Treatment with a neutrophil elastase inhibitor and ofloxacin reduces P. aeruginosa burden in a mouse model of chronic suppurative otitis media.

Chronic suppurative otitis media (CSOM) is a widespread, debilitating problem with poorly understood immunology. Here, we assess the host response to middle ear infection over the course of a month post-infection in a mouse model of CSOM and in human subjects with the disease. Using multiparameter flow cytometry and a binomial generalized linear machine learning model, we identified Ly6G, a surface marker of mature neutrophils, as the most informative factor of host response driving disease in the CSOM mouse model. Consistent with this, neutrophils were the most abundant cell type in infected mice and Ly6G expression tracked with the course of infection. Moreover, neutrophil-specific immunomodulatory treatment using the neutrophil elastase inhibitor GW 311616A significantly reduces bacterial burden relative to ofloxacin-only treated animals in this model. The levels of dsDNA in middle ear effusion samples are elevated in both humans and mice with CSOM and decreased during treatment, suggesting that dsDNA may serve as a molecular biomarker of treatment response. Together these data strongly implicate neutrophils in the ineffective immune response to P. aeruginosa infection in CSOM and suggest that immunomodulatory strategies may benefit drug-tolerant infections for chronic biofilm-mediated disease.

Current concepts in the pathogenesis and treatment of chronic suppurative otitis media.

Otitis media (OM) is an inflammation of the middle ear associated with infection. Despite appropriate therapy, acute OM (AOM) can progress to chronic suppurative OM (CSOM) associated with ear drum perforation and purulent discharge. The effusion prevents the middle ear ossicles from properly relaying sound vibrations from the ear drum to the oval window of the inner ear, causing conductive hearing loss. In addition, the inflammatory mediators generated during CSOM can penetrate into the inner ear through the round window. This can cause the loss of hair cells in the cochlea, leading to sensorineural hearing loss. Pseudomonas aeruginosa and Staphylococcus aureus are the most predominant pathogens that cause CSOM. Although the pathogenesis of AOM is well studied, very limited research is available in relation to CSOM. With the emergence of antibiotic resistance as well as the ototoxicity of antibiotics and the potential risks of surgery, there is an urgent need to develop effective therapeutic strategies against CSOM. This warrants understanding the role of host immunity in CSOM and how the bacteria evade these potent immune responses. Understanding the molecular mechanisms leading to CSOM will help in designing novel treatment modalities against the disease and hence preventing the hearing loss.

Attenuated TLRs in middle ear mucosa contributes to susceptibility of chronic suppurative otitis media.

The variability in the recovery of otitis media (OM) is not well understood. Recent data have shown a critical role for toll-like receptors (TLRs) in inflammatory responses to bacteria. It remains unclear whether TLRs-mediated mucosal immunity plays a role in the OM recovery. The etiology, pathological profile, expression levels of TLR2, TLR4, TLR5, TLR9 and proinflammatory cytokines were measured in human middle-ear mucosae sampled from three subject groups: non-OM group, chronic otitis-media (COM) group, and chronic suppurative otitis-media (CSOM) group. Of the 72 ears, 86.11% CSOM patients were positive for bacteria. The cellular makeup of the middle ear mucosa differs among the three groups. Mucosae from the CSOM group presented chronic inflammation or suppurative inflammation in the rudimentary stroma, mainly with infiltration of monocytes and macrophages. The mRNA and protein levels of TLR2, TLR4, and TLR5 exhibited no difference between the non-OM and COM groups but were significantly lower in the CSOM group. Conversely, there was no significant difference in the TLR9 level among the three groups. Furthermore, proinflammatory cytokines TNF-α, IL-1ß, IFN-γ, IL-6 were up-regulated in the CSOM group. This study provides evidence that the variability in clinical otitis media recovery might be associated with the variability in the expression of mucosal TLRs. Reduced TLR levels in the middle-ear mucosa might cause weak host response to bacteria, persistent inflammation and susceptibility to CSOM.

