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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.

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-1β (IL-1β), 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.

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 IKKβ 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.

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.

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.

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., (© FASEB.)

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.

Objective: To investigate the histopathological changes and the expression of vascular endothelial growth factor (VEGF), inducible NO-synthase (iNOS), endothelial NO-synthase (eNOS), interleukin (IL)-1β, and IL-17 in the rabbit middle ear mucosa after direct gastric content exposure., Methods: Exploratory controlled study in which histological and immunochemical features were studied after gastric content-induced inflammation was established in rabbits. Sixteen healthy rabbits were divided into two equal groups. Gastric contents of an animal were injected into the middle ear of the same animal for 20 days. Saline was injected into the middle ear of the animals in the control group. The rabbits were humanely killed on day 27. Inflammation was assayed by light microscopy. Immunochemical staining was performed for VEGF, iNOS, eNOS, IL-1β, and IL-17 expression. Experimental and control animals were examined using the same protocol., Results: The expression levels of VEGF, iNOS, IL-1β, and IL-17 differed significantly between the experimental and control groups (p=0.018, p=0.010, p=0.002, and p=0.002, respectively). The expression level of eNOS was not significantly different between the two groups (p=0.132)., Conclusion: This study demonstrates that gastroesophagial reflux induced middle ear inflammation is associated with increased expression of VEGF, IL-1β, IL-17, and iNOS., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

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.

Introduction: There are three different pneumococcal vaccines available for infants, each oriented to a specific set of serotypes. The vaccination of newborns will prevent pneumococcal disease in this vaccinated group via direct effects, and will also affect the non-vaccinated population through indirect or "herd" immunity., Objective: To develop a model that compares the health and economic consequences between the three vaccines., Method: We developed a simulation model for an entire population, providing vaccine to children less than 2 years of age. The vaccines varied by serotypes covered and included a 7- (4, 6B, 9V, 14, 18C, 19F and 23F), 10- (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F) and 13-valent (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) vaccines. The base case was PCV-7, and clinical and economic outcomes were estimated for the vaccinated persons and for other persons through assumptions about a herd effect. By comparison, clinical and economic outcomes for the population were also estimated for the 10 and 13 serotype vaccines., Results: In the base case (PCV-7), with the seven serotype vaccine, there were 9.38 cases of hospitalized pneumonia, 0.22 cases of meningitis, 3.69 cases of bacteremia, 60.19 cases of otitis media, and 373 cases of pneumonia, per 100,000 persons in the population, at all ages. With the 10-valent vaccine and a herd effect, invasive pneumonia fell to 8.71 cases, meningitis to 0.21 cases, and bacteremia to 3.39 cases. Otitis media fell to 57 cases and pneumonia to 344 cases. There were further reductions with the 13-valent vaccine, with invasive pneumonia falling to 8.37 cases, bacteremia to 3.33 cases, otitis media to 51.9 cases and all-cause pneumonia to 336.2 cases. Among the vaccines evaluated, PCV-13 was associated with the lowest health services costs and the greatest improved health outcomes., Conclusions: Increased serotype coverage of the 13-valent vaccine is expected to have a substantial public health and economic impact on infectious disease, when considering direct and indirect effects., (Copyright 2010 Elsevier Ltd. All rights reserved.)

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.

Phase variation in the colonial opacity phenotype of Streptococcus pneumoniae has been implicated as a factor in bacterial adherence, colonization, and invasion in the pathogenesis of pneumococcal otitis media (OM). The purpose of this study was to determine whether S. pneumoniae opacity variants influence the induction of gene expression for proinflammatory mediators in vivo using the rat model of OM. Both the opaque and transparent phenotype variants induced a significant up-regulation in gene expression for interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-10, tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) compared to saline sham-inoculated controls at both 4 and 24 h postinoculation (P < 0.05 in all cases). Furthermore, whereas a significant difference in gene expression was evident for only IL-6 (greater following challenge with the opaque variant) and IL-1beta (greater following challenge with the transparent variant) at 4 h, by 24 h the opaque variant cohort demonstrated a significant increase in gene expression for IL-1alpha, IL-1beta, IL-6, IL-10, and iNOS relative to animals inoculated with the transparent phenotype variant (P < 0.05 in all cases). Enzyme-linked immunosorbent assay results confirmed the gene expression data as determined by real-time PCR. Moreover, the concentrations of the opaque variant in the middle ear lavage fluid were a full log higher than those of the transparent variant. The aforementioned results indicate that the opaque phenotype variant is more efficient at survival and multiplication within the middle ear space, resulting in the accumulation of more inflammatory cells and the enhanced expression and production of inflammatory mediators. However, when the data were normalized to account for differences in middle ear bacterial titers, it became apparent that the transparent variant of S. pneumoniae is a more potent inducer of inflammation, triggering the accumulation of more inflammatory cells and substantially greater fold increases in the expression and production of inflammatory mediators. Data from this study indicate that S. pneumoniae opacity variants influence the temporal mRNA expression of inflammatory mediators within the middle ear.

