A rat model for pneumococcal otitis media

The middle ears of 29 male Sprague-Dawley rats were injected with viable pneumococci, type 3 or 6A, and changes were monitored by otomicroscopy and analysis of bacterial samples from middle ear effusions, blood, and the nasopharynx. Depending on the type of pneumococci and its concentration, three responses were noted: otitis media with purulent effusion, otitis media with serous effusion, or no reaction. The mortality rate was low and the animals recovered without permanent deterioration or otomicroscopically discernable change. The results of this study show the rat to be a suitable animal model for the study of bacterial otitis media.     

Effect of gammaglobulin on pneumococcal otitis media in the rat.

Previously it has been found possible to induce pneumococcal AOM that closely resembles human infections, in the rat. In this study, the rat model has been used to study the possibilities to affect the infection by administration of gammaglobulin. The gammaglobulin was administered either systemically by intravenous injection of 0.2 g/kg bodyweight in 15 rats, or topically when 8 mg was instilled into the middle ear cavity of 18 rats. Although intravenous administration in this model failed to provide protection from AOM, topical administration simultaneously with the bacterial challenge was successful.     

Early structural changes in the rat tympanic membrane during pneumococcal otitis media

The early inflammatory reaction in the rat tympanic membrane was studied during the first 36h following inoculation of Streptococcus pneumoniae type 3 in the middle ear cavity. Otomicroscopic examination showed only minor signs of inflammation in the early stages although changes at the light microscopic level were pronounced. This reaction differed significantly between the pars flaccida and pars tensa of the tympanic membrane. Three hours after inoculation, edema and infiltration with polymorphonuclear leukocytes and macrophages were found in the pars flaccida whereas in the pars tensa no polymorphonuclear leukocytes were noted until after 12 h. This reaction was most prominent after 36h. In the pars flaccida, mitoses occurred frequently among the cells of the simple squamous epithelium, which changed into a double-layered cuboidal epithelium. These findings demonstrate that an inflammatory reaction starts earlier in the pars flaccida than in the pars tensa of the tympanic membrane.     

大鼠实验性肺炎球菌性中耳炎的病程及病理变化

目的建立大鼠肺炎球菌型中耳炎模型,以观察其病程及病理变化.方法44只SD大鼠,向耳注入肺炎球菌悬液,定期观察,分批处死,观察中耳的病理变化.结果大部分实验鼠中耳炎表现为自限性疾病,但病程迁延较长.部分转为慢性中耳炎.鼓膜外观恢复正常的实验耳,于6个月时仍有炎症改变.结论大鼠肺炎球菌性中耳炎并非完全是自限性疾病,其病理变化与人化脓性中耳炎类似,是研究人化脓性中耳炎较好的动物模型.     

Maternal pneumococcal conjugate immunization protects infant chinchillas in the pneumococcal otitis media model

Infant immunization with pneumococcal polysaccharide-protein conjugate vaccines (PCVs) is unlikely to elicit protective serum antibody concentrations during the first 4-6 months of life, when recurrent pneumococcal otitis media (POM) often begins. We therefore investigated a maternal pneumococcal immunization strategy to prevent early infant POM. Pregnant chinchillas (dams) received injections of heptavalent PCV or saline. Post-partum maternal and infant (kits) blood samples were obtained, and kits were subsequently challenged by intranasal inoculation of a vaccine-type pneumococcal strain (19F). Anti-pneumococcal capsular polysaccharide IgG antibody (Ab) concentration was measured using an ELISA in maternal and kit serum samples. Immunized dams and their kits had significantly higher Ab titers than control dams and their kits. Antibody titer in kits declined with a half-life of 12 days. Maternal immunization significantly reduced both the incidence (p = 0.05) and severity (p <0.01) of experimental POM in chinchilla kits, and was 82% effective at preventing mortality from invasive pneumococcal disease. Pre-challenge serum Ab concentration in kits was the single best predictor of POM severity (r = -0.66). This experiment strongly supports the hypothesis that maternal immunization with PCV will reduce the burden of early infant POM and invasive pneumococcal disease.     

Prevention of otitis media in children by pneumococcal vaccination

A total of 3,340 infants, 95 per cent of them 7 to 9 months old, were randomly vaccinated in a double-blind fashion with either the 14-valent pneumococcal (Pn) polysaccharide vaccine or a saline placebo in three urban areas in Finland. The second dose of the vaccine was given 5 months later. Age and sex distribution, recruitment of infants, and their otitis-related treatment and follow-up were similar in the study areas. Side effects after vaccination were mild and fewer than among older children. Antibody responses to vaccine polysaccharides varied from type to type, but were generally poor, especially to types most prevalent in otitis media. After the first dose of vaccine, the occurrence of otitis visits among the Pn-vaccinated, as compared with controls, showed inter-area differences, but ranged from not more than a 30 per cent reduction at its best to an increase in some areas and in some clinical categories. The respective figures for children with acute otitis media were similar between the vaccination groups and the study areas. The effect of the vaccine on acute otitis media caused by specific Pn types/groups represented in the vaccine was variable but generally poor. Group 6 attacks especially seemed to behave problematically. The second dose of the vaccine did not give additional benefit serologically or clinically. The efficacy of currently available pneumococcal vaccine against otitis media seemed poor in infants.     