Allergic rhinitis and chronic suppurative otitis media.

Allergic inflammation in upper airways can act as a predisposing factor for infectious ear diseases. There are some evidences about the role of allergic rhinitis in chronic otitis media with effusion, but its role in establishing chronic suppurative otitis media (CSOM) has not been clearly shown. 68 adult patients with established CSOM, who were candidates for ear surgery, and 184 age- and sex-matched controls were evaluated for the presence of allergic rhinitis. Standard questionnaire was filled out for all participants. All patients and controls underwent skin prick test for 28 common regional aeroallergens, and serum total IgE was measured by means of ELISA method. Allergic rhinitis were defined as a positive responses to the questionnaire, positive skin prick test to at least one allergen, and/or high level of serum total IgE. Allergic rhinitis was diagnosed in 20 (29.41%) and 41 (22.28%) of patients and controls, respectively (P = 0.241) (OR = 1.28, CI = 0.69-2.36). Outdoor allergens, especially grass pollen, were the most prevalent allergens among both groups, but indoor allergens like mites and molds have a low prevalence. The study did not show a significant difference in the prevalence of AR in the CSOM patients compared to the controls. The intermittent nature of allergy and other less known intervening factors in the etiopathogenesis of CSOM make such a conclusion difficult.

[Immune status of patients with exacerbation of chronic suppurative otitis media and its correction].

The authors report results of a clinical and immunological study involving 38 patients with exacerbation of chronic suppurative otitis media. It has demonstrated high immunological and clinical efficiency of combined treatment with derinate (an agent stimulating local immunity) and high molecular-weight polypeptide polyoxidonium. It is concluded that inclusion of these drugs in combined therapy of chronic suppurative otitis media increases its immunological and clinical effectiveness and allows duration of patients' hospitalization to be reduced.

[Assessment of immune disorders in patients presenting with various forms of chronic suppurative otitis media in the postoperative period].

The present study was designed for immunological examination of 46 patients presenting with various forms of chronic suppurative otitis media. All the patients underwent surgical intervention on mastoid process (processus mastoideus). It was shown that exacerbation of catarrhal inflammation provoked a significant decrease of the total lymphocyte, T-helpers, and B-lymphocyte (CD22) count whereas the concentration of T-suppressors increased. In patients with the distractive form of chronic suppurative otitis media, the absolute number of total lymphocytes, T-lymphocytes, T-helpers, and B-lymphocytes was also reduced while the concentration of IgG was elevated. The level of serum cytokines (IL-1-beta, IL-6, TNF-alpha) increased in both forms of the disease although the rise in case of catarrhal inflammation was much more pronounced. These data suggest beneficial effect of supplemental corrective immunotherapy.

[Peculiarities of the treatment of patients presenting with chronic suppurative otitis media associated with Gram-negative microorganisms].

The objective of the present study was to determine the absolute and relative numbers of T-lymphocytes, B-lymphocytes, T-suppressors, T-helpers, phagocytes, immunoglobulins A, M, and G in blood sera of patients presenting with chronic suppurative otitis media who had undergone sanation surgery. The study included tests for the evaluation of immunological efficiency of combined treatment with imunofan and myelopid in 89 patients divided into two groups. Pretreatment immunological status of all patients was characterized by reduced B-lymphocyte, T-helper, and phagocyte counts coupled to the elevated levels of IgM and T-suppressors. Patients of group 1 received conventional antibacterial therapy, those in group 2 were treated with a combination of imunofan and myelopid. Conventional therapy failed to correct immune deficiency. Additional treatment with a combination of imunofan and myelopid following sanation surgery proved to be much more efficacious. This observation was confirmed by the results of clinical, cytological, and immunological studies.

Comparative analysis of middle ear immune response and micronutrient level between mucoid and purulent otitis media.