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.

The role of matrix metalloproteases and their regulation in the pathology of middle ear cholesteatoma is still unclear. Recently we have demonstrated that incubation of keratinocytes with cholesteatoma debris and granulation tissue extracts causes induction of gelatinase B (matrix metalloproteinase-9, MMP-9) secretion in vitro. Antibodies against a variety of growth factors revealed some inhibitory effect on MMP-9 induction, caused by debris or granulation tissue extracts. In order to investigate the coherence of growth factor expression and matrix metalloproteinase activity in vivo in middle ear cholesteatoma, we performed quantitative gelatin zymographic analysis with tissue homogenates of 37 cholesteatoma and nine external ear canal skin (EACS) samples. Furthermore we quantified levels of the cytokines IL-1alpha, IL-1beta, TNF-alpha, TGF-beta and EGF present in tissue extracts, using enzyme-linked immunosorbent assays (ELISA), and correlated cytokine concentrations with gelatinolytic activities. Zymographic analysis revealed a highly heterogeneous expression of gelatinase A and B in cholesteatoma specimens. As shown previously, MMP-9, but not MMP-2, was increased in cholesteatoma when compared to EACS samples. ELISA studies revealed a significantly elevated IL-1alpha level in cholesteatoma. Regression analysis involving gelatinolytic activity and cytokine concentrations in tissue homogenates showed no statistically significant correlation between expression of gelatinases and the cytokines IL1-alpha, IL1-beta, TNF-alpha, TGF-beta or EGF. The discrepancy between in vitro observations and the situation in vivo is discussed critically.

Superoxide dismutase has been known to play a role as an anti-oxidative system against oxidative injury during acute inflammation. To investigate the role of superoxide dismutase in eustachian tubal mucosa during acute otitis media (AOM), an animal model was made. Sprague-Dawley rats were inoculated with Streptococcus pneumoniae through the nasal cavity following development of virus-induced upper respiratory infection. The animals were divided into three groups according to their tympanic cavity conditions following bacterial inoculation; inoculated animals with no resultant AOM (no-AOM), animals with resultant AOM (AOM) and animals with resolving otitis media (recovery). The changes of superoxide dismutase in each tubal mucosa were compared with that of the normal control using immunohistochemistry and immunoblotting methods. On Western blot, there were little changes of optical density and surface area in no-AOM (213.5 +/- 22.4, 13.2 +/- 0.8 mm2) and recovery group (219.3 +/- 18.7, 14.8 +/- 0.7 mm2) compared to the normal control (223.5 +/- 26.2, 16.7 +/- 0.4 mm2). However, a marked decrease was found in the AOM model (167.6 +/- 19.3, 6.5 +/- 0.9 mm2). These findings suggest that superoxide dismutase may play a role in protecting tubal mucosa from free radical injury during AOM.

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.

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.

Moraxella catarrhalis is a normal resident of the human nasopharyngeal flora, but it is also isolated from middle ear fluid of acute otitis media and otitis media with effusion patients. To determine whether M. catarrhalis has direct pathogenicity in the middle ear, heat-killed M. catarrhalis was inoculated into the middle ear bullae of guinea pigs, and the inflammatory response was investigated. Middle ear mucosal histopathology observed in M. catarrhalis-inoculated ears included subepithelial edema, capillary dilatation, thickening of lamina propria mucosa, inflammatory cell and erythrocyte infiltration into the lamina propria mucosa. Inflammatory cell numbers, lysozyme and myeloperoxidase concentrations in the middle ear washing suspensions of M. catarrhalis-inoculated ears were significantly higher than control ears throughout the experiment. Therefore, nonviable M. catarrhalis induced middle ear inflammation and mucoperiosteal histopathology, which might be caused by direct injury of the nonviable bacteria (e.g. lipooligosaccharide or outer membrane proteins) and metabolic products of inflammatory cells.

