transforming-growth-factor-beta and Otitis-Media

The study objective was to assess the levels of VEGF-A and TGF-β cytokines in the children with adenoid hypertrophy concomitant with exudative otitis media (OME) and in children with adenoid hypertrophy (HA) alone.. The study material consisted of hypertrophic adenoids removed during adenoidectomy from 39 children (20 girls and 19 boys), aged 2-7 years suffering from OME. The reference group included 41 children (19 girls and 22 boys), aged from 3 to 9 years with adenoid hypertrophy. The levels of VEGF-A and TGF-β were determined in supernatants obtained from phytohemagglutinin-stimulated cell cultures of the adenoids using a commercial enzyme-linked immunosorbent assay kit.. The median VEGF-A and mean TGF-β concentrations in the study group were significantly higher than those in the reference group (503 pg/mL versus 201 pg/mL, P < 0.001 and 224 pg/mL versus 132 pg/mL, P < 0.001, respectively). ROC analysis revealed that the area under the curve (AUC) for VEGF-A was 0.952 with diagnostic sensitivity and specificity of 95%, whereas for TGF-β it was 0.902 with 60% sensitivity and the same specificity as for VEGF-A. There was no significant difference between the AUC for VEGF-A and TGF-β (P = 0.573).. The changes in the levels of VEGF-A and TGF-β may indicate bacterial pathogen as one of the causes of exudative otitis media in children. Determination of VEGF-A and TGF-β could be used as additional and objective tests to confirm the clinical diagnosis.

Topics: Adenoidectomy; Adenoids; Area Under Curve; Child; Child, Preschool; Exudates and Transudates; Female; Humans; Hypertrophy; Male; Otitis Media; Otitis Media with Effusion; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFβ) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFβ signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 (Tgif1). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1β in ear fluids. We also identified downstream effects on TGFβ signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFβ pathway. The identification and characterization of the Tgif mutant supports the role of TGFβ signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population.

Topics: Animals; Craniofacial Abnormalities; Cytokines; Disease Models, Animal; Ear, Middle; Epithelial Cells; Female; Genotype; Hair Cells, Auditory; Hearing Loss; Homeodomain Proteins; Homozygote; Male; Mice; Mice, Knockout; Mutation; Otitis Media; Phenotype; Placenta; Pregnancy; Repressor Proteins; Signal Transduction; Transforming Growth Factor beta

Genetic predisposition is recognized as an important pathogenetic factor in otitis media (OM) and associated diseases. Mutant Lmna mice heterozygous for the disheveled hair and ears allele (Lmna(Dhe/+)) exhibit early-onset, profound hearing deficits and other pathological features mimicking human laminopathy associated with the LMNA mutation. We assessed the effects of the Lmna(Dhe/+) mutation on development of OM and pathological abnormalities characteristic of laminopathy. Malformation and abnormal positioning of the eustachian tube, accompanied by OM, were observed in all of the Lmna(Dhe/+) mice (100% penetrance) as early as postnatal day P12. Scanning electronic microscopy revealed ultrastructural damage to the cilia in middle ears that exhibited OM. Hearing assessment revealed significant hearing loss, paralleling that in human OM. Expression of NF-κB, TNF-α, and TGF-β, which correlated with inflammation and/or bony development, was up-regulated in the ears or in the peritoneal macrophages of Lmna(Dhe/+) mice. Rugous, disintegrative, and enlarged nuclear morphology of peritoneal macrophages and hyperphosphatemia were found in Lmna(Dhe/+) mutant mice. Taken together, these features resemble the pathology of human laminopathies, possibly revealing some profound pathology, beyond OM, associated with the mutation. The Lmna(Dhe/+) mutant mouse provides a novel model of human OM and laminopathy.

