Vaccination against T-cell epitopes of native ApoB100 reduces vascular inflammation and disease in a humanized mouse model of atherosclerosis.

BACKGROUND AND OBJECTIVES: The T-cell response to low-density lipoprotein (LDL) in the vessel wall plays a critical role in atherosclerotic plaque formation and stability. In this study, we used a new translational approach to investigate epitopes from human apolipoprotein B100 (ApoB100), the protein component of LDL, which triggers T-cell activation. We also evaluated the potential of two selected native ApoB100 epitopes to modulate atherosclerosis in human ApoB100-transgenic Ldlr-/- (HuBL) mice. METHODS AND RESULTS: HuBL mice were immunized with human atherosclerotic plaque homogenate to boost cellular autoimmune response to tissue-derived ApoB100 epitopes. In vitro challenge of splenocytes from immunized mice with a library of overlapping native peptides covering human ApoB100 revealed several sequences eliciting T-cell proliferation. Of these sequences, peptide (P) 265 and P295 were predicted to bind several human leucocyte antigen (HLA) haplotypes and induced high levels of interferon (IFN)-γ. Vaccination of HuBL mice with these peptides mounted a strong adaptive immune response to native ApoB100, including high levels of epitope-specific plasma IgGs. Interestingly, P265 and P295 vaccines significantly decreased plaque size, reduced macrophage infiltration and increased IgG1 deposition in the plaques. Purified IgGs from vaccinated mice displayed anti-inflammatory properties against macrophages in vitro, reducing their response to LPS in a dose-dependent manner. CONCLUSION: We identified two specific epitopes from human native ApoB100 that trigger T-cell activation and protect HuBL mice against atherosclerosis when used in a vaccine. Our data suggest that vaccination-induced protective mechanisms may be mediated at least in part through specific antibody responses to LDL that inhibit macrophage activation.

Effectiveness of photodynamic therapy in Bowen's disease: a retrospective observational study in 423 lesions.

BACKGROUND: Photodynamic therapy (PDT) is a well-known technique that is often used for treating superficial precancerous and cancerous skin lesions. However, only a handful of studies, with a relatively small number of treated lesions, have been carried out on the effectiveness of PDT for Bowen's disease (BD). OBJECTIVES: This study aimed to assess the effectiveness and recurrence risk of PDT in the treatment of BD. The secondary objectives were to determine what factors affected the response rates and the cosmetic result of the treatment. METHOD: In this retrospective observational study, the electronic patient charts at Sahlgrenska University Hospital (SUH) in Gothenburg, Sweden, were searched to find all patients diagnosed with BD who were treated with PDT between 1 January 2002 and 31 December 2014. Data were collected regarding clinical response at the first follow-up visit, recurrences during later follow-up visits and other relevant patient and tumour characteristics. RESULTS: In total, 423 BD lesions in 335 patients were included in the study. The mean FU duration was 11.2 months (range 0.2-151 months). The complete response rate at the first FU visit was 77.5% for all BD lesions. During later FU visits, another 60 recurrences were observed, which resulted in a recurrence rate of 18.3%. Thus, the overall clearance rate after FU was 63.4% for all BD lesions. Significant risk factors for unsuccessful treatment in this study were large lesion size (>2 cm) and a single PDT session. CONCLUSION: This study shows that PDT is a relatively effective treatment modality for BD.

National assessment of validity of coding of acute mastoiditis: a standardised reassessment of 1966 records.

OBJECTIVES: To investigate the internal validity of the diagnosis code used at discharge after treatment of acute mastoiditis. DESIGN: Retrospective national re-evaluation study of patient records 1993-2007 and make comparison with the original ICD codes. SETTING: All ENT departments at university hospitals and one large county hospital department in Sweden. PARTICIPANTS: A total of 1966 records were reviewed for patients with ICD codes for in-patient treatment of acute (529), chronic (44) and unspecified mastoiditis (21) and acute otitis media (1372). MAIN OUTCOME MEASURES: ICD codes were reviewed by the authors with a defined protocol for the clinical diagnosis of acute mastoiditis. Those not satisfying the diagnosis were given an alternative diagnosis. RESULTS: Of 529 records with ICD coding for acute mastoiditis, 397 (75%) were found to meet the definition of acute mastoiditis used in this study, while 18% were not diagnosed as having any type of mastoiditis after review. Review of the in-patients treated for acute media otitis identified an additional 60 cases fulfilling the definition of acute mastoiditis. Overdiagnosis was common, and many patients with a diagnostic code indicating acute mastoiditis had been treated for external otitis or otorrhoea with transmyringeal drainage. CONCLUSIONS: The internal validity of the diagnosis acute mastoiditis is dependent on the use of standardised, well-defined criteria. Reliability of diagnosis is fundamental for the comparison of results from different studies. Inadequate reliability in the diagnosis of acute mastoiditis also affects calculations of incidence rates and statistical power and may also affect the conclusions drawn from the results.

