ModM DNA methyltransferase methylome analysis reveals a potential role for Moraxella catarrhalis phasevarions in otitis media.

Moraxella catarrhalis is a significant cause of otitis media and exacerbations of chronic obstructive pulmonary disease. Here, we characterize a phase-variable DNA methyltransferase (ModM), which contains 5'-CAAC-3' repeats in its open reading frame that mediate high-frequency mutation resulting in reversible on/off switching of ModM expression. Three modM alleles have been identified (modM1-3), with modM2 being the most commonly found allele. Using single-molecule, real-time (SMRT) genome sequencing and methylome analysis, we have determined that the ModM2 methylation target is 5'-GAR(m6)AC-3', and 100% of these sites are methylated in the genome of the M. catarrhalis 25239 ModM2 on strain. Proteomic analysis of ModM2 on and off variants revealed that ModM2 regulates expression of multiple genes that have potential roles in colonization, infection, and protection against host defenses. Investigation of the distribution of modM alleles in a panel of M. catarrhalis strains, isolated from the nasopharynx of healthy children or middle ear effusions from patients with otitis media, revealed a statistically significant association of modM3 with otitis media isolates. The modulation of gene expression via the ModM phase-variable regulon (phasevarion), and the significant association of the modM3 allele with otitis media, suggests a key role for ModM phasevarions in the pathogenesis of this organism.

C-Jun N-terminal kinase (JNK) isoforms play differing roles in otitis media.

BACKGROUND: Innate immunity and tissue proliferation play important roles in otitis media (OM), the most common disease of childhood. CJUN terminal kinase (JNK) is potentially involved in both processes. RESULTS: Genes involved in both innate immune and growth factor activation of JNK are upregulated during OM, while expression of both positive and negative JNK regulatory genes is altered. When compared to wildtypes (WTs), C57BL/6 mice deficient in JNK1 exhibit enhanced mucosal thickening, with delayed recovery, enhanced neutrophil recruitment early in OM, and delayed bacterial clearance. In contrast, JNK2-/- mice exhibit delayed mucosal hyperplasia that eventually exceeds that of WTs and is slow to recover, delayed recruitment of neutrophils, and failure of bacterial clearance. CONCLUSIONS: The results suggest that JNK1 and JNK2 play primarily opposing roles in mucosal hyperplasia and neutrophil recruitment early in OM. However, both isoforms are required for the normal resolution of middle ear infection.

TNFA deletion alters apoptosis as well as caspase 3 and 4 expression during otitis media.

BACKGROUND: Tumor necrosis factor (TNFA) is the canonical member of the TNF superfamily, which plays a major role in both inflammation and apoptosis. To evaluate the role of TNFs in otitis media (OM), the most common disease of childhood, we evaluated middle ear (ME) expression of genes encoding the TNF and TNF receptor superfamilies during bacterial OM in the mouse, characterized OM in TNFA-deficient mice, and assessed apoptosis during OM in normal versus TNF-deficient MEs. RESULTS: TNFs and TNF receptors were broadly regulated during OM, with TNFA showing the highest level of up-regulation. TNF deficient mice exhibited mucosal hyperplasia even in the absence of infection and exuberant growth of the mucosa during OM, including the formation of mucosal polyps. Mucosal recovery during OM was also delayed, in parallel with a delay in mucosal apoptosis and reduced caspase gene expression. CONCLUSIONS: The TNF and TNF receptor superfamilies mediate both inflammation and apoptosis during OM. TNF appears to be critical for the maintenance of mucosal architecture in both the normal and infected ME, since excessive accumulation of mucosal tissue is seen in TNFA-/- MEs both before and after bacterial inoculation of the ME. TNFA is also required for appropriate regulation of caspase genes.

The economic benefits of increasing breastfeeding rates in Spain.