Topics: Acoustic Impedance Tests; Animals; Calcium; Cell Count; Cell Movement; Cilia; Cytokines; Disease Models, Animal; Ear, Middle; Epithelial Cells; Eustachian Tube; Evoked Potentials, Auditory, Brain Stem; Gene Expression Regulation; Humans; Inflammation Mediators; Ions; Lamin Type A; Macrophages, Peritoneal; Mice; Mice, Mutant Strains; Otitis Media; Otoacoustic Emissions, Spontaneous; Phosphorus; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Otitis media (OM) is a common childhood disease characterised by middle ear inflammation and effusion. Susceptibility to recurrent acute OM (rAOM; ≥ 3 episodes of AOM in 6 months) and chronic OM with effusion (COME; MEE ≥ 3 months) is 40-70% heritable. Few underlying genes have been identified to date, and no genome-wide association study (GWAS) of OM has been reported.. Data for 2,524,817 single nucleotide polymorphisms (SNPs; 535,544 quality-controlled SNPs genotyped by Illumina 660W-Quad; 1,989,273 by imputation) were analysed for association with OM in 416 cases and 1,075 controls from the Western Australian Pregnancy Cohort (Raine) Study. Logistic regression analyses under an additive model undertaken in GenABEL/ProbABEL adjusting for population substructure using principal components identified SNPs at CAPN14 (rs6755194: OR = 1.90; 95%CI 1.47-2.45; P(adj-PCA) = 8.3 × 10(-7)) on chromosome 2p23.1 as the top hit, with independent effects (rs1862981: OR = 1.60; 95%CI 1.29-1.99; P(adj-PCA) = 2.2 × 10(-5)) observed at the adjacent GALNT14 gene. In a gene-based analysis in VEGAS, BPIFA3 (P(Gene) = 2 × 10(-5)) and BPIFA1 (P(Gene) = 1.07 × 10(-4)) in the BPIFA gene cluster on chromosome 20q11.21 were the top hits. In all, 32 genomic regions show evidence of association (P(adj-PCA)<10(-5)) in this GWAS, with pathway analysis showing a connection between top candidates and the TGFβ pathway. However, top and tag-SNP analysis for seven selected candidate genes in this pathway did not replicate in 645 families (793 affected individuals) from the Western Australian Family Study of Otitis Media (WAFSOM). Lack of replication may be explained by sample size, difference in OM disease severity between primary and replication cohorts or due to type I error in the primary GWAS.. This first discovery GWAS for an OM phenotype has identified CAPN14 and GALNT14 on chromosome 2p23.1 and the BPIFA gene cluster on chromosome 20q11.21 as novel candidate genes which warrant further analysis in cohorts matched more precisely for clinical phenotypes.

Topics: Case-Control Studies; Child; Child, Preschool; Chromosome Mapping; Cohort Studies; Female; Genetic Predisposition to Disease; Genome-Wide Association Study; Genomics; Genotype; Humans; Infant; Infant, Newborn; Male; Models, Genetic; Odds Ratio; Otitis Media; Phenotype; Polymorphism, Single Nucleotide; Pregnancy; Regression Analysis; Risk Factors; Surveys and Questionnaires; Transforming Growth Factor beta

Otitis media (OM) is a common childhood disease characterised by middle ear inflammation following infection. Susceptibility to recurrent acute OM (rAOM) and chronic OM with effusion (COME) is highly heritable. Two murine mutants, Junbo and Jeff, spontaneously develop severe OM with similar phenotypes to human disease. Fine-mapping of these mutants identified two genes (Evi1 and Fbxo11) that interact with the transforming growth factor β (TGFβ) signalling pathway. We investigated these genes, as well as four Sma- and Mad-related (SMAD) genes of the TGFβ pathway, as candidate rAOM/COME susceptibility genes in two predominantly Caucasian populations. Single-nucleotide polymorphisms (SNPs) within FBXO11 (family-based association testing Z-Score=2.61; P(best)=0.009) were associated with severe OM in family-based analysis of 434 families (561 affected individuals) from the Western Australian Family Study of OM. The FBXO11 association was replicated by directed analysis of Illumina 660W-Quad Beadchip data available for 253 cases and 866 controls (OR=1.55 (95% CI 1.28-1.89); P(best)=6.9 × 10(-6)) available within the Western Australian Pregnancy Cohort (Raine) Study. Combined primary and replication results show P(combined)=2.98 × 10(-6). Neither cohort showed an association with EVI1 variants. Family-based associations at SMAD2 (P=0.038) and SMAD4 (P=0.048) were not replicated. Together, these data provide strong evidence for FBXO11 as a susceptibility gene for severe OM.

Topics: Alleles; Australia; Child; Child, Preschool; DNA-Binding Proteins; F-Box Proteins; Genetic Predisposition to Disease; Haplotypes; Humans; Linkage Disequilibrium; MDS1 and EVI1 Complex Locus Protein; Otitis Media; Polymorphism, Single Nucleotide; Protein-Arginine N-Methyltransferases; Proto-Oncogenes; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

Granulation tissue is common in otitis media (OM), yet little is known about the signaling pathways in the formation of granulation tissue in response to infections. In this study, we sought to investigate the activation of the transforming growth factor beta (TGF-beta) signaling pathway in the formation of granulation tissue in response to middle ear pathogens.. Rat OM models were made by inoculating pneumococcus type 6A or nontypeable Haemophilus influenzae into the middle ear cavity or by obstructing the eustachian tube. Various pathway activities in the middle ear mucosa were analyzed with microarrays.. The TGF-beta signaling pathway was highly regulated in the middle ear cleft with bacterial OM, but not in the ears with eustachian tube obstruction. In ears with bacterial OM, the TGF-beta signaling pathway products were higher in Haemophilus-infected ears than in pneumococcus-infected ears.. Bacterial OM triggers granulation tissue to thrive in the middle ear cleft of rats. Nontypeable H influenzae is more potent than pneumococcus type 6A in the formation of granulation tissue. Eustachian tube obstruction alone did not contribute to granulation tissue formation in the middle ear.