Characterization of scrubber water discharges from ships using comprehensive suspect screening strategies based on GC-APCI-HRMS.

An extended suspect screening approach for the comprehensive chemical characterization of scrubber discharge waters from exhaust gas cleaning systems (EGCSs), used to reduce atmospheric shipping emissions of sulphur oxides, was developed. The suspect screening was based on gas chromatography coupled with high-resolution mass spectrometry (GC-HRMS) and focused on the identification of polycyclic aromatic hydrocarbons (PAHs) and their alkylated derivatives (alkyl-PAHs), which are among the most frequent and potentially toxic organic contaminants detected in these matrices. Although alkyl-PAHs can be even more abundant than parent compounds, information regarding their occurrence in scrubber waters is scarce. For compound identification, an in-house compound database was built, with 26 suspect groups, including 25 parent PAHs and 23 alkyl-PAH homologues. With this approach, 7 PAHs and 12 clusters of alkyl-PAHs were tentatively identified, whose occurrence was finally confirmed by target analysis using GC coupled with tandem mass spectrometry (GC-MS/MS). Finally, a retrospective analysis was performed to identify other relevant (poly)cyclic aromatic compounds (PACs) of potential concern in scrubber waters. According to it, 18 suspect groups were tentatively identified, including biphenyls, dibenzofurans, dibenzothiophenes and oxygenated PAHs derivatives. All these compounds could be used as relevant markers of scrubber water contamination in heavy traffic marine areas and be considered as potential stressors when evaluating scrubber water toxicity.

An offset-free MPC formulation for nonlinear systems using adaptive integral controller.

This paper investigates a novel offset-free control scheme based on a multiple model predictive controller (MMPC) and an adaptive integral action controller for nonlinear processes. Firstly, the multiple model description captures the essence of the nonlinear process, while keeping the MPC optimization linear. Multiple models also enable the controller to deal with the uncertainty associated with changing setpoint. Then, a min-max approach is utilized to counter the effect of parametric uncertainty between the linear models and the nonlinear process. Finally, to deal with other uncertainties, such as input and output disturbances, an adaptive integral action controller is run in parallel to the MMPC. Thus creating a novel offset-free approach for nonlinear systems that is more easily tuned than observer-based MPC. Simulation results for a pH-controller, which acts as an example of a nonlinear process, are presented to demonstrate the usefulness of the technique compared to using an observer-based MPC.

Smad7 mediates apoptosis induced by transforming growth factor beta in prostatic carcinoma cells.

Transforming growth factor beta (TGF-beta) is an important regulator of apoptosis in some cell types, but the underlying molecular mechanisms are largely unknown. TGF-beta signals through type I and type II receptors and downstream effector proteins, termed Smads. TGF-beta induces the phosphorylation of Smad2 and Smad3 (receptor-activated Smads) which associate with Smad4 and translocate to the nucleus, where they regulate gene transcription [1]. Smad7 protein is induced by TGF-beta1 and has been classified as an inhibitory Smad. Smad7 prevents phosphorylation of receptor-activated Smads, thereby inhibiting TGF-beta-induced signaling responses [1]. Smad7 expression is increased in rat prostatic epithelial cells undergoing apoptosis as a result of castration [2]. Here we have shown that TGF-beta1 treatment or ectopic expression of Smad7 in human prostatic carcinoma cells (PC-3U) induces apoptosis. Furthermore, TGF-beta1-induced apoptosis was prevented by inhibition of Smad7 expression, by antisense mRNA in stably transfected cell lines or upon transient transfection with antisense oligonucleotides in several investigated cell lines. These findings provide evidence for a new effector function for Smad7 in TGF-beta1 signaling.