BACKGROUND: Interventions aimed at promoting breastfeeding rates are among the most effective possible health policies available, with an estimated return of US$35 per dollar invested. Indeed, some authors found that a 10% increase in exclusive breastfeeding rates in the first two years of life led to a reduction in treatment costs of US$312 million in the US, US$7.8 million in the UK, US$30 million in China, and US$1.8 million in Brazil. Among high-income countries, Spain stands out for its low breastfeeding rate. METHODS: We calculated the savings that the Spanish National Health System would have benefited from had breastfeeding rates been higher in Spain, both from the time of hospital discharge and at 6 months postpartum. We followed the methods used in similar studies carried out in the US, Italy, Australia, the Netherlands, and the UK, to conservatively estimate these potential savings by considering only the lower thresholds in all our estimates. Here we approximated the benefits of having increased exclusive breastfeeding rates based on the lower incidence of infantile pathologies among exclusively breastfed infants. Robust evidence indicates that among breastfed infants there is a lower prevalence of otitis media, gastroenteritis, respiratory infections, and necrotising enterocolitis. We obtained the estimated monetary cost of these diseases by combining their prevalences with data about their economic costs for diagnosis-related groups. RESULTS: The estimated effects we calculated imply that the Spanish National Health System could have saved more than €5.6 million for every percentage point increase in exclusive breastfeeding rates in Spain during 2014. CONCLUSIONS: Breastfeeding is essential both for the health of mothers and the health and development of newborns but is rarely considered as an economic issue and remains economically invisible. In addition to the improved wellbeing of mothers and their infants, breastfeeding can positively impact society as a whole and should therefore be better defined in public policies. Thus, strategies aimed at increasing exclusive breastfeeding rates would likely contribute to lowering the fiscal burden of the Spanish National Health System. Moreover, the magnitude of these potential benefits suggests that such policies would likely be socially cost-effective.

Fisetin administration improves LPS-induced acute otitis media in mouse in vivo.

Acute otitis media is one of the most common infectious diseases worldwide in spite of the widespread vaccination. The present study was conducted to explore the effects of fisetin on mouse acute otitis media models. The animal models were established by lipopolysaccharide (LPS) injection into the middle ear of mice via the tympanic membrane. Fisetin was administered to mice for ten days through intragastric administration immediate after LPS application. Hematoxylin and eosin (H&E) staining was performed and the pro-inflammatory cytokines, including interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), IL-6 and VEGF, were measured through enzyme-linked immunosorbent assay (ELISA) method and RT-qPCR analysis. Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway was detected by immunoblotting assays. Reactive oxygen species (ROS) generated levels were determined through assessment of anti-oxidants, and TXNIP/MAPKs signaling pathways were explored to reveal the possible molecular mechanism for acute otitis media progression and the function of fisetin. Fisetin reduced mucosal thickness caused by LPS. In fisetin-treated animals, pro-inflammatory cytokine release was downregulated accompanied with TLR4/NF-κB inactivation. ROS production was significantly decreased in comparison to the LPS-treated group. The TXNIP/MAPKs signaling pathway was inactivated for fisetin treatment in LPS-induced mice with acute otitis media. The above results indicated that fisetin improved acute otitis media through inflammation and ROS suppression via inactivating TLR4/NF-κB and TXNIP/MAPKs signaling pathways.

Quercetin inhibits NTHi-triggered CXCR4 activation through suppressing IKKα/NF-κB and MAPK signaling pathways in otitis media.

Otitis media is one of the most common bacterial infections in children, contributing to hearing loss. A vital bacterial pathogen leading to otitis media development is the nontypeable Haemophilus influenzae (NTHi). Inflammation response is reported as an important characristic for otitis media. Chemokine CXC receptor 4 (CXCR4) is a 352-amino acid seven-span transmembrane G-protein coupled receptor, essential for inflammatory response. However, the possible molecular mechanism indicating the alteration of CXCR4 modulated by NTHi is poorly known. In the present study, NTHi enhanced CXCR4 expression through phosphorylation of IKKα and p38, which relied on nuclear factor-κB (NF-κB) translocation in vitro as well as in the middle ear of mice in vivo. Previously, quercetin, a natural production mainly isolated from rutin, has shown anti-inflammatory effects. Here, we report that quercetin suppressed NTHi-induced CXCR4 expression levels in vitro and in vivo. Quercetin blocked CXCR4 activation through direct IKKß phosphorylation inhibition, as well as of p38 MAPK restraining. Hence, identification of quercetin may be a potential therapeutic strategy for treating otitis media induced by NTHi through inflammation suppression.