Increasing evidence implicates free radicals in the pathogenesis of inflammatory disease, including otitis media. The anti-oxidant enzymes catalase, glutathione peroxidase and superoxide dismutase protect tissues from the destructive effects of free radicals. Our previous work has shown depressed levels of superoxide dismutase in the infected middle ears of a guinea pig model of otitis media in comparison with normal control ears. We studied the distribution and relative abundance of catalase in the middle ear of this animal model in an effort to elucidate the role free radicals play in the pathogenesis of otitis media. Catalase distribution was mapped immunohistochemically in the middle ears of guinea pigs with induced streptococcus otitis media, and compared with normal control ears. In the control ears, catalase was localized to the epithelium of the middle ear mucosa, with scant distribution in the submucosa. The infected ears demonstrated inflammatory cell invasion with hyperemia and submucosal edema. Catalase was localized to the epithelium and had scant distribution in the submucosa. This distribution was similar to that found previously with superoxide dismutase. Enzyme-linked immunosorbent assay of catalase demonstrated a mean value of 1.00 +/- 0.06 microgram/mg protein in the control ears, and 1.06 +/- 0.12 microgram/mg in the infected ears, but these two values were not statistically different.

Cu, Zn superoxide dismutase (SOD) is a metalloprotein that catalyzes the dismutation of the superoxide anion into O2- and H2O2, and therefore functions to maintain a low intracellular concentration of an otherwise toxic metabolite of oxygen. SOD protects living tissue from the destructive effects of free radicals. Increasing evidence implicates free radicals, including the superoxide radical (O2-), in the pathogenesis of disease, including otitis media. In an effort to elucidate the role free radicals play in the pathogenesis of otitis media, SOD was localized immunocytochemically to determine its cellular distribution in specimens of guinea pig middle ear. In normal ears, SOD was found concentrated in the epithelium of the middle ear mucosa. Low quantities were characteristic of connective tissue, bone, and cartilage. In streptococcus-infected ears, SOD localized similarly, concentrating in the epithelium. The infected ears had extensive submucosal edema which stained poorly and appeared to have less SOD than did normal ears. This was confirmed by an assay using laser densitometry of Western blots to quantify the amount of SOD in the mucosa of normal versus infected middle ears. This demonstrated a value of SOD in normal mucosa of 1.77 +/- 0.48 micrograms/mg of protein compared with 1.02 +/- 0.28 micrograms/mg in the infected mucosa. The two groups were significantly different at P < 0.05. These findings are discussed, and suggestions for future experimentation addressed.

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.

Most functional disturbances within the auditory tube can be shown and documented morphologically. In the case of a blocked tube we demonstrated changes on the tubal lining as well as in the adjacent salivary glands. Complete obliteration of the lumen is a very rare instance. A lack of ventilation already produces a vacuum within the tympanic cleft, followed by a typical effusion out of the altered mucosa. Anaerobic conditions favor hypertransformation and metaplastic transformation of the epithelium. The missing clearance makes the middle ear discharge a great challenge to the mucosa. Granulation tissue proliferates, an enzyme production starts, phagocytes help to eliminate the tympanic content. If the auditory tube is blocked long enough, the result is the atelectatic tympanum, filled by scar tissue, granulations with fatty degeneration or dystrophic calcification. The ossicular chain is fixed, if not destroyed or interrupted. A retraction pocket within the eardrum membrane, often a reason for secondary acquired cholesteatoma, is very dangerous.

A comparative study was made of granulocyte proteases and protease inhibitors in middle ear effusions (MEE) from patients with acute otitis media (OMA) and serous otitis media (SOM). The mean concentrations of the granulocyte proteases, neutral protease and elastase were three- to fourfold higher in OMA as compared to SOM effusions. The mean concentration of granulocyte elastase in SOM effusions was approximately 1 400, and in OMA effusions 5 500 times higher than the corresponding plasma concentrations. In OMA patients the individual MEE/plasma ratios of the main plasma protease inhibitors, alpha 1-antitrypsin, antichymotrypsin and alpha 2-macroglobulin, showed a positive uniform correlation with corresponding albumin MEE/plasma ratios. In SOM effusions, the concentration of alpha 2-macroglobulin was low, indicating a restricted membrane passage in SOM. In SOM effusions both alpha 1-antitrypsin and the locally produced antileukoprotease showed a residual inhibitory capacity. In OMA effusions the inhibitory capacity of anti-leukoprotease was saturated, whereas there was a residual inhibitory capacity of alpha 1-antitrypsin. However, in those OMA effusions, where large amounts of alpha 1-antitrypsin complexes with granulocyte proteases were found, most of the remaining free alpha 1-antitrypsin was inactive.