Interconnectivity imaged in three dimensions: Nano-particulate silica-hydrogel structure revealed using electron tomography.

We have used Electron Tomography (ET) to reveal the detailed three-dimensional structure of particulate hydrogels, a material category common in e.g. controlled release, food science, battery and biomedical applications. A full understanding of the transport properties of these gels requires knowledge about the pore structure and in particular the interconnectivity in three dimensions, since the transport takes the path of lowest resistance. The image series for ET were recorded using High-Angle Annular Dark Field Scanning Transmission Electron Microscopy (HAADF-STEM). We have studied three different particulate silica hydrogels based on primary particles with sizes ranging from 3.6nm to 22nm and with pore-size averages from 18nm to 310nm. Here, we highlight the nanostructure of the particle network and the interpenetrating pore network in two and three dimensions. The interconnectivity and distribution of width of the porous channels were obtained from the three-dimensional tomography studies while they cannot unambiguously be obtained from the two-dimensional data. Using ET, we compared the interconnectivity and accessible pore volume fraction as a function of pore size, based on direct images on the nanoscale of three different hydrogels. From this comparison, it was clear that the finest of the gels differentiated from the other two. Despite the almost identical flow properties of the two finer gels, they showed large differences concerning the accessible pore volume fraction for probes corresponding to their (two-dimensional) mean pore size. Using 2D pore size data, the finest gel provided an accessible pore volume fraction of over 90%, but for the other two gels the equivalent was only 10-20%. However, all the gels provided an accessible pore volume fraction of 30-40% when taking the third dimension into account.

Thyrotropin (TSH)-induced receptor internalization in nonthyroidal cells transfected with a human TSH-receptor complementary deoxyribonucleic acid.

TSH-induced desensitization was studied in nonthyroidal cells expressing functionally active TSH receptors (TSHR). Chinese hamster ovary (CHO) cells and mouse NIH 3T3 cells were stably transfected with a human TSHR cDNA. Stimulation of the CHO-TSHR and NIH-TSHR cells with 10 mU/ml TSH resulted in a decreased sensitivity to a second TSH stimulation only in the NIH-TSHR cells. A decrease in TSH-induced cAMP was present within 1 h and coincided with a decreased binding of [125I]TSH. The half-maximal effect was observed after a 3- to 4-h stimulation with TSH, and exposure of cells to TSH for 20 h led to a 70-80% inhibition of cAMP formation. After withdrawal of TSH, cells regained full responsiveness to TSH after 6 h. Moreover, the desensitization effect observed in NIH-TSHR cells was not mimicked by forskolin and, therefore, was not mediated by cAMP. Stimulation of the CHO-TSHR cells with TSH did not result in a desensitization toward a second TSH stimulation, nor did it reduce the binding of [125I]TSH. This difference between the two cell lines might be explained by a higher turnover rate of receptors in the CHO cells. Indeed, incubation of cells with [125I]TSH showed a more efficient internalization of ligand in the CHO-TSHR cells compared to the NIH-TSHR cells. In summary, the homologous desensitization observed in TSHR-transfected NIH 3T3 cells appears to be the result of ligand-induced receptor down-regulation.

Decreased growth rate and tumour formation of human anaplastic thyroid carcinoma cells transfected with a human thyrotropin receptor cDNA in NMRI nude mice treated with propylthiouracil.

The effect of the human TSH-receptor (TSHR) on the growth of human anaplastic thyroid carcinoma cells lacking the endogenous expression of TSHR, was studied both in vitro and in vivo in NMRI nude mice. Cells from a human anaplastic thyroid carcinoma cell line (C643) were transfected with a TSHR cDNA, and clones were isolated after neomycin selection. The expression of a functional receptor protein was ensured by analysis of the specific binding of 125I-TSH and measurement of TSH-induced cAMP. Incorporation of [3H]thymidine and increase in cell number was slightly inhibited by TSH in TSHR-expressing cells in vitro. In order to investigate whether the regained expression of a functional TSHR protein in C643 cells could influence the in vivo growth, cells were injected subcutaneously into NMRI nude mice. To manipulate the endogenous level of TSH, animals were given 6n-propyl-2-thiouracil (PTU; resulting in a high TSH level), T4 (a low TSH level) or no treatment (as a control). There seemed to be a TSH induced inhibition of tumour growth, since tumours in mice treated with PTU grew after a longer take rate and with a slower growth rate. The present results suggest a TSH-mediated growth inhibition in the TSHR-transfected C 643 anaplastic thyroid carcinoma cells.

Lack of responsiveness to TGF-beta1 in a thyroid carcinoma cell line with functional type I and type II TGF-beta receptors and Smad proteins, suggests a novel mechanism for TGF-beta insensitivity in carcinoma cells.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine. In the present study we have investigated the expression of TGF-beta receptors (TbetaR's) and SMAD proteins in non-neoplastic and neoplastic thyroid follicle cells. We found expression of all TbetaR's (type I, II and III) and SMAD proteins analysed (Smad2, Smad3, Smad4, Smad6 and Smad7). Five out of six human anaplastic thyroid carcinoma cell lines were growth inhibited by addition of TGF-beta1, and therefore considered to be TGF-responsive. One cell line however, HTh 7, did not respond to TGF-beta1 with growth inhibition, induction of the extracellular matrix protein fibronectin or immediate early genes junB, Smad6 and Smad7 mRNA. Analysis of the TGF-beta intracellular signalling pathway in HTh 7 cells showed that receptors were capable of signalling, e.g. Smad2 phosphorylation and SMAD nuclear translocation. In summary, our data shows abundant expression of TGF-beta signalling components in thyroid follicle cells, and the escape from TGF-beta sensitivity in one anaplastic thyroid carcinoma despite an apparently functional TGF-beta/SMAD-signalling pathway, indicating a novel mechanism for TGF-beta insensitivity.

Mucosal immunoreactivity in experimental pneumococcal otitis media.

A rat model was used to investigate immunological events in the middle ear mucosa during pneumococcal acute otitis media (AOM). Twelve healthy male Sprague-Dawley rats were challenged in the right middle ear with Streptococcus pneumoniae type 3, the presence of AOM being confirmed by otomicroscopy. Randomly selected rats, four at a time, were sacrificed on days 7, 14 and 56 after bacterial challenge. Immunohistochemical staining for IgG, secretory IgA (SIgA), and IgM was performed on tissue specimens from four separate locations in the middle ear and tubal mucosa. Immunoglobulins, especially IgG, were found around blood vessels in the middle ear mucosa. Immunoreactive lymphoid cells of all three Ig classes investigated, undetectable before bacterial challenge, appeared within 7-14 days after middle ear challenge, and the location of these cells in the middle ear mucosa suggests the presence of IgG, SIgA, and IgM, respectively. On the other hand, reactivity with anti-SIgA (but not with anti-IgG or anti-IgM) in Eustachian epithelial cells, and also in the subepithelial glands of tubal mucosa was present both before and after challenge. The results suggest that the middle ear mucosa is an immunoreactive site only after it has been activated with pathogens. In contrast, the tubal mucosa exhibits immunological activity also prior to the antigenic stimulation of present interest.

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

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

Intra- and interstrain differences of virulence among nontypeable Haemophilus influenzae strains.

Nontypeable Haemophilus influenzae (NTHi) is sometimes the causative agent of invasive diseases, and it has been suggested that there may be differences in virulence among NTHi strains. Whilst studying clinical isolates of NTHi in a rat model of acute otitis media, intra- and interstrain differences in virulence were observed. Two strains with suddenly reduced capacity to cause middle ear infections and one highly virulent strain with dose requirements comparable only to encapsulated H. influenzae strains were further investigated, together with 15 other H. influenzae strains. The strains were characterized by analyzing the lipopolysaccharide, the outer membrane proteins, the hemagglutinating ability, and the polymerase chain reaction products after amplification of a gene sequence associated with encapsulation. The pathogenic capacity was assessed in two different in vivo models. It was found that the two strains with reduced pathogenic capacity could regain their virulence after animal passage. The LPS analysis and the results from the chicken embryo model suggested that the observed change in virulence might be associated with the lipopolysaccharide. For the non-animal-passaged strain 3655 there were indications that an undefined factor(s) contributed to its relatively potent virulence. As all three strains lacked genes necessary for encapsulation, in no case could any part of the increased virulence be attributed to the expression of small amounts of capsule.