Genetic polymorphisms affecting antioxidant enzymes are present in tympanosclerosis patients.

BACKGROUND: This study investigated genetic polymorphisms affecting the inducible nitric oxide synthase, superoxide dismutase and catalase enzymes in chronic otitis media patients with and without tympanosclerosis, and the role of genetic susceptibility in the disease aetiology. METHODS: A total of 162 patients who underwent surgery for chronic otitis media were divided into two study groups: a tympanosclerosis group and a chronic otitis media group. A third, the control, group comprised 188 healthy volunteers. Venous blood samples were evaluated using reverse transcriptase polymerase chain reaction. RESULTS: There was a significant difference in GG genotype distribution of the -277A>G polymorphism in the NOS2 gene between the tympanosclerosis and control groups (p T) polymorphism in the SOD2 gene (p > 0.05). There were significant differences in the TT genotype distribution of the -21A>T polymorphism in the CAT gene between the tympanosclerosis and control groups, and between the chronic otitis media and control groups (p < 0.05). CONCLUSION: These results suggest that genetic predisposition may play a role in the aetiopathogenesis of tympanosclerosis.

Is there a relationship between myeloperoxidase activity and conductive hearing loss in chronic otitis media complicated by cholesteatoma?

We conducted a prospective, controlled study of patients with chronic otitis media and cholesteatoma (1) to examine the expression of myeloperoxidase (MPO) using immunohistochemical staining techniques and (2) to investigate the relationship between MPO activity and the degree of conductive hearing loss in these patients. Our study population included 51 adults-26 men and 25 women, aged 18 to 58 years (mean: 37.5)-who had been diagnosed with chronic otitis media and cholesteatoma by physical examination and computed tomography (study group). Another 30 patients-13 men and 17 women, aged 18 to 52 years (mean: 32.7)-who had chronic otitis media without cholesteatoma served as the control group. Following audiometric evaluations, all patients underwent appropriate surgery. Postoperatively, cholesteatoma samples were analyzed by immunostaining for MPO positivity as a marker for acute inflammation. We found that MPO activity was present in all 51 study patients (100%) but in only 10 controls (33.3%); the difference was statistically significant (p< 0.01). In the study group, the degree of MPO activity was slight in 6 patients (11.8%), moderate in 24 patients (47.1%), and intense in 21 patients (41.2%), while in the control group, all 10 MPO-positive cases showed only a slight degree of activity. We also found a statistically significant association in the study group between the degree of MPO activity and the degree of conductive hearing loss (χ(2) = 13.518; p < 0.001). We encourage further study of all steps in the process of cholesteatoma formation.

A comparative study of intratympanic steroid and NO synthase inhibitor for treatment of cochlear lateral wall damage due to acute otitis media.

We studied the damage to the cochlear lateral wall induced by otitis media and the therapeutic effects of intratympanic administration of steroid and nitric oxide (NO) synthase inhibitor. In Sprague-Dawley rats, right middle ear cavities were inoculated with lipopolysaccharide, followed after 30 min by intratympanic administration of dexamethasone, NOS-inhibitor or PBS. Twenty-four hours after lipopolysaccharide inoculation, cochlear blood flow was measured by laser-Doppler flowmetry. Prostaglandin E(1) was topically applied to the round window membrane of the right ear and changes in cochlear blood flow were calculated. Changes of cochlear blood flow were significantly different among the three groups. Increases in cochlear blood flow following PGE(1) application were higher in the group that received NOS-inhibitor. Electron microscopic examination revealed that changes in the stria vascularis were less severe in rats treated with dexamethasone or NOS-inhibitor. Our results show the effectiveness of intratympanic dexamethasone or NOS-inhibitor in treating cochlear lateral wall damage caused by otitis media.

Bone resorption factors in chronic otitis media.

