α in autoimmune, infectious, and neoplastic diseases [review]. J Inflamm.1995;45:1-12.
31.
Dinarello  CA Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int Rev Immunol.1998;16:457-499.
32.
Mackowiak  PA The normal microbial flora. N Engl J Med.1982;307:83-93.
33.
Ruuskanen  OHeikkinen  T Viral-bacterial interaction in acute otitis media. Pediatr Infect Dis J.1994;13:1047-1049.
×