OBJECTIVE: To determine the differences in the outcome of treatment, the middle ear immune response, and micronutrient status between patients with purulent (POM) and mucoid (MOM) otitis media. DESIGN: Children with acute otitis media were recruited, treated, and followed up for between 6 and 10 months. The sera and middle ear secretion (MES) were analyzed for immunoglobulins and zinc using enzyme-linked immunoassay and flame atomic absorption spectrophotometry, respectively. The parameters were compared with selected healthy controls. SUBJECTS: There were 399 participants: 228 children with acute otitis media selected using the American Academy of Otolaryngology-Head and Neck Surgery criteria and 171 healthy controls, with ages ranging between 6 months and 9 years (mean 7 years; SD 2.32 years). RESULT: POM accounted for 126 of 228 patients, and MOM accounted for 102. The chronicity of otitis media (using the 3-month duration cutoff) was seen in 87 subjects (46%): 31 of 126 POM subjects and 56 of 102 MOM subjects (p = .002). The serum IgA to IgG ratios were 0.03 and 0.07 among POM and MOM, respectively; however, the MES IgA to IgG ratios were higher, 0.3 and 0.2, for POM and MOM, respectively. This gives a MES to serum ratio of IgA to IgG value of 0.3 to 0.03 (10) for POM and 0.2 to 0.07 (2.67) for MOM, which showed a significant statistical difference (p = .001). The MES to serum ratios of IgE were 0.89 and 1.3 among POM and MOM, respectively (p = .03). There was a significant difference in the serum IgG (p = .019), serum zinc (p = .009), and MES IgA (p = .028) and IgG (p = .01). CONCLUSION: Chronicity was greater in MOM than in POM; POM had a higher middle ear immune response (measured by the MES to serum ratio of IgA to IgG) and serum zinc than MOM but a lower MES IgE.

[Influence of dentoalveolar pathology on the efficiency of conservative treatment of chronic suppurative otitis].

The study was designed to analyse immunoreactivity in patients with chronic suppurative otitis and results of its treatment. A separate group was comprised of patients with dentoalveolar pathology. It is concluded that the efficacy of therapy of chronic suppurative otitis depends on the presence of concomitant dentoalveolar abscess and carious lesions.

Serum and middle ear immunoglobulins in suppurative otitis media.

BACKGROUND AND PURPOSE: Despite the increasing occurrence of suppurative otitis media (SOM), the content of immunoglobulin fractions in the middle ear secretion (MES) has still not been determined. METHOD: The serum and MESwere analyzed for IgG and M using enzyme-linked immunoassay. RESULT: The subjects and controls were 30 males and 22 females, between 6 months and 9 years old, with a mean age of 6 years (SD = 3.26). The patient group included 20 chronic SOM (CSOM) and 17 acute SOM (ASOM) cases, and sera of 15 subjects made the control group. The mean serum IgG levels were for controls 1,051 mg/dl, ASOM 666.1 mg/dl and CSOM 1,321.1 mg/dl; the MES levels were for ASOM 203.4 mg/dl and CSOM 511.5 mg/dl. The mean serum IgM levels were for controls 35 mg/dl, ASOM 64.1 mg/dl and CSOM 40 mg/dl; the MES levels were for ASOM 22.59 mg/dl and CSOM 3.44 mg/dl. The mean MES:serum ratios for IgG and IgM were between 0.1 and 0.4 in ASOM and CSOM. The ratio of serum IgG levels of controls to ASOM cases was 0.66 while that of controls to CSOM was 1.3. The corresponding ratios of IgM were 1.6 and 0.88. Multivariate analysis revealed a significant correlation between serum IgG levels of ASOM and CSOM (p = 0.043) and MES IgG (p = 0.02) in ASOM and CSOM but no correlation between serum IgG levels in controls and ASOM (p = 0.25), serum IgM levels in controls and CSOM (p = 0.62) and serum IgM levels in controls and ASOM (p = 0.73), ASOM and CSOM (p = 0.064) and MES IgM levels of ASOM and CSOM (p = 0.06). CONCLUSIONS: Monitoring of the serum and MES IgG in ASOM may provide a useful index to assess the possibility of progression to chronicity. This forms a database for the immunological status of SOM patients.