Collagenase was identified within naturally occurring rat chronic otitis media by the use of an immunohistochemical technique with peroxidase-antiperoxidase to stain the paraffin. Collagenase was found in fibroblasts, mononuclear cells, and osteoclast cells in the bone-resorbing area. Collagenase was found only in fibroblasts in contact with epithelial basal cells. Macrophages from rat peritoneum were cultured with different concentrations of a lipopolysaccharide. The prostaglandin E2 level reached a maximum during the 12- to 24-hour period in the presence of endotoxin. This prostaglandin E2 was confirmed by immunofluorescent staining. The endotoxin-activated macrophage produced an insignificant amount of collagenase. These findings suggest that activated macrophages may be able to stimulate fibroblast collagenase production through the chemical mediator prostaglandin E2. Also, the interaction between fibroblasts and epidermal cells appears to encourage and enhance the biochemical events resulting in bone resorption in chronic otitis media.

Analysis of protease activity in human otitis media.

Chronic otitis media is a common problem associated with a nonintact tympanic membrane frequently involving Staphylococcus aureus and Pseudomonas aeruginosa. The virulence of Pseudomonas bacteria is related to the production of two matrix metalloproteinases, elastase and alkaline protease. Serine proteases, such as neutrophil elastase, are produced by the host inflammatory response. These proteases are thought to contribute to tissue destruction and assist bacterial invasion during infection. This preliminary study was done to identify protease activity in otorrhea samples from patients with otitis media and a nonintact tympanic membrane and to examine the ability of selective protease inhibitors to decrease protease activity. Ilomostat (galardin) is a synthetic, specific inhibitor of matrix metalloproteinases including P. aeruginosa elastase and alkaline protease, whereas alpha1-antitrypsin inhibits serine proteases including neutrophil elastase. Samples were collected and cultured from 20 patients with otorrhea resulting from tympanic membrane perforations or pressure-equalization tubes. A protease assay that used azocasein as the substrate was used to quantify protease activity, with and without addition of selective protease inhibitors. Cultures revealed P. aeruginosa alone in 7 samples, P. aeruginosa plus other organisms in 10, and S. aureus alone in 3. Protease activity was detected in 15 (75%) of the samples. A statistically significant (p < 0.05) decrease in protease activity was seen with the addition of alpha1-antitrypsin or Ilomostat plus alpha1-antitrypsin, but not with Ilomostat alone. Analyzing the 10 samples with the highest protease activity, a statistically significant decrease in activity was seen with Ilomostat or alpha1-antitrypsin alone and with both Ilomostat and alpha1-antitrypsin together. Bacteriologic type, source of sample, age and gender of the subject, and duration of infection were not significantly related to protease activity. This is the first study to quantify protease activity and inhibition by selective protease inhibitors in human otorrhea. Protease inhibitors effectively decrease protease activity in most cases and in addition to standard antibiotic therapy might prove beneficial in the treatment of otitis media with a nonintact tympanic membrane. This study supports future clinical investigations into the role of proteases and inhibition of protease activity in the treatment of otitis media.

Lysosomal hydrolases in middle ear effusions.

Biochemical studies of middle ear effusions have demonstrated generally higher levels of certain hydrolytic and oxidative enzymes in mucoid fluids when compared to serous. We have extended these studies by analyzing middle ear effusions for the content of a large number of lysosomal hydrolases. The mean specific activity for alpha-glucosidase in mucoid fluids was found to be ten times that for serous fluids while alpha-mannosidase, beta-glucuronidase, hexosaminidase, acid phosphatase, beta-galactosidase, alkaline phosphatase, and lactate dehydrogenase were found to be three to five times greater in mucoid than serous effusions. In this study the specific enzyme activities for lysosomal hydrolases from purulent effusions were found to be intermediate between the activities in serous and mucoid effusions. No significant correlation was found between the specific activities of lysosomal hydrolases and the presence or absence of bacteria in mucoid or serous middle ear effusions. The hexosaminidase isozyme distribution was found to be identical for serous and mucoid fluids and similar to that found in human serum. However, the isozyme pattern of beta-glucuronidase in mucoid effusions was significantly different than that in normal human serum as mucoid fluids contain a large amount of an anionic isoenzyme of beta-glucuronidase that is barely detectable in human serum.

