How can the hooded seal dive to a depth of 1000 m without rupturing its tympanic membrane? A morphological and functional study.

Recent studies using a satellite-linked dive recorder have shown that the hooded seal (Cystophora cristata), a common Arctic pinniped, can dive to a depth of > 1000 m and stay submerged for close to 1 h. At these depths the water pressure reaches 100 atm, entailing obvious risk of serious damage to the hearing apparatus, mainly the tympanic membrane (TM) and middle ear (ME). We dissected and photodocumented the temporal bones of five newborn and three adult hooded seals in order to study the temporal bone structure and reveal its protective mechanisms for extreme pressure changes. Specimens were sectioned and stained for light microscopy. The thicknesses of the pars tensa and pars flaccida were found to average 60 and 180 microm, respectively. The ME cavity hosts a cavernous tissue of thin-walled vessels beneath the modified respiratory epithelium. The ME and external ear canal (EAC) volumes can be altered appreciably by filling/emptying the cavernous tissue with blood. The ossicles were fixed by contracting the tensor tympani and stapedius muscles simultaneously with complete occlusion of the EAC. According to Boyle's law, the volume of the gas-filled ME cavity at a depth of 1000 m is only 1% of its volume at the surface of the sea. Ascent from such a depth allows the gas in the ME cavity to expand, causing the TM to bulge laterally. This movement is counteracted by a reduction in the blood volume inside the cavernous sinuses, action in the tensor tympani and stapedius muscles and discharge of gas through the Eustachian tube. The presence of a firm, broad-based exostosis in the floor of the EAC lateral to the TM helps to obstruct the EAC.

Remarkable attachment of lactoferrin to Streptococcus pyogenes during acute pharyngotonsillitis.

The purpose of this study was to establish whether lactoferrin (hLf) attached to Streptococcus pyogenes, one causative agent of acute pharyngotonsillitis (AT), during the course of the disease. Bacterial samples were obtained from the tonsillar surfaces of 7 patients (6 females, 1 male; median age 26 years; range 16-50 years) suffering from AT who were culture-positive for S. pyogenes and from 5 healthy adult controls who were culture-negative for this pathogen. Using gold-labelled antiserum against S. pyogenes and hLf, this pathogen and other bacteria on the tonsillar surfaces coated with hLf could be identified by tracing the gold particles in a transmission electron microscope. In healthy adults, 8% (median value; range 6-12%) of the surface tonsillar bacteria were coated with hLf. In AT patients, 59% (median value; range 42 67%) of S. pyogenes were coated with hLf, in contrast to 9% (median value; range 0-26%) of all other bacteria (p < 0.01). This study hints that hLf might participate in recovery from AT in several ways, e.g. by binding to the S. pyogenes pathogens, in addition to its well-known virtue of iron-binding capacity.

Bacterial coating with immunoglobulins on the palatine tonsils during infectious mononucleosis: immunocytochemical study with gold markers.

Epithelial cells and bacteria were sampled from the tonsillar surfaces of seven patients (six males, one female; median age 16 years, range 10 to 21 years) suffering from acute infectious mononucleosis with concomitant pharyngotonsillitis. By using gold-labelled antiserum to human IgG and secretory IgA (sIgA), micro-organisms on the tonsillar surfaces coated with these immunoglobulins could be identified by tracing the gold particles in the transmission electron microscope. The patients harboured significantly fewer bacteria coated with immunoglobulins than did healthy controls. More bacteria were coated with IgG immunoglobulins than with sIgA. Reduced immunoglobulin-coating of the bacteria on the tonsillar surfaces during infectious mononucleosis can explain their tendency to immense local colonization and proneness to penetrate into the epithelial cells.

Bacterial penetration into tonsillar surface epithelium during infectious mononucleosis.

Bacterial penetration into epithelial cells, scraped from the palatine tonsils of 14 patients (10 males, four females; median age 16 years) with current infectious mononucleosis and concomitant membranous tonsillitis, was studied using the transmission electron microscopic (TEM) technique. Bacteria were seen to adhere to and penetrate the epithelial cells, some of which were completely filled with bacteria. This finding suggests intracellular proliferation of bacteria. Epstein-Barr virus, the causative agent of infectious mononucleosis, especially when associated with growth of beta-haemolytic streptococci on the palatine tonsils, induces bacterial penetration into tonsillar tissue, that in turn might be a causative mechanism in the development of peritonsillar abscess.

Exostoses and cavernous venous formation in the external auditory canal of the hooded seal as a functional physiological organ.

Exostoses of the external auditory canal (EAC) develop after protracted mechanical, chemical or thermal irritation in particular. This is a common disorder among aquatic sportsmen and has been considered unique to Man. We dissected and photodocumented the EACs of 5 newborn and 3 adult Hooded Seals (Cystophora cristata). Serial sections of the EACs were prepared for light microscopic evaluation after staining with haematoxylin-eosin or toluidine blue. All EACs exhibited a firm, broad-based. mountain peak-shaped exostosis on the floor of the meatus, lateral to the eardrum. In addition, the meatal skin of the bony EAC harboured large venous sinuses. The exostosis and venous sinuses of the seal EAC participate in the protection of the sensitive hearing apparatus, particularly the pars tensa portion of the drum, during divine.