Topics: Animals; Collagen; Down-Regulation; Ear, Middle; Epithelial Cells; Eustachian Tube; Fibroblasts; Fibronectins; Gene Expression Profiling; Granulation Tissue; Haemophilus Infections; Microarray Analysis; Models, Animal; Otitis Media; Pneumococcal Infections; Rats; Rats, Sprague-Dawley; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA; Signal Transduction; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta; Up-Regulation

Helicobacter pylori is a Gram-negative bacterium that is recognized as one of the key factors in gastric diseases such as gastritis, peptic ulcer and gastric cancer. Recent studies have shown relationships between H. pylori and extra-digestive diseases, and the presence of H. pylori in the middle ear and upper respiratory tract has been reported. However, the role of H. pylori in middle ear disease remains unclear. The present study demonstrated that H. pylori whole-cell protein directly induces macrophage migration inhibitory factor, macrophage inflammatory protein 2, interleukin 1 beta and tumor necrosis factor alpha in middle ear epithelium in mice, and severe proliferation of inflammatory cells was observed in middle ear cavity inoculated with H. pylori whole-cell protein. In addition, trans-tympanic injection of macrophage migration inhibitory factor up-regulated expression of macrophage inflammatory protein 2 in the middle ear. These findings indicate that H. pylori infection causes immunological inflammation in middle ear epithelium, and H. pylori may play a significant role in otitis media.

Topics: Animals; Antigens, Bacterial; Biomarkers; Chemokine CXCL2; Ear, Middle; Helicobacter Infections; Helicobacter pylori; Interleukin-10; Interleukin-1beta; Macrophage Migration-Inhibitory Factors; Male; Mice; Mice, Inbred C57BL; Models, Animal; Otitis Media; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Chronic otitis media (COM) is an inflammatory process involving the middle ear mucosa and the tympanic membrane. The healing and epidermization is mostly impaired by immunological response of the host. Investigating the activity and the function of immunological response elements one can learn the immunological mechanisms taking place in chronic otitis media. The ultrastructural investigations of the tympanic membrane were done on its fragments obtained from 19 patients with COM during middle ear surgery, performed at ENT Department of Medical University of Gdańsk in the years 1997-1999. Immunohistochemical investigations were performed using monoclonal antibodies against tenascin, S-100 protein, Ki 67, CD 31, F VIII, HLA-DR, TGFbeta1 and EGFR. The control group was 11 healthy tympanic membranes from cadavers. The presence of tenascin was proven in all COM tympanic membranes and in 45.5% of those from control group. S-100 protein was present in 88.9% of the patients with COM and absent in control group. Ki 67 was observed in 44.4% of the patients with COM and in 27.3% of the healthy tympanic membranes. Angiogenesis factors (CD 31 and FVIII) were present in 77.8% of the investigated COM tympanic membranes, in control group in 45.5%. HLA-DR expression was observed in 90% COM patients, in control group in 72.7%. Growth factor TGFbeta1 was present in the all cases in mucous and fibrous layer and in 54.5% of healthy tympanic membranes. EGF receptor was present in 60% of COM patients, mainly in epithelial layer of tympanic membrane and in 54.5% of those from control group. The presented investigations confirm the immunological activity of tympanic membrane in chronic otitis media.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Case-Control Studies; Chronic Disease; ErbB Receptors; Female; HLA-DR Antigens; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Otitis Media; Platelet Endothelial Cell Adhesion Molecule-1; Poland; S100 Proteins; Tenascin; Time Factors; Transforming Growth Factor beta; Tympanic Membrane; von Willebrand Factor