Neuraminidase activity in middle ear effusions.

Analyses of Streptococcus pneumoniae culture filtrates and middle ear effusions (MEE) containing S pneumoniae for various hydrolytic enzymes have demonstrated substantial levels of neuraminidase activity when measured employing a sensitive fluorometric assay. S pneumoniae neuraminidase exhibits optimum activity near neutral pH (6.0 to 6.5), and catalyzes the cleavage of sialic acid residues from glycoproteins, gangliosides and mucopolysaccharides. S pneumoniae begins secreting large amounts of neutral neuraminidase (mean [means] = 43.3 units/mL culture filtrate) when cells enter the stationary phase. Nearly all (96%) human chronic MEEs yielding positive cultures for S pneumoniae contain neuraminidase activity (means = 0.200 units/mg protein), while only 21.1% to 45.5% of all other effusions contain the enzyme. Middle ear effusions obtained from S pneumoniae infected-chinchillas contained large amounts of neuraminidase activity (approximately 200 units/mL), which decayed exponentially in vivo with an apparent half-life of 8 1/2 days. Three neuraminidase isoenzymes (designated I-III) were identified in S pneumoniae culture filtrates, as well as in MEEs from chinchillas infected with the organism, using a combination of ion-exchange and gel filtration chromatography. With 4-methylumbelliferyl-N-acetylneuraminic acid serving as substrate, preparation I from both culture filtrates and MEEs was characterized by a high Michaelis constant (Km), while forms II and III had low Km values. Preferred substrates were orosomucoid and neuramin-lactose; gangliosides, thyroglobulin, and bovine submaxillary mucin were poorer substrates.

Hydrolase activity in acute otitis media with effusion.

Biochemical studies of middle ear effusions (MEE) from patients with chronic or recurrent otitis media with effusion (OME) have demonstrated the presence of significant levels of certain hydrolytic and oxidative enzymes. We have examined MEE from patients with acute OME for the content of a number of lysosomal hydrolases and find no significant differences in the mean values for acid phosphatase, alpha-mannosidase, beta-galactosidase, beta-glucuronidase, hexosaminidase, and neuraminidase between purulent and serous effusions. In every case, the mean activities of these enzymes were greater in culture-positive than in culture-negative effusions although this difference was significant only in the case of neuraminidase. Neuraminidase activity was detected in 78% of those MEEs from which Streptococcus pneumoniae could be cultured and in only 32% to 64% of all other effusions. No correlation was observed between the level of neuraminidase released into the extracellular growth medium and the infectivity of various strains of S pneumoniae.

Secretory lysozyme of the human middle ear mucosa: immunocytochemical localization.

Lysozyme was demonstrated by an immunocytochemical technique in the biopsied mucosa obtained from the promontory of the fifteen patients who had chronic middle ear effusions. Lysozyme was localized in the mucigen granules of the secretory cells, as well as in the specific granules of the polymorphonuclear neutrophilic leukobytes (PMN) and macrophages. The specimens obtained from patients with mucous effusion showed numerous secretory cells that contained lysozyme, in sharp contrast to the serous type in which only a few secretory cells could be found. The present morphological finding was in agreement with the biochemical finding which demonstrated higher lysozyme level in mucous effusions than that of the serous type. It was concluded that human middle ear mucosa provided lysozyme and that its secretion was active in serous otitis media, particularly of mucoid type.

Expression of acute otitis media after receptor blockade of platelet activating factor, thromboxane, and leukotrienes in the chinchilla.