SIgA- and IgG-coated Streptococcus pyogenes on the tonsillar surfaces during acute tonsillitis.

Bacterial swabs were collected from the tonsillar surfaces of eight patients with current acute tonsillitis, culture-positive for Streptococcus pyogenes. Using gold-labelled antiserum to S. pyogenes, these micro-organisms could be localized in the samples by transmission electron microscopy. S. pyogenes pathogens were further characterized with gold-labelled antiserum to human IgG and SIgA antibodies. Roughly 90% of the pathogens were found coated with IgG antibodies, irrespective of the duration of the disease, whereas the proportion of SIgA-coated pathogens increased with disease duration. Insufficient IgA coating of pathogens might well be a contributory cause of the induction of tonsillar infection, probably due to inadequate prevention of the attachment of the S. pyogenes bacteria to the tonsillar surface epithelium.

Where are the receptors for Streptococcus pyogenes located on the tonsillar surface epithelium?

Streptococcus pyogenes is the most frequent causative agent of acute pharyngotonsillitis (AT). The first events in the etiopathogenesis of an AT infection caused by these bacterial pathogens are their penetration through the mucus film covering the oropharyngeal mucosa, and their attachment to the surface epithelium. Adherence of S. pyogenes to tonsillar epithelial cells is a precondition for bacterial colonisation, for triggering off cell activation, internalising of bacteria into the epithelial cells and cytokine release from the epithelial cells with subsequent induction of an inflammatory reaction in underlying tissues. Scanning and transmission electron microscopic studies revealed that the surface epithelium of the human palatine tonsils consisted of a weakly keratinized, stratified squamous epithelium built up of pentangular cells where the apical cell surface formed an irregular pattern of microridges. The distance between two adjacent microridges was roughly one-third of the diameter of a S. pyogenes bacterium. By using gold-labelled antiserum to S. pyogenes, we showed that the target region for these pathogens on the epithelial cells during an on-going AT infection was located on the crests of the microridges where bacterial pili made adhesin-receptor contact with the tonsillar surface epithelium.

Non-specific and specific immunity to bacterial invasion of the middle ear cavity.

The role of the non-specific (mucociliary clearance, mucus, lactoferrin, lysozyme, fibronectin, lactoperoxidase, complement, phagocytosis) and specific (immunoglobulins) immune systems in combating bacterial pathogens invading the middle ear cavity is discussed. The non-specific system is present from birth, acts promptly against a broad spectrum of microorganisms, has no memory, and is present for life. In contrast, immunoglobulins act specifically. Secretory IgA antibody prevents bacteria from attaching to the epithelium, has no pro-inflammatory effect and does not activate complement. IgG antibodies can opsonize the bacteria for phagocytosis and eventually prevent the bacteria from penetrating the epithelium. IgG is pro-inflammatory and can activate a complement cascade.

Human beta-defensin-1 mRNA is transcribed in tympanic membrane and adjacent auditory canal epithelium.

The external auditory canal is less susceptible to infections than the sensitive middle-ear cavity. Since recent research has provided insight to the production of potent antimicrobial peptides from various surface epithelia, we wanted to investigate whether protection of the external auditory canal in part could be explained by the production of human beta-defensin-1 (HBD-1). This particular peptide is known to be constitutively expressed in various surface epithelia, such as airway, skin, and urogenital tissues. By reverse transcriptase PCR we demonstrate HBD-1 mRNA in the pars tensa and pars flaccida of the tympanic membrane and in the meatal skin. In situ hybridization studies localized the HBD-1 mRNA to the epidermal layer of these tissues. The HBD-1 transcripts were also evident in the sebaceous glands and in hair follicles of the meatal skin. In contrast, HBD-1 mRNA was not detected in the tympanal epithelium of the eardrum. The widespread presence of mRNA encoding for this broad-spectrum antimicrobial peptide in the meatal skin and tympanic membrane suggests that HBD-1 participates in the innate antimicrobial defense of the external auditory canal and middle-ear cavity.

Initial events in the pathogenesis of acute tonsillitis caused by Streptococcus pyogenes.

Bacterial and epithelial cell samples were obtained, within 24 h of onset of pharyngeal symptoms, from the palatine tonsils of nine patients (four female and five male; age range 10-40 years, median age 23) with acute tonsillitis, culture-positive for Streptococcus pyogenes. The specimens were examined using fluorescein isothiocyanate- (FITC) and gold-labelled antiserum to S. pyogenes and fluorescence, scanning electron and transmission electron microscopy. S. pyogenes could be identified both in the mucous layer covering the tonsils and attached to the surface epithelial cells. Long chains of coccus-shaped bacteria could be seen encroaching on the epithelial cell borders. S. pyogenes can apparently penetrate the mucous barrier, attach to the epithelial cells, spread from cell to cell and possibly penetrate into the outermost layer of the epithelial cells. These events in turn provoke cytokine production and/or complement activation, which induce inflammatory reaction in the tonsillar tissue.