Immunization and protection in pneumococcal otitis media studied in a rat model.

The recent and growing problem of bacterial resistance to common antibiotics has generated great interest in different methods for prevention of infections. The treatment of the pathogens causing upper airway infections and especially acute otitis media (AOM) is especially interesting in this context because these infections are a common cause of prescription of antibiotics all over the world. Both in AOM and recurrent AOM, Streptococcus pneumoniae, the most frequently occurring bacterium is isolated in 30-50% of all AOM attacks. In the last decade, multiresistant S. pneumoniae have emerged as a major problem. Thus, it is important to explore possibilities that immunization may protect against pneumococcal OM. In a well-defined animal model using Sprague-Dawley rats, we have investigated the effects of different routes of immunization with different antigens and whole cells. Together with otomicroscopical evaluation of middle ear (ME) status, samples for bacterial cultivation as well as for studies of histopathological changes have been collected. Antibody titers have been followed during and after pneumococcal AOM by an enzyme-linked immunosorbent assay (ELISA) method.

Bone modeling dynamics in acute otitis media.

OBJECTIVE: A number of middle ear diseases are associated with pathologic bone modeling, either formative or resorptive. As such, the pathogenesis of a sclerotic mastoid has been controversial for decades. Experimental studies on acute middle ear infection have shown varying degrees of both osteoresorption and osteoneogenesis. This study presents data on the dynamics of bone modeling in a rat model of acute pneumococcal otitis media, studied longitudinally from day 1 through 6 months after inoculation. RESULTS: Qualitative, as well as quantitative histopathology revealed initial osteoresorption, followed by increasing apposition of new bone in the middle ear cavity, initiated at the outer periosteum. Measured bone thickness in four anatomically distinct locations peaked 3 months after inoculation, followed by some degree of normalization. However, bone thickness was still massively increased 6 months after the acute incident. Except in perilymphatic spaces of the otic capsule, resorptive and formative activity were found in all bone tissue structures surrounding the middle ear cavity, including the bony external auditory canal and the ossicles. CONCLUSION: These findings may support the existence of a perilymphatic barrier of specialized bone and suggest that even a single episode of acute infection may alter properties of ossicular chain conduction. The authors conclude that acute otitis media is accompanied by massive and progressing net osteoneogenesis, already evident at 3 days and peaking 3 months after inoculation, followed by some degree of normalization. This is conceivably in support of the environmental theory of mastoid pneumatization, claiming inflammatory disease as the cause of a sclerotic mastoid.

Pathogenesis of middle ear adhesions.

Middle ear adhesions are well-known to the ear surgeon, although data on etiology, pathogenesis, and significance are lacking in current literature. This study on experimental acute otitis media presents histopathological data on these aspects. Pneumococci were inoculated in the right middle ear bulla of 25 rats; the left ear served as control. At days 4, 8, 16, 90, and 180, respectively, 5 rats were decapitated, and the bullae were removed, opened, and stained with periodic acid-Schiff (PAS)/alcian blue. The entire middle ear mucosae were dissected from the bone, embedded as whole mounts in colophonium chambers, and examined by light microscopy. Representative parts of the mucosae were sectioned and examined in the same way. All inoculated ears from day 8 and later (20 in total), contained mucosal adhesions of various sizes, shapes, and locations. None were found in control ears. The site of predilection for the development of adhesions was the hypotympanum, followed by the anterior epitympanum, the attic, the drum, the interossicular spaces, and the tubal orifice. Based on present histopathological findings, we conclude that the middle ear adhesion is a pathological phenomenon caused by infection, and we propose a six-stage hypothesis of pathogenesis: 1. Localized epithelial rupture; 2. Prolapse of subepithelial tissue; 3. Epithelialization of the prolapse; resulting in a polypous/fold-like prominence; 4. Growth and elongation of the prominence; 5. Fusion of the end/tip of the prominence with another part of the mucosa; 6. Formation of an adhesion.

Relationship between the microstructure and the mechanical and barrier properties of whey protein films.

