muramidase and Otitis-Media

The sterility of the eustachian tube and tympanic cavity of normal individuals is maintained not only by the adaptive immune system, but also by the mucociliary system and the antimicrobial molecules of innate immunity. Mucin production and periciliary fluid homeostasis are essential for normal mucociliary function and dysfunction of this system is an important risk factor for otitis media. The secreted antimicrobial molecules of the tubotympanum include lysozyme, lactoferrin, beta defensins, and the surfactant proteins A and D (SP-A, SP-D). Defects in the expression or regulation of these molecules may also be the major risk factor for otitis media.

Topics: Animals; Aquaporins; beta-Defensins; Child; Child, Preschool; Cilia; Disease Susceptibility; Epithelial Cells; Eustachian Tube; Gene Expression Profiling; Glycoproteins; Homeostasis; Humans; Immunity, Innate; Infant; Lactoferrin; Mice; Mucins; Mucus; Muramidase; Otitis Media; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Protein D; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Rabbits; Rats; Tympanic Membrane

Otitis media, commonly known as middle ear inflammation, is among one of the most common maladies and results in significant morbidity such as loss of hearing. In view of the bacteria invasion such as Staphylococcus aureus causes the majority forms of otitis media, drug treatment generally uses antibacterial by topical or systematic approach. However, the effectiveness of antibacterial is diminishing because of the rapid emergence of antibiotic-resistant bacterial strains. Here, we designed and fabricated a silver nanoparticle (AgNPs)-based multicomponent hybrid nanocomposite termed as TSIIA @ CS/Lys @ AgNPs, which was comprised of a AgNPs core, a chitosan (CS) or lysozyme (Lys) middle layer, and a Tanshinone IIA (TSIIA) inclusion outlayer. Coating of CS or Lys to AgNPs through electrostatic interaction probably produced a core-shell nanocomplex resembling the endocarp of walnut. This design could reduce the dosage of AgNPs while maintaining antibacterial activity possibly due to the favorable interactions between nanocomplex and bacteria. The deposition of Chinese herb active component TSIIA by inclusion complexation formed the out layer of hybrid nanocomposite towards an improved antibacterial performance, which showed a therapeutic effect against acute otitis media of guinea pig comparable to the clinical commercial-used ofloxacin administrated by injection. The hybrid nanocomposite, when dispersed in poly (lactic-co-glycolic acid)/N-methyl-2-pyrrolidone (PLGA/NMP) solution as an in-situ organogel, not only maintained the therapeutic effectiveness, but also possessed the advantage of lower injection frequency compared with solution formulation. In addition, no obvious toxicity to the basilar membrane and epithelia tissue was observed after the healthy guinea pigs were treated with hybrid nanocomposite or organogel. This study provides a promising strategy to develop hybrid nanocomposite with enhanced antibacterial efficacy and also opens a new way for the establishment of efficient therapeutic systems with reduced administration frequency as substitute of antibiotics to treat otitis media.

Topics: Abietanes; Animals; Anti-Bacterial Agents; Bacteria; Chitosan; Female; Guinea Pigs; Male; Metal Nanoparticles; Muramidase; Nanocomposites; Otitis Media; Silver

In our model system using the THP-1 monocytic cell line, whole heat-killed cells of Alloiococcus otitidis elicited several pro-inflammatory cytokines identified in ear effusions of children with otitis media (OM). Levels of these cytokines were equivalent to or greater than those elicited by a standard Gram-positive otopathogen, Streptococcus pneumoniae. The current study examined the hypothesis that extracellular material produced by A. otitidis might also contribute to the inflammatory responses in OM. Cell-free culture filtrates of recent A. otitidis isolates (n = 39) were tested for induction of pro-inflammatory cytokines from THP-1 cells primed with IFN-γ. The highest responses were from IL-8 followed by IL-1β, and the lowest from IL-6. Filtrates from nine isolates were treated with lysozyme or proteinase K to assess the nature of the extracellular stimulants. Peptidoglycan was not a major component eliciting the responses. There was no correlation between colony type or β-haemolysin production. Proteinase K treatment indicated extracellular proteins might induce the inflammatory responses, particularly the 70-75 ku band. Further studies on the role of the extracellular proteins of A. otitidis and cytokine responses in pathogenesis of ear infections are needed.