To determine the role of inflammatory products of phospholipid metabolism in acute otitis media (AOM), we infected 128 chinchillas with Streptococcus pneumoniae and randomly assigned them to one of four equal-sized treatment groups receiving intramuscular ampicillin sodium (control) or intramuscular ampicillin plus receptor blockers of platelet activating factor (WEB 2086, 5 mg/d orally), of leukotriene (MK 571, 0.5 mg/d orally), or of thromboxaneA2 (GR 32191B, 5 mg/d orally). All treatments were begun on day 2 postinoculation and continued for 10 days. On days 3, 6, 9, and 12, 8 animals from each group were sacrificed. Effusions were recovered for biochemical assay, and the right middle ears were prepared for histologic study. Differences among groups in the number of ears with effusion or in effusion volume were not statistically significant. In comparison to the control group, mucosal thickness and the number of ears with histopathologic signs of inflammation were significantly less in the GR and WEB treatment groups, but not the MK group. Also, effusion concentrations of free fatty acids, protease, and hydrolytic enzymes were significantly less in those groups. These results show that the addition of a receptor blocker for either platelet activating factor and/or thromboxane to ampicillin in the treatment of AOM reduces mucosal inflammation and decreases the production of other inflammatory chemicals. The failure of a receptor blocker of leukotrienes to moderate disease expression suggests either a less important role for these chemicals in AOM or an insufficient bioavailability of the specific MK 571 inhibitor. These results confirm that platelet activating factor and thromboxane are active mediators of inflammation in AOM.

Bone resorption in chronic otitis media. A light-microscopical and histochemical investigation of acid phosphatase activity.

As a continuation of our previous work, where we have demonstrated that in chronic otitis media the picture in the submucosa-bone marginal zone is dominated by capillary proliferation and occurrence of a mononuclear, histiocyte-like cell containing lysosome-like cytoplasmatic bodies, we now report the presence of considerable activity of acid phosphatase in close relation to the eroded bone. The activity was localized both extracellularly, spread along the bony surface, as well as intracellularly in mononuclear, histiocyte-like cells. The acid phosphatase is the "marker" enzyme for lysosomes, and cells with these lysosomes guarantee the presence of enzymatic activity capable of attacking bone collagen. It is difficult to avoid the conclusion that the lysosomes and their enzymes are directly involved in the processes of bone resorption.

Localization of antileukoprotease in middle ear mucosa.

The localization of antileukoprotease was studied immunohistologically in normal middle ear mucosa specimens obtained at autopsy and in chronically inflamed middle ear mucosa specimens obtained at middle ear surgery for chronic otitis media. In the sections of normal as well as in the sections of chronically inflamed middle ear mucosa, antileukoprotease localization was confined to PAS-positive goblet cells of surface epithelium and to PAS-positive goblet-like cells of submucosal glands and crypts, whereas ciliated mucosal cells and stratified squamous epithelial cells were devoid of anti-leukoprotease. In comparison with normal middle ear mucosa, an increased number of goblet cells--and thus an increased number of cells containing antileukoprotease--was present in the chronically inflamed middle ear mucosa. Since antileukoprotease is a potent inhibitor of granulocyte elastase and Cathepsin G, it was concluded that this proliferation of the respiratory epithelium during inflammatory processes in the middle ear indicates an increased activity of the biologic defence system against the action of granulocyte proteases.

Effect of neuraminidase on receptor-mediated adherence of Streptococcus pneumoniae to chinchilla tracheal epithelium.

The trachea whole organ perfusion technique was used to study the effect of the disruption of the Streptococcus pneumoniae neuraminidase nanA gene on bacterial adherence and alteration of the carbohydrate surface structures of respiratory epithelial cells. Six different lectin probes were used to examine alterations of the cell surface carbohydrates in chinchilla tracheal epithelium incubated in vitro with S. pneumoniae deltaNA1, a neuraminidase-deficient mutant, or its D39 parent strain. The labeling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated WGA was significantly increased in the luminal surface of the trachea in the D39-incubated cohort compared with the uninfected control, which indicated that GlcNAc and D-galactose residues were exposed. Concurrently, decreased labeling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the tracheal epithelium subsequent to incubation with D39. The deltaNA1 neuraminidase-deficient mutant, however, did not induce any significant changes in the lectin labeling patterns, which were comparable to those of the control cohort. Moreover, adherence data expressed as colony-forming units (CFU) of S. pneumoniae per millimeter of trachea indicated a significant decline in the ability of deltaNA1 to adhere in vitro. We propose that products of the nanA gene have a significant impact on changes in the carbohydrate moieties in the tracheal epithelium, and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.