This work was focused on the relationship between the microstructure and the mechanical and barrier properties of whey protein isolate (WPI) films. Sorbitol (S) and glycerol (G) were used as plasticizers and the pH was varied between 7 and 9. The films were cast from heated aqueous solutions and dried in a climate room at 23 degrees C and 50% relative humidity for 16 h. The microstructure of the films was found to be dependent on the concentration, the plasticizers, and the pH. When the concentration increased, a more aggregated structure was formed, with a denser protein network and larger pores. This resulted in increased water vapor permeability (WVP) and decreased oxygen permeability (OP). When G was used as a plasticizer instead of S, the microstructure was different, and the moisture content and WVP approximately doubled. When the pH increased from 7 to 9, a denser protein structure was formed, the strain at break increased, and the OP decreased.

Effects of pH and the gel state on the mechanical properties, moisture contents, and glass transition temperatures of whey protein films.

The mechanical properties, moisture contents (MC), and glass transition temperature (T(g)) of whey protein isolate (WPI) films were studied at various pH values using sorbitol (S) as a plasticizer. The films were cast from heated aqueous solutions and dried in a climate chamber at 23 degrees C and 50% relative humidity (RH) for 16 h. The critical gel concentrations (c(g)) for the cooled aqueous solutions were found to be 11.7, 12.1, and 11.3% (w/w) WPI for pH 7, 8, and 9, respectively. The cooling rate influenced the c(g), in that a lower amount of WPI was needed for gelation when a slower cooling rate was applied. Both cooling rates used in this study showed a maximum in the c(g) at pH 8. The influence of the polymer network on the film properties was elucidated by varying the concentration of WPI over and under the c(g). Strain at break (epsilon(b)) showed a maximum at the c(g) for all pH values, thus implying that the most favorable structure regarding the ability of the films to stretch is formed at this concentration. Young's modulus (E) and stress at break (sigma(b)) showed a maximum at c(g) for pH 7 and 8. The MC and epsilon(b) increased when pH increased from 7 to 9, whereas T(g) decreased. Hence, T(g) values were -17, -18, and -21 degrees C for pH 7, 8, and 9, respectively. E and sigma(b) decreased and epsilon(b) and thickness increased when the surrounding RH increased. The thickness of the WPI films also increased with the concentration of WPI.

Aging of whey protein films and the effect on mechanical and barrier properties.

This work focuses on the aging of whey protein isolate (WPI) films plasticized with glycerol (G) and sorbitol (S). The films were cast from heated aqueous solutions at pH 7 and dried at 23 degrees C and 50% relative humidity (RH) for 16 h. They were stored in a climate room (23 degrees C, 50% RH) for 120 days, and the film properties were measured at regular intervals. The moisture content (MC) of the WPI/G films decreased from 22% (2 days) to 15% (45 days) and was thereafter constant at 15% (up to 120 days). This affected the mechanical properties and caused an increased stress at break (from 2.7 to 8.3 MPa), a decreased strain at break (from 33 to 4%), and an increased glass transition temperature (T(g)) (from -56 to -45 degrees C). The barrier properties were, however, unaffected, with constant water vapor permeability and a uniform film thickness. The MC of the WPI/S films was constant at approximately 9%, which gave no change in film properties.

Mucosal changes induced by experimental pneumococcal otitis media are prevented by penicillin V.

Penicillin V (pcV) was administered to 50 rats, either before bacterial challenge (prevention group), or after bacterial challenge but before fulminant purulent acute otitis media (AOM) was established (early treatment group). Five animals from each group were killed on days 4, 8, and 12, and 2 and 6 months after challenge. Middle ear mucosa was sampled at six different sites and studied in the light microscope. Untreated pneumococcal AOM in the rat has been shown to cause persistent structural changes of the middle ear mucosa. Both in the early treatment group and in the prevention group, the structural changes were diminished, as compared with those of untreated infected controls. The persistent structural changes seen after 6 months in untreated controls were not seen in animals that had received pcV in conjunction with the AOM episode. Though the beneficial effect on the mucosal changes during the first 2 weeks was more pronounced when pcV was given prophylactically, its use as early treatment would seem to be almost as effective in preventing the persistence of mucosal changes.