Topics: Bacterial Proteins; Carnobacteriaceae; Cell Line; Cytokines; Endopeptidase K; Hemolysin Proteins; Humans; Hydrolysis; Inflammation; Interferon-gamma; Monocytes; Muramidase; Otitis Media

Antimicrobial agents (AMAs) and mast cells in chronic otitis media (COM) may play a protective role in the pathogenesis of COM. Among them, our results indicated that lysozyme may be the most important parameter of the mucosa infectivity in COM.. AMAs include alpha 1-antitrypsin (α1-AT), lysozyme, and lactoferrin and exhibit innate immune activity that is known to play a critical role in mucosal defenses. In addition, proteases expressed in mast cells contribute to mucosal defenses. The aim of this study was to identify AMAs responsible for COM as well as their association with middle ear mucosal diseases.. Middle ear mucosa (MEM) was intraoperatively collected by biopsy from patients, diagnosed as COM without or with cholesteatoma, and immediately processed for immunohistochemical study to evaluate expression of AMAs and mast cell proteases (chymase and tryptase).. The AMAs and mast cell enzymes were observed prominently in the chronic middle ear diseases whereas immunoreactivity of α1-AT and lysozyme was not significantly increased in the COM with cholesteatoma compared with that in COM without cholesteatoma. Lactoferrin was not detected in the diseased MEMs.

Topics: Adult; alpha 1-Antitrypsin; Biomarkers; Biopsy; Cholesteatoma, Middle Ear; Female; Humans; Immunohistochemistry; Lactoferrin; Male; Mast Cells; Middle Aged; Mucous Membrane; Muramidase; Otitis Media

Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity.. Immunohistochemistry was performed to localize lysozyme in the Eustachian tube. Lysozyme expression was compared between the wild type and the lysozyme M-/- mice using real time quantitative RT-PCR and western blotting. Muramidase activity and bactericidal activity of lysozyme was measured using a lysoplate radial diffusion assay and a liquid broth assay, respectively. To determine if depletion of lysozyme M increases a susceptibility to pneumococal otitis media, 50 CFU of S. pneumoniae 6B were transtympanically inoculated to the middle ear and viable bacteria were counted at day 3 and 7 with clinical grading of middle ear inflammation.. Immunolabeling revealed that localization of lysozyme M and lysozyme P is specific to some/particular cell types of the Eustachian tube. Lysozyme P of lysozyme M-/- mice was mainly expressed in the submucosal gland but not in the tubal epithelium. Although lysozyme M-/- mice showed compensatory up-regulation of lysozyme P, lysozyme M depletion resulted in a decrease in both muramidase and antimicrobial activities. Deficiency in lysozyme M led to an increased susceptibility to middle ear infection with S. pneumoniae 6B and resulted in severe middle ear inflammation, compared to wild type mice.. The results suggest that lysozyme M plays an important role in protecting the middle ear from invading pathogens, particularly in the early phase. We suggest a possibility of the exogenous lysozyme as an adjuvant therapeutic agent for otitis media, but further studies are necessary.

Topics: Animals; Disease Susceptibility; Eustachian Tube; Gene Expression Profiling; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Muramidase; Otitis Media; Pneumococcal Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA; Streptococcus pneumoniae

Topics: Acute Disease; Administration, Intranasal; Animals; Disease Models, Animal; Female; Humans; Influenza, Human; Mice; Mice, Inbred BALB C; Muramidase; Orthomyxoviridae; Otitis Media; Pneumococcal Infections; Streptococcus pneumoniae

Despite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and beta defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable Haemophilus influenzae (NTHi) 12, Moraxella catarrhalis 035E, and Streptococcus pneumoniae 3, and 6B.. Two assay methods, the radial assay and the liquid broth assay, were employed for testing the antimicrobial activity of the molecules. This was done in order to minimize the possibility that the observed effects were artifacts of any single assay system employed. Also, transmission electron microscopy (TEM) was employed to evaluate the effect of antimicrobial innate immune molecules on OM pathogens. For the statistical analysis of the data, Student's t-test was performed.. Results of the radial diffusion assay showed that beta defensin-2 was active against all four OM pathogens tested, while treatment with beta defensin-1 appeared to only affect M. catarrhalis. The radial assay results also showed that lysozyme was quite effective against S. pneumoniae 3 and 6B and was partially bacteriostatic/bactericidal against M. catarrhalis. Lysozyme however, appeared not to affect the growth of NTHi. Thus, lysozyme seems to have a more pronounced impact on the growth of the Gram-positive S. pneumoniae as compared to that of Gram-negative pathogens. Lactoferrin on the other hand, enhanced the growth of the bacteria tested. The results of the radial assays were confirmed using liquid broth assays for antimicrobial activity, and showed that lysozyme and beta defensin-2 could act synergistically against S. pneumoniae 6B. Moreover, in the liquid broth assay, beta defensin-1 showed a modest inhibitory effect on the growth of S. pneumoniae 6B. As assessed by ultrastructural analysis, lysozyme and beta defensin-2, and to a much lesser extent, beta defensin-1, appeared to be able to cause damage to the bacterial membranes.. Here we report that lysozyme and the beta defensins can inhibit the growth of clinical isolates of otitis media pathogens - namely NTHi strain 12, S. pneumoniae strains 3 and 6B and M. catarrhalis strain 035E - and cause ultrastructural damage to these pathogens. Moreover, we demonstrate that lysozyme and beta defensin-2 can act synergistically against S. pneumoniae. These findings are consistent with the concept that secreted antimicrobial peptides and other components of innate immunity constitute the first line of defense protecting host mucosal surfaces, including the tubotympanal (eustachian tube and middle ear cavity) mucosa, against pathogens.

Topics: Anti-Infective Agents; beta-Defensins; Haemophilus influenzae; Humans; Microbial Sensitivity Tests; Microscopy, Electron, Transmission; Moraxella catarrhalis; Muramidase; Otitis Media; Streptococcus pneumoniae

A common complication of virus-induced upper respiratory tract infections is acute otitis media caused by bacterial pathogens. Simultaneously, increased bacterial colonization in the nasopharynx occurs. Our intention in this study was to identify the causes of this increased colonization of bacteria by evaluating their coating with the antibacterial substances lysozyme, lactoferrin and immunoglobulins IgG, S-IgA and IgM and their ability to penetrate epithelial cells during infectious mononucleosis (IM) caused by Epstein-Barr virus.. Cellular samples were collected from the oropharynx of 21 patients (16 males, five females; age range 10-21 years) with current IM. An immunocytochemical assay using gold-labelled antiserum to human lysozyme, lactoferrin, IgG, S-IgA and IgM followed by gold particle and epithelial cell tracing in the transmission electron microscope.. A significant reduction in bacterial coating with IgG (P<0.05) and S-IgA (P<0.01) was noted, whereas there was a significant increase in coating with lactoferrin (P<0.01) and IgM (P<0.01). No significant change in lysozyme coating of the bacteria was noted, compared with healthy controls. Bacterial penetration into epithelial cells was seen particularly in patients culture-positive for beta-haemolytic streptococci.. Reduced bacterial coating with IgG and S-IgA immunoglobulins, combined with bacterial penetration into epithelial cells, may exacerbate the bacterial colonization on oropharyngeal mucosal membranes observed during IM.

Topics: Acute Disease; Adolescent; Adult; Case-Control Studies; Child; Data Interpretation, Statistical; Epithelial Cells; Female; Herpesvirus 4, Human; Humans; Immunity, Mucosal; Immunoglobulin A, Secretory; Immunoglobulin G; Immunoglobulin M; Infectious Mononucleosis; Lactoferrin; Male; Microscopy, Electron; Muramidase; Nasal Mucosa; Nasopharynx; Otitis Media; Pharyngitis

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.

Topics: Animals; Ear, Middle; Edema; Epithelium; Guinea Pigs; Moraxella catarrhalis; Muramidase; Otitis Media; Peroxidase

Haemophilus influenzae is one of the most frequent pathogens of acute otitis media. To determine the middle ear response during the early stage of acute inflammation, a small amount of H. influenzae was inoculated into the bullae of guinea pigs through the tympanic membrane. The bullae were harvested at 6, 12, 24, 36, and 48 hours after H. influenzae inoculation and washed with phosphate-buffered saline (PBS). The number of viable H. influenzae and inflammatory cells, the concentrations of myeloperoxidase (MPO) and lysozyme in the washing suspensions were measured, and compared with those in PBS-inoculated control ears. The number of viable H. influenzae increased very rapidly from 6 to 12 hours after inoculation and remained stationary up to 48 hours. The number of inflammatory cells and the MPO concentration were significantly higher in the H. influenzae-inoculated ears than in the control ears from 12 to 48 hours after inoculation. The lysozyme concentration was already significantly higher at 6 hours in the H. influenzae-inoculated ears; the lysozyme was released in the middle ear before the accumulation of inflammatory cells and degranulation of MPO from inflammatory cells. The results indicated that inflammatory reactions were present already at 6 hours after bacterial inoculation, and were rapidly accelerated during the subsequent hours. Consequently, acute middle ear inflammatory responses were seen immediately following inoculation of viable bacteria, and these responses originated in direct responses of middle ear mucosa, and oxidative and non-oxidative neutrophil metabolic products, which may cause tissue injury.

Topics: Acute Disease; Animals; Ear, Middle; Guinea Pigs; Haemophilus influenzae; Muramidase; Otitis Media; Peroxidase; Time Factors

Most Streptococcus pneumoniae strains are killed by very low concentrations of penicillin and other beta-lactam antibiotics, yet middle ear inflammation and effusion persist for days to weeks after treatment in most cases of pneumococcal otitis media. To study the effect of beta-lactam antibiotic treatment on pneumococci and the middle ear inflammatory response during pneumococcal otitis media, we measured concentrations of pneumococci, inflammatory cells, and lysozyme in middle ear fluid (MEF) by using the chinchilla model. Procaine penicillin G given intramuscularly 12 and 36 h after inoculation of pneumococci into the middle ear caused a significant acceleration in the MEF inflammatory cell concentration compared with that in untreated controls, with a significant peak in the inflammatory cell concentration 24 h after pneumococcal inoculation. The lysozyme concentration in MEF also increased more rapidly in treated than in control animals. Viable pneumococci were not detected in MEF after the second dose of penicillin, but the total pneumococcal cell concentration remained unchanged for at least 45 days. Therefore, penicillin treatment accelerated middle ear inflammation while killing pneumococci, but treatment did not accelerate clearance of the nonviable pneumococcal cells from MEF. Further studies will need to define the contribution of these responses to acute and chronic tissue injury.

Topics: Animals; Chinchilla; Muramidase; Otitis Media; Penicillins; Pneumococcal Infections

The pathogenesis of middle ear inflammation caused by Streptococcus pneumoniae was explored in the chinchilla model with different pneumococcal cell wall (CW) preparations, including isolated native CW, M1 muramidase CW (M1-CW) digest, amidase CW digest, and M1 peptidoglycan (M1-PG) digest. Inflammatory cell and lysozyme concentrations in middle ear fluid (MEF) were measured between 6 and 72 h after the middle ears were inoculated with one of the preparations or sterile saline. Middle ear histopathology was measured quantitatively at 72 h. Native CW, M1-CW digest, and amidase-CW digest caused significantly more inflammatory cell influx and lysozyme accumulation in MEF than saline did. M1-PG digest also caused more inflammatory cell influx and lysozyme accumulation in MEF than saline did but caused less inflammation than native CW or either CW digest. Epithelial metaplasia was significantly greater in ears inoculated with native CW than in ears inoculated with the CW or PG digest or with saline. Pneumococcal CW is, therefore, the principal factor that initiates middle ear inflammation in acute pneumococcal otitis media, and CW teichoication seems to be important in initiating this response.

Topics: Amidohydrolases; Animals; Cell Wall; Chinchilla; Dose-Response Relationship, Immunologic; Muramidase; Otitis Media; Streptococcus pneumoniae; Time Factors

Streptococcus pneumoniae is an important bacterial pathogen in the pathophysiology of otitis media. To elucidate the inflammatory responses that occur during pneumococcal otitis media, the kinetics of the biochemical and cytologic middle ear responses to heat-killed encapsulated and nonencapsulated pneumococci were studied in the chinchilla model. Inoculation of the middle ear cavity with at least 10(6) S pneumoniae cells induced an early, brief vascular response with leakage of small (albumin) followed by larger (alpha 2-macroglobulin) proteins, followed by sustained influx of acute inflammatory cells and lysozyme. The threshold for a sustained lysozyme response was 1,000 times lower for nonencapsulated than for encapsulated pneumococci. These results indicate that nonviable S pneumoniae organisms with an intact envelope initiate the middle ear inflammatory response. Therefore, interventions that enhance the clearance of pneumococcal cells from the middle ear may reduce the inflammatory response and prevent chronic middle ear inflammation.

Topics: Albumins; alpha-Macroglobulins; Animals; Chinchilla; Muramidase; Neutrophils; Otitis Media; Otitis Media, Suppurative; Streptococcal Infections

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.

Topics: Animals; Chinchilla; Muramidase; Otitis Media; Otitis Media with Effusion; Otitis Media, Suppurative; Pneumococcal Infections; Time Factors

Topics: Adolescent; Adult; Aged; Animals; Bacterial Infections; Child; Child, Preschool; Dogs; Ear, Middle; Eustachian Tube; Food Hypersensitivity; Gases; Humans; Hypersensitivity; Immunoglobulin E; Infant; Lymph; Middle Aged; Mucus; Muramidase; Otitis Media; Otitis Media with Effusion; Perilymph

Topics: Animals; Child; Child, Preschool; Chinchilla; Exudates and Transudates; Female; Humans; Infant; L-Lactate Dehydrogenase; Male; Muramidase; Otitis Media; Otitis Media with Effusion; Otitis Media, Suppurative; Penicillins

Topics: Animals; Exudates and Transudates; Humans; L-Lactate Dehydrogenase; Muramidase; Otitis Media; Otitis Media, Suppurative

Topics: Complement System Proteins; Eustachian Tube; Humans; Immunoglobulins; Lymphocytes; Muramidase; Otitis Media; Prostaglandins

147 samples of punctured middle ear effusion fluid from cases of otitis media with effusion and 150 samples from patients with acute purulent otitis media were tested for lysozyme activity. In otitis media with effusion the concentration was 182.0 U/ml, in acute otitis 433.8 U/ml. The lysozyme concentration in otitis media with effusion depended upon the nature of the effusion. Serous fluid showed an activity of 124.8 U/ml and mucoid 311.6 U/ml, respectively. In culture-positive cases of acute otitis media the lysozyme level was 423.4 U/ml. Culture-negative cases showed about the same concentration, 438.3 U/ml. The possible role of lysozyme in defence systems of the middle ear is discussed.

Topics: Adolescent; Adult; Aged; Albumins; Child; Child, Preschool; Humans; Immunoglobulins; Infant; Middle Aged; Muramidase; Otitis Media; Otitis Media, Suppurative

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.

Topics: Acid Phosphatase; Alkaline Phosphatase; Exudates and Transudates; Humans; Hydrolases; L-Lactate Dehydrogenase; Leucyl Aminopeptidase; Malate Dehydrogenase; Muramidase; Olfactory Mucosa; Otitis Media; Oxidoreductases

Middle ear effusions from 100 patients (ages 6 months to 10 years) with serous otitis media were examined. The IgA, IgG, and lysozyme were demonstrated at a higher level in the effusions than the corresponding sera, indicating local production. The mucoid type contained higher level of immunoglobulins and lysozyme compared to serous type effusions. Bacteria were found in 77 percent of the effusions by means of a smear, and 52 percent yielded positive bacterial culture. The incidence of positive culture in effusions of the patients less than 6 years of age was 60 percent, while the group older than 6 years old was 32%, and the group over 8 was only 22 percent. Bacterial recovery rate was inversely related to the dramatic increase with age of IgA and IgG and lysozyme levels in effusions.

Topics: Age Factors; Biological Assay; Child; Child, Preschool; Chronic Disease; Exudates and Transudates; History, 19th Century; Humans; Immunodiffusion; Immunoglobulin A; Immunoglobulin G; Infant; Leukocytes; Mucus; Muramidase; Otitis Media

Topics: Animals; Dog Diseases; Dogs; Drug Therapy, Combination; Edetic Acid; Female; Male; Muramidase; Otitis Externa; Otitis Media; Pseudomonas aeruginosa; Pseudomonas Infections; Therapeutic Irrigation; Tromethamine

Topics: Antibody-Producing Cells; Antigens, Viral; Autoradiography; Biopsy; Blood Proteins; Carbon Isotopes; Exudates and Transudates; Fluorescent Antibody Technique; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; In Vitro Techniques; Lactoferrin; Mucous Membrane; Muramidase; Otitis Media; Proteins

Topics: Child; Deoxyribonucleases; Humans; Lysostaphin; Muramidase; Otitis Media; Oxygen Consumption; Species Specificity; Staphylococcal Infections; Staphylococcus

Topics: Aerosols; Humans; Muramidase; Otitis Media; Otosclerosis; Papain