Role of elevated immunoglobulin E levels in suppurative otitis media.

BACKGROUND AND OBJECTIVES: An association between suppurative otitis media (SOM) and allergy has been reported in about 80% of patients with allergy. However, there is controversy regarding their relationship and the concept of middle-ear allergic response. We test the hypothesis that increased secretion of IgE in the middle ear is higher in chronic than in acute SOM. METHODS: Allergy skin testing and enzyme-linked immunoassay of specimens of middle-ear secretions and sera were analysed. RESULTS: Paired sera and middle-ear secretions (MES) from 37 subjects with SOM, 20 chronic (CSOM) and 17 acute (ASOM), and sera of 15 controls selected from children without otitis media were analysed. There were 30 males and 27 females aged between 6 months and 9 years, mean (SD) 6 years (3.26). A history of allergy and skin test positive to one or more of dust, house dust mite, mould, cockroach and poultry feathers were found in 80% of CSOM, 47% ASOM and 33% controls. The mean IgE levels in sera were: controls 52.1 mg/dL, ASOM 63.9 mg/dL and CSOM 79.2 mg/dL; the MES levels were: AOM 60.4 mg/dL and CSOM 102.0 mg/dL. The MES to serum IgE ratios were 0.75 for ASOM and 1.4 for CSOM. The serum IgE ratio of controls to ASOM was 1.22 and to CSOM was 1.5. Multivariate analysis of the mean showed significant correlation between IgE level of MES in ASOM and CSOM (p=0.04) but no correlation between IgE levels in control and ASOM sera (p=0.10), control and CSOM sera (p=0.7) or AOM and CSOM sera (p=0.3). CONCLUSION: Allergy appears to play a contributory role in CSOM and elevated IgE in the MES suggests a likely mucosal response.

Toll-like receptors 2, 3 and 4 (TLR-2, TLR-3 and TLR-4) are expressed in the microenvironment of human acquired cholesteatoma.

Human toll-like receptors (TLR 1-10) are crucial in the induction and activation of innate immunity in the course of an infection. They are expressed mainly on the cells of the immune system, and also on some epithelia and endothelia. Their ligands so called pathogen associated molecular patterns are abundant on invading microbes. TLR-ligand binding results in cell signal transduction and subsequent production of various proinflammatory cytokines such as IL-1 and TNF-alpha. Acquired cholesteatoma is formed during chronic otitis media in the proportion of cases. It has adverse effects on ear structures, resulting in osteolysis and bone resorption. Its formation and pathogenesis are not fully understood. The current study attempted to search the possible role of TLRs in this somewhat awkward pathological condition. Surgical specimens of human acquired cholesteatoma (n=15) and normal external auditory canal skin (n=5, control tissues) were tested by immunohistochemistry for the presence of TLRs. Three TLRs were examined: TLR-2, TLR-3 and TLR-4. All TLRs tested were demonstrated in matrix (layer of keratinizing epithelium) and perimatrix (granulation tissue) of this inflammatory tumour. Expression of particular TLRs within the keratinizing epithelium was distinct and uneven. In the perimatrix, numerous T (CD3+) cells were seen and relatively few macrophages (CD11c+, HLA-DR+). There was a weak expression of all TLRs on normal (non-inflammatory) skin. Expression of TLR-3 both on the epithelium and some cells within the perimatrix and the presence of T cells may suggest that apart from innate immune responses, mechanisms of adaptive immunity also operate in cholesteatoma. Weak expression of these receptors on normal skin may also suggest the important role of TLRs in the etiopathogenesis of cholesteatoma.

Lipopolysaccharide concentration and bone resorption in cholesteatoma.