Molecular and cellular mechanisms in chronic otitis media (COM) with cholesteatoma have not been clearly enough known so far. Investigations on cholesteatoma are focused on its immunological and morphological status. The authors presented the results of immunomorphological evaluation of 31 patients with COM with cholesteatoma, divided into three groups. In the first group there were 4 patients with congenital cholesteatoma, in the second group 19 patient with primary acquired cholesteatoma and in the third group 8 patient with secondary acquired cholesteatoma. Immunohistochemical investigations were performed using antibodies for identification the tenascin, S-100 protein, antigens Ki 67, CD 31, FVIII, HLA-DR, and growth factors TGFbeta1 and EGFR. The immunological activity was assessed for three types and matrix and perimatrix of cholesteatoma. High expression of tenascin was proven in the perimatrix of cholesteatoma and protein S-100 was highly expressed in the cholesteatoma matrix. Ki 67 antigen was seen rarely and mostly was present in basal cells of the matrix. The presence of endothelial cells was proven mainly in the connective and vascular tissue of perimatrix (CD 31, FVIII). The high expression of HLA-DR in matrix confirms the presence of Langerhans cells. The presence of growth factors TGFbeta1 and EGFR was observed in peribasal layer of the epithelium and keratinocytes of cholesteatoma matrix. Activity of immunological processes in COM with cholesteatoma confirms their important role in pathophysiology of chronic otitis media with cholesteatoma.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Child; Child, Preschool; Cholesteatoma, Middle Ear; ErbB Receptors; Female; HLA-DR Antigens; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Otitis Media; Platelet Endothelial Cell Adhesion Molecule-1; Poland; S100 Proteins; Tenascin; Transforming Growth Factor beta; von Willebrand Factor

Acute otitis media (AOM) is the most common reason for outpatient antimicrobial therapy. Mixed infections pose a potential problem, since the first-line drug used for the treatment of AOM, amoxicillin, can be neutralized by beta-lactamase-producing pathogens of the upper respiratory tract. To study the effects of a 5-day course of amoxicillin on a mixed middle ear infection, rats were challenged with Streptococcus pneumoniae alone or in combination with beta-lactamase-producing nontypeable Haemophilus influenzae. Amoxicillin was introduced at the clinical peak of the infection. Local and systemic changes were monitored by otomicroscopy, bacterial culture, and analysis of histological changes and the expression of the transforming growth factor beta (TGF-beta) gene. beta-Lactamase-producing H. influenzae did not demonstrate an ability to protect S. pneumoniae. Amoxicillin eradicated the pneumococci in all treated animals but increased to some degree the ability of H. influenzae to persist at the site of infection. Thus, only an insignificant acceleration of the resolution of the AOM caused by a mixture of pathogens was observed during treatment. Moderate to major morphological changes could not be avoided by treatment of the mixed infections, but a slight downregulation of TGF-beta expression was observed. In contrast to infections caused by a single pathogen, the mixed infections induced white plaques in the tympanic membrane at a remarkably high frequency independent of treatment. These experimental findings constitute support for further studies of antimicrobial drugs and AOM caused by bacteria with and without mechanisms of antibiotic resistance.

Topics: Amoxicillin; Animals; beta-Lactamases; Culture Media; Ear, Middle; Haemophilus Infections; Haemophilus influenzae; Male; Otitis Media; Penicillins; Pneumococcal Infections; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Streptococcus pneumoniae; Transforming Growth Factor beta; Treatment Outcome

Acute otitis media (AOM) elicits potent inflammatory responses from the cells of the middle ear mucosa as well as from infiltrating leukocytes. To explore host responses during experimental AOM induced by Streptococcus pneumoniae type 3 and nontypeable Haemophilus influenzae (NTHi), otomicroscopy findings and expression of cytokine genes in the middle ear were monitored up to 1 month postinoculation. The mucosa and infiltrating cells responded rapidly to the bacterial challenge. Otomicroscopically, AOM appeared 1 day after NTHi inoculation and 3 days after pneumococcus inoculation. Pneumococcal AOM was more severe than NTHi otitis, but in general, lower transcript levels were detected in pneumococcus-infected than in NTHi-infected animals. Interleukin-6 (IL-6) mRNA levels peaked at 3 to 6 h for both pneumococcus-infected and NTHi-infected animals. IL-1alpha, tumor necrosis factor alpha, and IL-10 mRNA levels peaked at 6 h for NTHi otitis and 1 to 3 days for pneumococcal otitis. Comparing otomicroscopy with expression profiles, it would appear that the majority of cytokine mRNAs had passed their peak before the AOM diagnosis could be made clinically. Only transforming growth factor beta mRNA followed a slower time course, peaking very late and continuing expression even after the AOM was otomicroscopically resolved. IL-2 and IL-4 mRNAs were not detected in any animal at any time. Most of the investigated cytokines are very early markers for AOM and may be involved in initiation of inflammation, but they would be poor targets for pharmacological manipulation since their levels decline before clinical signs appear.

Topics: Acute Disease; Animals; Base Sequence; Cytokines; Disease Models, Animal; DNA Primers; Gene Expression; Haemophilus Infections; Haemophilus influenzae; Humans; Interleukins; Kinetics; Male; Otitis Media; Pneumococcal Infections; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha