interleukin-8 and Otitis-Media

Cell culture models are valuable tools for investigation of the molecular pathogenesis of diseases including otitis media (OM). Previous study indicates that age-, sex-, and race-associated differences in molecular signaling may impact disease pathophysiology. Currently, a singular immortalized middle ear epithelial (MEE) cell line exists, HMEEC-1, derived from an adult without known middle ear disease. In this study, HMEEC-1 and primary MEE cultures from pediatric patients with and without OM were stimulated with inflammatory cytokines or OM-pathogenic bacterial lysates to examine differences in the response of molecules associated with OM pathogenesis.. Case-control series.. MEE cultures were established from patients aged <6 years: two with recurrent OM (ROM), two with OM with effusion (OME), and one patient without OM who was undergoing cochlear implant surgery control undergoing cochlear implantation (Peds CI). Primary MEE cultures and HMEEC-1 cells were stimulated with tumor necrosis factor-α, interleukin (IL)-1β, or nontypeable Haemophilus influenzae lysate. TNFA, IL1B, IL6, IL8, IL10, and MUC5B were assayed via quantitative polymerase chain reaction. IL-8 was assayed by enzyme-linked immunosorbent assay.. Gene/protein target expressions were frequently higher in pediatric OM lines than in HMEEC-1 and Peds CI. HMEEC-1 cells were frequently less responsive to stimuli than all pediatric lines. OME lines were often more responsive than ROM lines.. OM may be associated with specific molecular phenotypes that are retained in primary cell culture. Adult-derived HMEEC-1 cells differ significantly in baseline expression and response of OM-associated molecules relative to pediatric MEE cells. Work is underway to immortalize pediatric OM MEE cultures as improved tools for the OM research community.. 4 Laryngoscope, 131:410-416, 2021.

Topics: Case-Control Studies; Cell Culture Techniques; Cell Line; Child; Cytokines; Ear, Middle; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Female; Haemophilus influenzae; Humans; Interleukin-10; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Mucin-5B; Otitis Media; Real-Time Polymerase Chain Reaction; Signal Transduction; Tumor Necrosis Factor-alpha

Streptococcus pneumoniae (Spn) is a common respiratory pathogen and a frequent cause of acute otitis media (AOM) in children. The first step in bacterial pathogenesis of AOM is the establishment of asymptomatic colonization in the nasopharynx. We studied Spn bacterial burden in conjunction with neutrophil recruitment and inflammatory gene transcription and cytokine secretion in samples of nasal wash collected from normal and otitis-prone children during health, viral upper respiratory infection without middle ear involvement (URI) and AOM. We found no significant associations between otitis-prone status and any of the measured parameters. However, Spn bacterial burden was significantly correlated with neutrophil recruitment, transcription of IL-8, TNF-α and SOD2, and secretion of TNF-α. We also found that transcription of IL-8 and TNF-α mRNA by neutrophils was significantly correlated with the secretion of these cytokines into the nasopharynx. We conclude that Spn bacterial burden in the NP is a major determinant of neutrophil recruitment to the NP and activity during URI and AOM, and that neutrophils are contributors to the secretion of IL-8 and TNF-α in the NP when the Spn burden is high.

Topics: Acute Disease; Asymptomatic Diseases; Bacterial Load; Cell Movement; Child, Preschool; Cytokines; Ear, Middle; Humans; Infant; Inflammation; Inflammation Mediators; Interleukin-8; Nasopharyngeal Diseases; Neutrophils; Otitis Media; Pneumococcal Infections; RNA, Messenger; Streptococcus pneumoniae; Superoxide Dismutase; Tumor Necrosis Factor-alpha

Acute otitis media (AOM) causes an inflammatory response in the middle ear. We assessed differences in innate immune responses involved in bacterial defense at onset of AOM in children who were stringently defined as otitis prone (sOP) and children not otitis prone (NOP).. Innate immune genes analysis from middle ear fluid (MEF) samples of children.. Genes of toll-like receptors (TLR), nod-like and retinoic acid-inducible gene-I-like receptors, downstream effectors important for inflammation and apoptosis, including cytokines and chemokines, were studied from MEF samples by using a real-time polymerase chain reaction array. Protein levels of differentially regulated genes were measured by Luminex.. Gene expression in MEF among children who were sOP was significantly different in upregulation of interleukin 8, secretory leukocyte peptidase inhibitor, and chemokine (C-C motif) ligand 3, and in downregulation of interferon regulatory factor 7 and its related signaling molecules interferon alpha, Toll-like receptor adaptor molecule 2, chemokine (C-C motif) ligand 5, and mitogen-activated protein kinase 8 compared with children who were NOP. Differences in innate gene regulation were similar when AOM was caused by Streptococcus pneumoniae or nontypeable Haemophilus influenzae.. Innate-immune response genes are differentially regulated in children who were sOP compared with children with NOP.

Topics: Adolescent; Chemokine CCL3; Chemokine CCL5; Child; Ear, Middle; Female; Gene Expression Regulation; Genetic Predisposition to Disease; Haemophilus parainfluenzae; Humans; Immunity, Innate; Interferon Regulatory Factor-7; Interferon-alpha; Interleukin-8; Male; Otitis Media; Prospective Studies; Secretory Leukocyte Peptidase Inhibitor; Streptococcus pneumoniae; Toll-Like Receptor 2

Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.

Topics: Animals; Inflammation; Influenza A virus; Interleukin-6; Interleukin-8; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Otitis Media; Pneumococcal Infections; Viral Load

Alloiococcus otitidis is a newly discovered organism frequently detected in otitis media. However, the association of the organism with the development of otitis media has not been disclosed in detail yet. In the middle ear, proinflammatory cytokines and chemokines are released in association with infection by pathogens, and these cytokines contribute to the induction of an inflammatory reaction. To investigate the profile of inflammation-related cytokines in the acute phase of A. otitidis infection, we analyzed the release of proinflammatory cytokines and chemokines in middle ear effusions of acute otitis media due to A. otitidis, in comparison with acute otitis media due to the well-known Gram-positive middle ear pathogen Streptococcus pneumoniae. The amounts of proinflammatory cytokines (IL-8, IL-1beta, IL-6, TNF-alpha) and CXC chemokines (IP-10, I-TAC) were significantly increased in the A. otitidis group as well as in the S. pneumoniae group. Various inflammation-related cytokines/chemokines were induced in the A. otitidis-infected middle ear, and the profile of cytokines was very similar to that in S. pneumoniae infection. This preliminary study suggests that A. otitidis has the potential to induce these cytokines, contributing to the development of an inflammatory reaction in the middle ear cavity in a similar manner to S. pneumoniae.

Topics: Acute Disease; Bacterial Infections; Chemokine CXCL11; Chemokine CXCL9; Child; Child, Preschool; Ear, Middle; Female; Gram-Negative Bacteria; Humans; Infant; Interleukin-8; Male; Otitis Media; Tumor Necrosis Factor-alpha

The results suggest that the anti-inflammatory effect of caffeic acid phenethyl ester (CAPE) is due to its inhibition of tumor necrosis factor (TNF)-alpha expression and interleukin (IL)-8 production. The anti-inflammatory effect of CAPE is possibly through the inhibition of nuclear factor (NF)-kappaB via the suppression of inhibitor-kappaB-alpha (IkappaB-alpha) degradation.. CAPE is a biologically active component of propolis, a resinous material obtained from bee hives, which originates from conifer bark. The effect of CAPE on lipopolysaccharide (LPS)-induced inflammatory reactions is not known. The aim of this study was to evaluate the anti-inflammatory effect of CAPE on cultured human middle ear epithelial cells (HMEECs).. The effect of CAPE on LPS-induced TNF-alpha expression was evaluated in HMEECs by real-time reverse transcription polymerase chain reaction (RT-PCR). LPS-induced IL-8 production was determined by enzyme-linked immunosorbent assay (ELISA), and LPS-induced IkappaB-alpha degradation was followed by Western blot analysis.. CAPE significantly inhibited LPS-induced up-regulation of TNF-alpha in a dose-dependent manner. IL-8 production by LPS was significantly suppressed by the CAPE pretreatment. Furthermore, LPS-induced IkappaB-alpha degradation was suppressed by the CAPE pretreatment.

Topics: Caffeic Acids; Cell Line; Gene Expression; Humans; Inflammation Mediators; Interleukin-8; Lipopolysaccharides; NF-kappa B; Otitis Media; Phenylethyl Alcohol; Tumor Necrosis Factor-alpha

Alloiococcus otitidis is a newly recognized species of gram-positive bacteria frequently associated with otitis media. Although immunostimulating activity of this organism has been investigated, little is known about the signaling pathways of chemokine/cytokine induction by this organism. We investigated the role of NF-kappaB and mitogen-activated protein (MAP) kinases in chemokine interleukin-8 (IL-8) production by human peripheral blood mononuclear cells (PBMCs) after stimulation with A. otitidis. The organism could induce in vitro IL-8 production in human PBMCs. I kappa B alpha, NF-kappaB, p38 MAP kinase, p44/42 MAP kinase (ERK1/2) became phosphorylated in PBMCs after stimulation with A. otitidis. And, inhibitors of NF-kappaB (caffeic acid phenylethyl ester), p38 (SB 203580), or ERK1/2 (PD 98059) significantly reduced IL-8 induction by the organism. These results were similar to findings in IL-8 induction by Streptococcus pneumoniae, another gram-positive major middle ear pathogen. Our preliminary study suggests that multiple pathways including NF-kappaB, p38, and ERK1/2 were simultaneously activated, and were associated with IL-8 induction by A. otitidis in human PBMCs.

Topics: Blotting, Western; Enzyme-Linked Immunosorbent Assay; Gram-Positive Bacterial Infections; Humans; Interleukin-8; Leukocytes, Mononuclear; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Otitis Media; p38 Mitogen-Activated Protein Kinases; Signal Transduction; Streptococcus pneumoniae

To investigate the expression of recently identified human mucin genes in human middle ear epithelial (MEE) specimens from in vivo middle ear (ME) tissue and to compare this mucin gene expression with mucin gene expression in an immortalized cell culture in vitro source of human MEE.. Human MEE was harvested as in vivo specimens, and human MEE cell cultures were established for in vitro experimentation. RNA was extracted from MEE and primers designed for reverse-transcription polymerase chain reaction to assess for mucin gene MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC11, MUC12, MUC13, MUC15, MUC16, MUC18, MUC19, and MUC20 expression. Mucin gene expression in the in vivo and in vitro ME tissue was compared against tissues with known expression of the mucin genes in question.. Mucin genes MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC8, MUC9, MUC11, MUC13, MUC15, MUC16, MUC18, MUC19, and MUC20 were identified and expressed in both the in vivo and in vitro samples of MEE. Mucin genes MUC6, MUC12, and MUC17 were not identified in either tissue samples.. Many of the mucin genes that have been recently identified are expressed in human MEE. These genes are expressed in a similar manner in both in vivo and in vitro models. Understanding the mechanisms in which these genes regulate the physiology and pathophysiology of MEE will provide a more thorough understanding of the molecular mechanics of the MEE and disease conditions such as otitis media.

Topics: Cells, Cultured; DNA Primers; DNA, Complementary; Ear, Middle; Epithelium; Gene Expression; Humans; Interleukin-1beta; Interleukin-8; Mucins; Otitis Media; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Respiratory syncytial virus (RSV) is an important cause of upper respiratory infections and is known to play a causal role in the pathogenesis of rhinitis, sinusitis, acute otitis media, and pneumonia. RSV appears to prime the respiratory tract to secondary inciting events, such as bacterial or antigen challenges. To study the proinflammatory priming effects of RSV infection, cytokine expression was measured in well-differentiated human nasal epithelial cells (WD-NE) after RSV infection alone or after subsequent tumor necrosis factor (TNF)-alpha stimulation.. In vitro investigation.. Human nasal epithelial cells were obtained from surgical specimens and allowed to differentiate in air-liquid interface cultures until ciliation and mucus production were evident. Two experimental paradigms were used. First, accumulation of cytokines in the media was measured by real-time, quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay after RSV infection alone. In the second set of experiments, cytokines were also measured after TNF-alpha stimulation in both RSV-infected and uninfected cultures.. RSV infection of WD-NE resulted in significant accumulations of interleukin (IL)-6, IL-8, and RANTES when compared with findings in control samples. Real-time, quantitative RT-PCR demonstrated significant increases in IL-8 gene expression following RSV infection when compared to controls. Secondary TNF-alpha stimulation following well-established (i.e., 72 h) RSV infection induced marked increases in IL-6, IL-8, and RANTES when compared with both RSV infection alone and TNF-alpha stimulation alone.. These findings suggest that RSV infection primes nasal epithelial cells to secondary proinflammatory challenge, resulting in a hyperimmune response. RSV-induced priming of a hyperimmune response may be important in the pathogenesis of sinusitis, acute otitis media, and pneumonia.

Topics: Chemokine CCL5; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; Nasal Mucosa; Otitis Media; Pneumonia; Respiratory Syncytial Virus Infections; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

Real-time PCR and enzyme-linked immunosorbent assay were used to evaluate the ability of influenza A virus and Streptococcus pneumoniae opacity variants, either alone or in combination, to induce cytokine and chemokine genes in primary cultures of human middle ear epithelial (HMEE) cells. Following treatment with influenza A virus, the induction of gene expression, which occurred in a dose- and time-dependent manner, was strong for macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta; moderate for tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8; and weak for IL-1 beta and monocyte chemotactic peptide 1 (MCP-1). Except for TNF-alpha, all the gene products were detected in the cell culture supernatants. In contrast, infection of HMEE cells with S. pneumoniae alone induced low levels of mRNA expression of MIP-1 alpha and MIP-1 beta and did not significantly induce the transcription of the other cytokines and chemokines examined. However, both S. pneumoniae opacity variants increased mRNA expression of MIP-1 alpha, MIP-1 beta, IL-6, and MCP-1 in HMEE cells activated by a prior influenza A virus infection compared to levels in cells treated with either agent alone. Up-regulation of IL-6, IL-8, and MCP-1 mRNA expression and production by the virus in combination with opaque S. pneumoniae was two- to threefold higher than that induced by the virus combined with the transparent S. pneumoniae variant. These data indicate that the activation of HMEE cells by influenza A virus enhances the induction of cytokine and chemokine gene transcripts by S. pneumoniae and that this effect appears to be most pronounced when S. pneumoniae is in the opaque phase.

Topics: Cells, Cultured; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Chemokines; Cytokines; Ear, Middle; Epithelial Cells; Gene Expression; Genetic Variation; Humans; Immunity, Innate; Influenza A virus; Interleukin-1; Interleukin-6; Interleukin-8; Macrophage Inflammatory Proteins; Otitis Media; RNA, Messenger; Streptococcus pneumoniae; Tumor Necrosis Factor-alpha

Nontypeable Haemophilus influenzae (NTHI) is an important etiological agent of otitis media (OM) and of exacerbated chronic obstructive pulmonary diseases (COPD). Inflammation is a hallmark of both diseases. Interleukin-8 (IL-8), one of the important inflammatory mediators, is induced by NTHI and may play a significant role in the pathogenesis of these diseases. Our studies demonstrated that a soluble cytoplasmic fraction (SCF) from NTHI induced much greater IL-8 expression by human epithelial cells than did NTHI lipooligosaccharides and envelope proteins. The IL-8-inducing activity was associated with molecules of < or =3 kDa from SCF and was peptidase and lipase sensitive, suggesting that small lipopeptides are responsible for the strong IL-8 induction. Moreover, multiple intracellular signaling pathways were activated in response to cytoplasmic molecules. The results indicated that the p38 mitogen-activated protein kinase (MAPK) and Src-dependent Raf-1-Mek1/2-extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) pathways are required for NTHI-induced IL-8 production. In contrast, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway did not affect IL-8 expression, although this pathway was concomitantly activated upon exposure to NTHI SCF. The PI3K-Akt pathway was also directly activated by IL-8 and significantly inhibited by an antagonist of IL-8 receptors during NTHI stimulation. These results indicated that the PI3K-Akt pathway is activated in response to IL-8 that is induced by NTHI and may lead to other important epithelial cell responses. This work provides insight into essential molecular and cellular events that may impact on the pathogenesis of OM and COPD and identifies rational targets for anti-inflammatory intervention.

Topics: Base Sequence; Cell Line; DNA, Complementary; Haemophilus Infections; Haemophilus influenzae; HeLa Cells; Humans; Interleukin-8; Lipase; Lipopolysaccharides; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Models, Biological; NF-kappa B; Otitis Media; p38 Mitogen-Activated Protein Kinases; Peptide Hydrolases; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Pulmonary Disease, Chronic Obstructive; Up-Regulation

Topics: Humans; Interleukin-1; Interleukin-6; Interleukin-8; Otitis Externa; Otitis Media; Tumor Necrosis Factor-alpha

To characterize the local response in acute otitis media, courses of interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)alpha in middle ear fluid (MEF) of the guinea pig otitis media model induced by nonviable Haemophilus influenzae were investigated with enzyme-linked immunosorbent assay (ELISA) kits. The IL-1beta concentration in H. influenzae-inoculated ears peaked 24 hours after inoculation. The IL-8 concentration was significantly higher in H. influenzae-inoculated ears than in controls 48 and 96 hours after inoculation. The TNF-alpha concentration in H. influenzae-inoculated ears had an initial peak 6 hours after inoculation and had significant late increases 48 and 96 hours after inoculation. The results suggest that IL-1beta and TNF-alpha were produced by middle ear mucosa in the early stage of the experiment by stimulation of bacterial inoculation, which caused subsequent inflammatory cell accumulation, and that IL-8 and TNF-alpha were produced in the late stage by accumulating inflammatory cells.

Topics: Acute Disease; Animals; Antibodies, Bacterial; Cross Reactions; Disease Models, Animal; Ear, Middle; Exudates and Transudates; Guinea Pigs; Haemophilus Infections; Haemophilus influenzae; Interleukin-1; Interleukin-6; Interleukin-8; Otitis Media; Tumor Necrosis Factor-alpha

The importance of interleukin (IL)-8 in the chemotactic activity of middle ear effusions (MEEs) was evaluated. There was a significantly higher IL-8 concentration in MEEs of children with acute otitis media (AOM) (n = 17; 136 ng/mL) than in children with otitis media with effusion (OME) (n = 28; 65 ng/mL). The IL-8 concentration in MEEs with bacteria (149 ng/mL) was significantly higher than in MEEs without bacteria (66 ng/mL). MEEs from children with AOM and OME had equally higher chemotactic activity than the diluent alone (23.3% and 24.8% vs. 9.2%). The chemotactic activity was not altered by the presence of bacteria nor did it correlate with IL-8 concentration. Fractionation of MEEs by gel chromatography demonstrated that the main chemotactic activity could clearly be separated from the IL-8 activity, thus excluding IL-8 as a main chemotactic component in MEEs.

Topics: Bacterial Infections; Chemotaxis; Child; Child, Preschool; Chromatography, Gel; Ear, Middle; Female; Humans; Infant; Interleukin-8; Male; Neutrophils; Otitis Media; Otitis Media with Effusion

Interleukin-8 (IL-8), a potent inflammatory mediator that is thought to control leukocyte recruitment and activation during inflammatory reactions, has been implicated in a variety of inflammatory diseases. Recent studies in our laboratory have demonstrated the presence of IL-8 in chronically inflamed human middle ear effusions. These data have led us to hypothesize that IL-8 is responsible for the leukocyte recruitment seen in otitis media. Because the effect of IL-8 on the middle ear mucosa has not been investigated and therefore its role in middle ear inflammation is not known, we investigated the ability of IL-8 to directly induce inflammation in the murine middle ear. For these studies, ICR mice were used to test the hypothesis that IL-8 could directly induce inflammation in the middle ear. Murine middle ears received 8-mL transtympanic injections of human IL-8 (25 mg/mL) in saline, heat-killed Streptococcus pneumoniae (1 x 10(8)/mL), or normal saline. Temporal bones were removed at 1, 4, 8, 24, and 48 hours, decalcified, and processed for histologic examination. Noninjected murine temporal bones served as controls. Normal saline-injected ears demonstrated little to no change as compared with temporal bones from noninjected ears. IL-8-injected ears histologically demonstrated thickening of the epithelial layer and subepithelial space (SES), with inflammatory cell infiltration in the SES peaking at 4 to 8 hours and resolving by 48 hours. Bacteria-injected ears demonstrated findings similar to, although not as extensive as, those found in IL-8-injected ears (i.e., inflammatory reactions peaked at 8 to 24 hours and resolved by 72 hours). Our results demonstrate that IL-8 is a potent inducer of middle ear inflammation and support the concept that IL-8 may be one of the key cytokines responsible for the leukocyte accumulation and activation seen in otitis media.

Topics: Animals; Female; Humans; Interleukin-8; Lymphocyte Activation; Mice; Mice, Inbred ICR; Neutrophils; Otitis Media; Pneumococcal Infections; Temporal Bone

In order to evaluate the role of polymorphonuclear leukocytes (PMNs) in acute otitis media (AOM), levels of leukotriene B4 (LTB4), a potent inflammatory product of PMNs, and interleukin-8 (IL-8), a PMN chemotactic cytokine, were measured in 271 middle ear fluid (MEF) samples from 106 children with AOM. Forty-two percent of the patients had evidence of respiratory viral infection. At the time of diagnosis, levels of both LTB4 and IL-8 were higher in the MEFs from patients with AOM associated with bacterial or bacterial and viral infection than those MEFs containing no pathogen (p < .05). Antibiotic treatment was not associated with a significant change in levels of LTB4 or IL-8 in the MEFs obtained 2 to 5 days into treatment, compared to those obtained at diagnosis. Bacteriologic failure after 2 to 5 days of treatment was associated with high LTB4 levels in the initial MEFs (p = .05). Recurrence of AOM within 1 month was associated with high IL-8 levels in the initial MEF (p = .04). Our findings suggest that LTB4 and IL-8 are produced during acute infection of the middle ear, and these PMN-related inflammatory substances may play an important role in delaying recovery or in recurrence of AOM. Effective treatment of AOM may require eradication of bacteria by antibiotics, as well as pharmacologic agents that modulate PMN functions.

Topics: Bacterial Infections; Enzyme-Linked Immunosorbent Assay; Female; Humans; Infant; Interleukin-8; Leukotriene B4; Male; Neutrophils; Otitis Media; Recurrence; Respiratory Tract Infections; Virus Diseases