HYPOTHESIS: There is a relationship between the local lipopolysaccharide (LPS) concentration in cholesteatoma and local bone resorption in chronic otitis media (COM) with cholesteatoma. BACKGROUND: During the past decade, it has become known that the recruitment of osteoclasts is the main causative factor that induces bone destruction in COM with cholesteatoma. Cellular inflammation factors like cytokines may trigger the osteoclast. Sequel to this, LPS is able to up-regulate cytokines. This makes it of interest to study whether the local LPS concentration is related to bone resorption in cholesteatoma. MATERIALS AND METHODS: Twenty-four cholesteatoma samples and control tissue from COM patients without cholesteatoma were collected. During surgery, the degree of bone resorption was established and classified. Retrospectively, the authors checked whether patients had chronic purulent otorrhea. LPS concentration of the tissue samples was measured by the limulus amebocyte lysate test. The one-way analysis of variance test was used to determine the relation between LPS concentration, otorrhea, and local bone resorption. RESULTS: A significantly higher concentration of LPS was measured in samples from patients with cholesteatoma with bone resorption and otorrhea compared with cholesteatoma without bone resorption and control tissue. There were no significant differences between the LPS levels of the different groups of patients with bone resorption. CONCLUSION: It is suggested that LPS is one of the first factors in the cascade of bone resorption in COM with cholesteatoma.

[Some indices of cellular and humoral immunity in infants with acute purulent otitis media]. / Nekotorye pokazateli kletochno-gumoral'nogo immuniteta u detei rannego vozrasta, bol'nykh ostrym gnoinym srednim otitom

The examination of 32 infants with acute otitis media purulenta identified shifts in immunological indices: T-cell immunity appeared 1.5-times weaker (up to 31.5 +/- 2.6%) than in healthy controls, synthesis of serum immunoglobulins intensified (IgA, IgM and IgG levels rose to 161 +/- 18 mg%, 124 +/- 11 mg% and 963 +/- 47 mg%, respectively). Thus, infants with acute otitis media purulenta need not only conventional therapeutic measures but also correction of disturbed cellular and humoral immunity.

Cellular proliferation of mucosa-associated lymphoid tissue with otitis media: a preliminary study.

Histopathologic and immunohistochemical analyses were used to investigate the cellular proliferation of mucosa-associated lymphoid tissue (MALT) in human temporal bones with and without evidence of otitis media (OM). Anti-proliferating cell nuclear antigen (PCNA) antibody (clone PC10) was applied after the antigen retrieval procedure. Positive PCNA expression was observed in temporal bones that had been stored for 10 to 31 years in 80% ethanol. In specimens with purulent OM, the MALT had faint germinal centers (GCs). Positive PCNA expression in the MALT was moderate and scattered. In specimens with mucous OM, the MALT had complete GCs. Positive PCNA expression in the MALT was moderate to strong, and the distribution of PCNA-positive cells was associated predominantly in the GCs, the mucosal epithelial layer, and/or the subepithelial layer. In specimens with serous OM, the MALT also had complete GCs. However, the PCNA expression was weak and scattered, and appeared to be similar to that of the MALT in the temporal bones without OM. These results indicate that the cellular proliferation of MALT in the temporal bone might reflect the activity that produces secretory IgA against invasion of foreign antigens. However, further studies are needed to elucidate whether the PCNA expression within MALT in the eustachian tube and middle ear is associated with a mucosal immune response to inflammation as in OM.

Proliferating macrophages, dendritic cells, natural killer cells, T and B lymphocytes in the middle ear and Eustachian tube mucosa during experimental acute otitis media in the rat.

Although many studies focus on the increase of immunocompetent cells within the middle ear mucosa during acute otitis media it is poorly understood how this increase is mediated. The differentiation between two possible causes, i.e. immigration and local proliferation, would help to better understand the pathophysiology of this disease. Therefore, the number of proliferating macrophages, dendritic cells, natural killer cells and T and B lymphocytes was studied during acute otitis media in the rat middle ear mucosa (ME mucosa) and Eustachian tube mucosa (ET mucosa) by labelling proliferating leucocytes with the DNA precursor bromodeoxyuridine (BrdU). By removing the middle ear and Eustachian tube 24 h after BrdU injection, the contribution of immigrated newly formed cells was estimated. At this timepoint, many leucocytes in the ME and ET mucosa had incorporated BrdU (between 15 and 25% within the subsets). By analysing these tissues one hour after BrdU injection, the local proliferation rate was determined (between 2 and 9% within the subsets). Thus, the inflamed ME and ET mucosa are the destination of immunocompetent cells and, as our data show, the inflamed microenvironment supports local proliferation of immunocompetent cells.

[Pathogenesis and treatment of chronic purulent otitis media and its complications: immunological aspects]. / Immunologicheskie aspekty patogeneza i lecheniia khronicheskogo gnoinogo srednego otita i ego oslozhnenii.

Clinicoimmunological, microbiological and cytological examinations were made in 96 patients with otitis media purulenta chronica (OMPC) and otogenic intracranial complications. It was found that in different clinical forms of OMPC nonspecific defense and immunity indices vary. In OMPC and chronic purulent mesotympanitis the indices change insignificantly. In chronic purulent epitympanitis and otogenic intracranial complications there was suppression in T-cell immunity and nonspecific defense. Middle ear secretion contained less SIgA and lysozyme, blood contained more amount of IgA and IgM and less of IgG. Deficiency of general and local defense antiinfectious mechanisms shows significance of immune and nonspecific resistance in transformation of ear inflammation into chronic phase and development of complications. Alterations in immunity, nonspecific defense and tympanic cavity depend on the causative agent of OMPC. Association of St. aureus with gramnegative flora or anaerobic infection cause the greatest immune deficiency. Temporal bone surgery and standard treatment for 21 days fail to eradicate initial immune disorders in the blood and middle ear. Thymalin immunocorrection improves nonspecific defense and immunity, attenuates inflammation and stimulates reparative processes in the trepanation wound of the ear.

p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours.

p73 (ref. 1) has high homology with the tumour suppressor p53 (refs 2-4), as well as with p63, a gene implicated in the maintenance of epithelial stem cells. Despite the localization of the p73 gene to chromosome 1p36.3, a region of frequent aberration in a wide range of human cancers, and the ability of p73 to transactivate p53 target genes, it is unclear whether p73 functions as a tumour suppressor. Here we show that mice functionally deficient for all p73 isoforms exhibit profound defects, including hippocampal dysgenesis, hydrocephalus, chronic infections and inflammation, as well as abnormalities in pheromone sensory pathways. In contrast to p53-deficient mice, however, those lacking p73 show no increased susceptibility to spontaneous tumorigenesis. We report the mechanistic basis of the hippocampal dysgenesis and the loss of pheromone responses, and show that new, potentially dominant-negative, p73 variants are the predominant expression products of this gene in developing and adult tissues. Our data suggest that there is a marked divergence in the physiological functions of the p53 family members, and reveal unique roles for p73 in neurogenesis, sensory pathways and homeostatic control.

Moraxella catarrhalis-induced purulent otitis media in the rat middle ear. Structure, protection, and serum antibodies.

To study the effects of viable and heat-killed Moraxella catarrhalis bacteria on the middle ear mucosa and to evaluate the protection after whole-cell immunizations, Sprague-Dawley rats were challenged and rechallenged with four different M. catarrhalis strains. The animals were monitored by clinical observations, bacterial and histological samples from middle ears, and serum IgG levels. Only viable bacteria at a high concentration induced purulent otitis media, which was culture positive in 58% of the cases on day 4. The infection was characterized by a mild acute reaction lasting otomicroscopically about 8 days, together with quantitative and qualitative changes of the goblet cells. Structurally the mucosal effects of the heat-killed bacteria were less pronounced in the early phase compared to the viable bacteria, but similar at the end of the experiment at 6 months. The intrabullar and subcutaneous immunizations evoked an IgG antibody response in all animals, and the protection rate after immunization was 50% or more. The induced protection was not strain-specific. The study showed the rat to be a possible alternative for the study of different aspects of M. catarrhalis otitis media, an infection that is clinically and structurally different from that elicited by Streptococcus pneumoniae and Haemophilus influenzae in the rat.