Pneumococcal bacteriology after pneumococcal otitis media with special reference to pneumococcal antigens

Fifty-four ears with evidence of pneumococcus (Pn) in the first acute otitis media (AOM) in 38 infants and with prolongation or recurrence of the MEF during the follow-up were observed for 7-22 months for the presence of Pn by culture or of pneumococcal antigen (Pn-ag) by counterimmunoelectrophoresis or latex agglutination methods in their MEFs. During the first three 1-month observation periods, Pn and/or Pn-ags were detected in 24% to 9% of these ears, always of the initial type/group. Later on new Pn types/groups appeared also. In two of the 9 MEFs persisting for greater than or equal to 3 months, initial Pn-ag, with culturable Pn, was repeatedly found but not for longer than 5 months. Of the 12 ears resulting in secretory otitis media (SOM) only one showed initial Pn-ag (and Pn) in the MEF of SOM. Pneumococcal type/group pattern associated with prolongation or recurrence of infection did not differ from that of initial AOM. In another series of 151 SOM ears in 97 children, Pn-ags were detected in 7 MEFs. Four of them grew Pn, each of the corresponding group. Our studies suggest that the persistence of Pn-ags in the middle ear after AOM is limited and their occurrence in the MEFs of SOM is rare. Thus, the role of the persistence of Pn-ags in prolonged, recurrent or secretory otitis media seems questionable.     

Experimental pneumococcal otitis media: longitudinal studies in the gerbil model

In order to ascertain that the Mongolian gerbil, Meriones unguiculatus, is an acceptable model for studying the development of chronic from acute otitis media, we used previously published methods for experimental otitis media in a longitudinal study of the acute disease and sequellae. The gerbil was found to be susceptible to as few as 30 viable Streptococcus pneumoniae type 3 cells, indicating that only a few viable pathogens are able to cause the disease. Untreated experimental infections with S. pneumoniae type 23 resulted in a mild, self-limiting disease with little permanent sequellae, while S. pneumoniae type 3 produced severe disease characterized by an acute phase of from 2 to 3 weeks, followed by the development of new bone formation and a vascularized granulation tissue which persisted throughout the 13-week study. Viable pneumococci could be recovered from the middle ears for only two weeks. We conclude that the gerbil is a useful model for otitis media.     

Middle ear fluid lysozyme source in experimental pneumococcal otitis media

Middle ear infection with Streptococcus pneumoniae is important in the pathogenesis of acute and chronic otitis media, and lysozyme in middle ear fluid (MEF) is an important inflammatory mediator in this disease. To determine the source of lysozyme during the early period of acute pneumococcal otitis media, chinchillas were irradiated to induce neutropenia, and their middle ears were inoculated with heat-killed, encapsulated pneumococci. The number of inflammatory cells and concentration of lysozyme were measured in MEF between 6 and 72 hours after inoculation. In pneumococcus-inoculated ears, the mean number of inflammatory cells but not lysozyme was significantly lower in MEF from irradiated animals than that from nonirradiated animals at 6 hours. Since lysozyme accumulated in MEF before the influx of inflammatory cells in irradiated animals, the initial release of this inflammatory mediator is most likely not from inflammatory cells; and mucosal epithelial cells, the only other known source of lysozyme in the middle ear environment, were the probable source induced by the direct stimulation of pneumococci. Inflammatory cells may contribute lysozyme later in the inflammatory response, since cellular and lysozyme concentrations in irradiated and nonirradiated animals were similar between 24 and 72 hours. These results suggest that future therapeutic interventions to limit middle ear inflammation in acute otitis media may need to recognize the direct action of pneumococcal cells or their envelope components on middle ear epithelium.     

Influence of pneumococcal conjugate vaccines on acute otitis media in Japan

Objective

This study investigated: (i) changes in the incidence of acute otitis media (AOM) following introduction of public funding for free inoculation with 7- and 13-valent pneumococcal conjugate vaccines (PCV7 and PCV13, respectively) and (ii) changes in the rate of myringotomies for AOM (MyfA) in children 1 year following the publication of the first edition of the clinical practice guidelines for the diagnosis and management of AOM in children in Japan.

Interleukin-8 expression in otitis media

Based on recent studies in the authors' laboratory on the correlation of cytokines and inflammation in otitis media (OM), the authors hypothesized that in chronic otitis media with effusion (COME) interleukin-8 (IL-8) is responsible for 1. the accumulation of leukocytes in the middle ear cleft and 2. in situ leukocyte activation with subsequent tissue damage. Additionally, the authors hypothesized that IL-8 expression is at least in part under the control of interleukin-1 (IL-1) and tumor necrosis factor (TNF). To begin to test this hypothesis, middle ear effusions (MEE) obtained from children ages 2 to 90 months (mean age, 29 months) undergoing tympanostomy tube placement for the presence of these inflammatory cytokines were analyzed. For these studies, IL-8, interleukin-1 β (IL-1β), tumor necrosis factor-α (TNF-α), and tumor necrosis factor-β (TNF-β) were measured in MEE by radioimmunoassay (RIA) or enzyme-linked immunoassay (ELISA). IL-8, IL-1β, TNF-α, and TNF-β were present in 92%, 67%, 77%, and 0% of effusions, respectively. The mean (± SEM) values for IL-8, IL-1β, and TNF-α were 4805 (± 913) pg/mg, 4076 (± 1510) pg/mg, and 163 (± 90) pg/mg. Further analysis indicated that levels of IL-8 correlated with IL-1β (R² = .500, P = .000) and TNF-α (R² = .387, P = .023). Thus the authors' studies clearly demonstrate that IL-8 is consistently present in the MEE of children with COME and is strongly correlated with levels of IL-1β and TNF-α, both known inducers of IL-8 production. These results support the authors' hypothesis that IL-1β, TNF-α, and IL-8 are intimately involved in the inflammatory cascade in the middle ear and suggest regulation of these cytokines as possible sites of future therapeutic intervention in otitis media with effusion (OME).     

Oral vaccine therapy for pneumococcal otitis media in an animal model

We investigated whether mucosal IgA response in the middle ear cavity against Streptococcus pneumoniae type 19F is enhanced by use of enteric capsules, and whether the resulting mucosal immunity can prevent pneumococcal otitis media. Adult Hartley guinea pigs were employed. With intratympanic inoculation of 10(5) and 10(6) live S pneumoniae, the occurrence of pneumococcal otitis media significantly decreased in guinea pigs that received intraduodenal and intragastric immunization by enteric capsules. In these guinea pigs, the values of salivary IgA antibody titers against S pneumoniae were significantly greater, and histologic changes of the middle ear mucosa were also slighter than those of control guinea pigs. These findings indicate that oral vaccination by enteric capsules elicits mucosal IgA responses, as well as intraduodenal immunization, to prevent pneumococcal otitis media. These results suggest the possibility of clinical application of oral vaccination by enteric capsules for the prevention of middle ear infection.     

The influence of pneumococcal otitis media on the cochlear lateral wall.

The cochlear influence of otitis media was investigated in order to identify damaged regions causing cochlear malfunction. BALB/c mice were challenged with viable Streptococcus pneumoniae into the middle ear cavity and were killed 1 day to 1 month later for immunohistochemical analysis. Otitis media was induced in all of the animals, and some showed inflammatory cells in the cochlea. Although other changes were not obvious by hematoxylin and eosin staining, immunohistochemistry showed the presence of fibrinogen in the cochlea, mainly in the lower portion of the spiral ligament and in the spiral limbus. Immunostaining for connexin 26 was decreased in the spiral ligament, accompanied by marked fibrinogen staining. Immunostaining for sodium-potassium-adenosine triphosphatase in the stria vascularis and in the type II fibrocytes of the spiral ligament was not affected obviously. The presence of fibrinogen in the cochlea suggests disruption of the blood-labyrinth barrier caused by the middle ear inflammation. Changes in connexin 26 staining suggest the possibility that the spiral ligament could be among the regions responsible for the cochlear malfunction.     

Toll样受体4信号途径在大鼠中耳积液中的表达

目的探讨Toll样受体4（Toll like receptor 4,TLR4）信号途径在中耳积液大鼠的作用机制。方法采用听泡内注入纤维蛋白封闭剂和脂多糖制作大鼠中耳积液模型。分别于3、5、7、9、11、13天处死大鼠,每次5只。取中耳黏膜检测TLR4、诱生型一氧化氮合酶（inducible nitric oxide synthase,iNOS）、白细胞介素8（interleukin-8,IL-8）mRNA的表达;免疫组化检测核因子κB（nuclear factor kappa B,NF-κB）表达情况。结果对照组中耳黏膜内有少量的TLR4、iNOS和IL-8mRNA的表达,中耳积液时表达量明显增高（P〈0.05）。免疫组化检测显示大鼠中耳黏膜内NF-κBp65的表达在中耳积液时明显提高。结论中耳积液时TLR4表达明显升高,并伴有NF-κB活化,促进下游IL-8等的表达,可能是引发大鼠分泌性中耳炎的机制之一。