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.

Proteolytic enzymes such as pepsine or papaine are able to split IgG antibodies into large fragments in vitro. These immunoglobulin fragments (IgG, IgA, IgM) were now detected in vivo from the purulent secretions of cholesteatoma, chronic otitis media and radical mastoid cavities. During chronic otitis media the intact immunoglobulins are split due to the proteolytic activity of neutral proteinases. These fragments were qualitatively and quantitatively investigated by means of various immunological procedures. After the immunoelectrophoretic separation of the purulent middle-ear-secretions and after diffusion against anti-IgG-, anti-IgA- and anti-IgM- serum double precipitate lines could be observed especially in middle-ear-secretion with a bacterial flora of pseudomonas aeruginosa (pyocyanea) and of the proteus-providencia-group. This was the first proof of the presence of split products of the immunoglobulins. The exact demonstration of these split products could be carried out by gel-filtration and fractionation of the intact and split immunoglobulins. During chronic otitis media intact immunoglobulins are split by leucocytic and extracellular bacterial proteinases into fragments of different molecular weight. The most malignant extracellular proteinases with the greatest proteolytic activity against intact immunoglobulins are the bacterial proteinases of pseudomonas aeruginosa. These proteinases can not be inhibited by the other serum proteinaseinhibitors except for alpha-2-macroglobulin of the human blood serum. This inhibitor has a very high molecular weight so that we can not find it in a higher concentration in the middle-ear-secretion. We can liberate this inhibitor by injuring the blood vessels during a tympanoplasty. In this way we get an inhibitory effect against these proteinases and combined with an appropriate antibiotic therapy we can cure a chronic otitis media.

Bone resorption is a significant component of chronic inflammatory ear disease. Bone is a unique tissue requiring both demineralization and collagen degradation for breakdown. Mineral removal probably occurs prior to collagen destruction. A localized change in pH may be one of the mechanisms that induces demineralization. Localized accumulations of lysosomal acid hydrolases could provide the acidic environment necessary for mineral removal. The present study utilizes the electron microscope to localize the lysosomal enzyme acid phosphatase in specimens removed from patients with chronic otitis media. Areas of localized bone resorption exhibited mononuclear inflammatory cells attached to the resorption margin. These cells contained abundant acid phosphatase, as did isolated fibroblasts within the zone of inflammation at the resorption margin. Extra cellular acid phosphatase was seen in these areas. Within the bone, osteocytes adjacent to resorption displayed increased cytoplasmic organelle patterns and contained the acid phosphatase reaction. The localization of destructive enzymes in mononuclear inflammatory cells and osteocytes explains the bony destructive changes observed in human chronic otitis media in the absence of multinucleated osteoclasts.

Middle ear effusions can cause the fibrinolytic dissolution of human fibrin clots. The data presented herein indicate that the highest level of fibrinolytic activity and general proteolytic activity is found in the serous effusions. The mucoid effusions show the lowest levels of fibrinolytic activity. The significance of the fibrinolytic activity in middle ear fluids is not fully understood. Plasmin, the active protease of the fibrinolysin system, is capable of activating a number of proteolytic systems, including the intrinsic coagulation system, the vasoactive peptide system, and the complement system. All the inflammatory mediator systems can promote increased vascular permeability, tissue edema, and leukocytic migration. The relation of these systems to inflammation in the middle ear may have a profound influence on the possible conversion of a simple middle ear effusion into chronic otitis media.

Among the three investigated proteinase inhibitors alpha-1-antitrypsin ("acute phase protein") possesses the highest antiproteolytic capacity. Alpha-2-macroglobulin reaches as an average one fifth of the preoperative level of alpha-1-antitrypsin and postoperatively one quater of this inhibitor level. The average concentrations of the low molecular acid stable proteinase inhibitor of the mucous membrane in the preoperative phase are about equivalent to the concentrations of alpha-2-macroglobulin. In the postoperative phase the concentrations of the low molecular acid stable inhibitor are a slightly lower than those of alpha-2-macroglobulin. The samples of the middle ear secretions from cases with chronic otitis media or with cholesteatoma do not differ essentially in their concentrations of the different proteinase inhibitors.

Bone destruction, commonly associated with chronic otitis media, requires collagen degradation. Collagenase, a neutral protease, appears to be an essential component in the process of collagen breakdown. Collagenase was identified within chronically inflamed and normal guinea pig temporal bones using an immunohistochemical technique with fluorescein isothyocyanate and peroxidase-antiperoxidase labels. Localization of the enzyme identifies sites of matrix resorption. Collagenase was found in osteoclasts, osteocytes, mononuclear inflammatory cells, and at resorbing margins. Inflammation increased the intracellular collagenase content of inflammatory bone osteocytes when compared to normal osteocytes using a microspectrofluorometer. It appears that the inflammatory process directly influences bone destruction through the action of mononuclear inflammatory cells and indirectly by stimulating bone cells to increase their proteolytic enzyme production.

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.

The proteolytic activity of purulent middle ear secretions from patients with cholesteatoma, chronic otitis media, acute otitis media and radical mastoid cavities were investigated. The proteolytic activity in the secretions from cases with cholesteatoma is higher than from patients with other chronic otitis media. The collagenolytic effect of the matrix of cholesteatoma combined with the high level of the bacterial and leukocytic proteinases in the middle ear secretions is undoubtedly one of the main factors in the destruction of the ossicular bones.

Non-specific acid alpha-naphthyl acetate esterase (ANAE) activity of the lymphocytes was investigated in smears made of middle ear secretions and peripheral blood from 61 children with secretory otitis media. On the average, 20.1% (right ear) and 2.1% (left ear) of the lymphocytes in the ear samples showed distinctive ANAE-positive spots in their cytoplasms, by the method used. The mean percentage of ANAE-positive cells in the blood was 47.8 and it was significantly (P less than 0.001) higher than the respective percentages of the ear samples. In normal childrne, 54.9% of the lymphocytes, on the average, were ANAE-positive. This difference in blood between patients and control group was significant (P less than 0.001). Consequently, the present study suggests that the relationship of T- and non-T-lymphocytes, as a marked with ANAE, are disturbed in children suffering from secretory otitis media. The importance of this finding and the validity of the method are discussed.

The kallikrein-kinin system was studied in middle ear effusions from 28 ears suffering from otitis media with either a purulent, mucopurulent, serous or seromucinous, or mucoid effusion. Kinin, kininogen, kininase, kallikrein, prekallikrein, and protein were measured in these secretions. The findings suggest that the concentration of kinin is greatest in the serous and seromucinous effusions and lowest in the mucoid effusion. This is also true for kininogen and total protein. These studies suggest that the vasoactive polypeptide system is present in most middle ear effusions and that the concentration of the components of this system are different for different effusions.

The second documented case of prolidase deficiency is presented. Clinical manifestations include chronic otitis media and sinusitis, dermatitis, and splenomegaly. Prolidase is undetectable in the white blood cells of the patient and near or less than the lower range of normal in each parent. The peptide chromatographic pattern of the urine is similar to that of the previously reported patient with prolidase deficiency. The quantity of amino acids excreted in urine per 24 hours is at least three times that of the upper range of normal (of these same amino acids) for the patients age group. More than 80% of the total amino acids excreted are in peptide form. The proline-to-hydroxyproline ratio suggests that the dipeptides are the catabolic products of other proteins in addition to collagen.

Thick mucosa removed from the promontory in cases with chronic otitis media showed prominent PAS-positive glands and epithelial secretory cells. Alcian blue positivity was less pronounced, contrary to the mucosa from glue ears. Enzyme activity in the epithelium and propria was comparable to that in glue ears, with some increase in alkaline phosphatase and some decrease in proprial lactate dehydrogenase and malate dehydrogenase activity. Removal of thick, permanently altered mucoas is recommended even in the absence of squamous epithelium. Steps should be taken to allow regrowth of thin, normal middle ear epithelium on the promontory.

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.

The sequential cytologic and biochemical events of middle ear effusion (MEE) were studied in experimental models of serous otitis media (SOM) and purulent otitis media (POM) in chinchilla. In the SOM model, the initial appearance of neutrophils was followed by macrophages. In the POM model, neutrophils were the predominant cells in MEE and the number of neutrophils was about 100-fold higher than in the SOM model. The activity of lysozyme in MEE was higher in POM than in SOM and correlated with the number of neutrophils and mononuclear phagocytes. The results of the present study suggest that neutrophils and mononuclear phagocytes are one of the main sources for lysozyme levels in MEE during otitis media.

The ceruminous glands have been studied histologically and histochemically in twenty patients suffering from chronic suppurative otitis media with discharging ears, and in ten patients with dry central perforations, in comparison with normal ears. The present study revealed hyperactivity of the glands initially; later on, atrophy and fibrosis took place. Thus the diminution of wax may be due to: (1) The washing effect of the discharge. (2) Atrophy of the glandular structures. (3) Periglandular fibrosis. As the discharge stops, regeneration of the glands occurs.

In characterizing the immunochemical and biochemical nature of middle ear effusions (MEE) of chronic serous otitis media (SOM), we have previously shown that MEE contain all major immunoglobulin classes, increased levels of several lysosomal enzymes, decreased total complement, but increased levels of C3. This report extends these observations by showing that MEE also contains C3 proactivator (C3PA) that can be activated by those organisms involved in acute otitis media, which confirms a functional alternate complement pathway. Furthermore, we have demonstrated the presence of soluble immune complexes in MEE using the Raji cell test. These data further support an immune complex mediated etiopathogenesis for chronic SOM.

No activity of the plasmin and plasminogen activators could be detected in middle ear effusion (MEE). A streptokinase proactivator, which was not plasminogen and markedly enhanced the activation of plasminogen by streptokinase, could be detected. MEE had an apparent inhibitory activity against both thiol protease (papain) and serine proteases (plasmin and trypsin). All these findings indicate that MEE contains unsaturated protease inhibitors and is in a state of predominance of inhibitors over fibrinolytic activity.

Activities of various oxidative (LDH, MDH) and hydrolytic (LAP, alkaline- and acid phosphatase, and lysozyme) enzymes in serous middle ear effusions (MEE) and serum from patients with serous otitis media were studied. The ratio of enzyme activity between MEE and serum (MEE/serum) was greater than one for all enzymes studied indicating a higher activity of these enzymes in MEE than in serum. These findings are consistent with a hypothesis suggesting the release of enzymes from inflammatory processes in the middle ear cavity. These enzymes presumably originate from 1) enzymes normally present in blood, 2) release of enzymes from inflamed middle ear mucosa, 3) release of enzymes from inflammatory cells present in the effusions.

Hydrolytic activity of cathepsin B-like thiol proteases and collagenolytic proteases was measured in middle ear effusions (MEE) from pediatric patients with acute and chronic otitis media with effusion (OME). Both activities were significantly higher in MEEs from acute OME than those from chronic OME (p less than 0.01). The ratio of hydrolytic activity in the extracellular portion to the total activity in MEEs from chronic OME was also analysed in both proteases. The ratio ranged from 20 to 100% in individual cases, indicating that the degree of the release of lysosomal thiol proteases including collagenolytic proteases from leukocytes is variable in MEEs with chronic OME. The kinetics of lysosomal thiol proteases including collagenolytic proteases in acute OME seems to be much more active than that in chronic OME, and the presence of these thiol proteases appears to be an important factor leading to chronic OME.

The proteolytic activity of different proteinases during chronic otitis media can be inhibited by alpha-2-macroglobulin and alpha-1-antitrypsin. A new low molecular (13,000) acid stable and polyvalent proteinase inhibitor could be investigated in the middle ear secretion from patients with cholesteatoma and chronic otitis media. We believe that this inhibitor is identical with the low molecular inhibitor of bronchial mucus and the nasal fluid. This inhibitor shows a high anti proteolytic capacity and can inactive trypsin, chymotrypsin, pronase and leucocytic preoteinases. The inhibitor is not detectable in any case. We could find it in 55 cases, three specimens of middle ear secretions obtained no acid stable inhibitor. It is present in the secretion in a masked form by in situ-reaction with leucocytic proteinases. By denaturating deproteinizing it is liberated out of the complex with proteinases and can be measured. The investigations demonstrate that the level of the inhibitor varies during the course of a chronic otitis media. In the postoperative phase the inhibitor concentrations were clearly higher than preoperatively. A steep drop of inhibitor can be observed in cases of chronic otitis with the symptomatology of an acute inflammation. In cases with a chronic inflammation the inhibitor level seems to remain low. The decrease of the inhibitor is explained as a using up effect during reaction between inhibitor and leucocytic proteinases. We believe that this inhibitor in the middle ear secretion results from a limited proteolysis and splitting of inter-alpha-trypsin inhibitor by a proteolytic enzyme, possibly by kallikrein.