Immunoglobulin- and complement-coated bacteria in pus from peritonsillar abscesses.

Fifty-five samples of pus were collected from 51 acute, non-perforated, two spontaneously ruptured and two recurrent peritonsillar abscesses (35 males and 18 females; median age 18 years) and analysed regarding (i) aerobic and anaerobic bacteria (standard culturing), (ii) morphology of bacteria and inflammatory cells (direct microscopy of acridine orange-stained material), and (iii) the percentage of bacteria coated with immunoglobulins IgG, secretory IgA (SIgA) and IgM and complement cleavage product C3b (immunofluorescence assay). Seventy-one per cent of the abscesses harboured a mixed bacterial flora of various aerobes and anaerobes. In none of the cases with a single bacterial species (27 per cent) could immunoglobulin- or complement-coated bacteria be found. In abscesses with a mixed flora, 18 per cent harboured IgG-coated, 15 per cent SIgA-coated, five per cent IgM-coated and five per cent C3b-coated bacteria, respectively. All pus samples contained inflammatory cells in abundance but they were mostly deformed and only occasionally could intracellular bacteria be recognized. Insufficient immunoglobulin-coating of bacteria might be an important aetiopathogenic factor in the development of a peritonsillar abscess. Bactericide in the abscesses is accomplished chiefly by protective mechanisms not dependent on antigen recognition by antibodies.

Selective attachment of beta-haemolytic streptococci group A to oropharyngeal epithelium in health and disease.

Localization and semiquantification of beta-haemolytic streptococci, Group A (GABHS), GABHS attachment and general bacterial attachment to epithelial cells (bacterial number and morphology) were studied during GABHS-positive acute tonsillitis and pharyngitis infections and among healthy GABHS carriers. Samples were collected from various areas of the oropharynx (palatine tonsils, posterior oropharyngeal wall, palatoglossal arch and buccal mucosa). During acute tonsillitis and pharyngitis, GABHS grew in samples obtained from the palatine tonsils and posterior oropharyngeal wall. The ratio of GABHS colonies to other aerobic colonies increased, and GABHS became attached to epithelial cells of both palatine tonsils and posterior oropharyngeal wall. In GABHS carriers, GABHS were found mainly on the palatine tonsils, but these microorganisms were not attached to the epithelium. Overall bacterial attachment to tonsillar and oropharyngeal epithelial cells increased during active tonsillitis and pharyngitis.

Direct microscopy of effusions obtained from peritonsillar abscesses as a complement to bacterial culturing.

Effusion material was aspirated from 51 consecutive peritonsillar abscesses (34 male, 17 female; age range eight to 46 years) and subjected to direct microscopy after staining with acridine orange. Bacteria were counted per ml effusion material and their morphology was analyzed. In addition, aerobic and anaerobic bacterial culturing was performed. Effusions containing beta-haemolytic streptococci Group A, which appeared as a single species contained fewer bacteria (8 x 10(6) per ml, median value) than effusions harbouring a mixed flora (7 x 10(8) bacteria per ml, median value). Direct microscopy of effusions obtained from peritonsillar abscesses makes possible rapid identification of a single or mixed flora, which is of importance for the antibiotic treatment of the disease.

Immunoglobulin-coated bacteria in effusions obtained during chronic maxillary sinusitis.

Local protection of the maxillary sinuses against bacterial invasion takes both specific and non-specific forms. The present study is intended to evaluate the participation of the specific protective factors, immunoglobulins IgG, secretory IgA, IgM and complement, in protecting the maxillary sinuses during chronic maxillary sinusitis (CMS). We collected 47 sinus effusion samples from 37 patients (17 male, 20 female) with current CMS of at least 3 months' duration. Patients' ages ranged from 3 to 80 years. The effusion material was subjected to qualitative and quantitative bacteriological analyses, while bacterial coating with IgG, SIgA, IgM and C3b was determined using an immunofluorescence technique. Detectable bacteria were harboured by 55% of the samples, the most common species being Moraxella catarrhalis, Streptococcus pneumoniae and Staphylococcus aureus. The bacterial counts ranged from 0 to 10(9) per ml effusion. Half of the samples hosting detectable bacteria showed microorganisms coated with protective immunoglobulins. Antibacterial factors had completely eradicated the microorganisms in 45% of the CMS cases and coated the organisms with specific immunoglobulins in a further 28%.

Identification of Streptococcus pyogenes on tonsillar epithelium during infection.

Epithelial cells were swabbed from the tonsillar surfaces of 5 patients with acute tonsillitis culture-positive for Streptococcus pyogenes. By using 10 nm gold particles conjugated to antiserum to S. pyogenes it was possible to trace the actual microorganisms when examined in a transmission electron microscope. The S. pyogenes bacteria, usually in pairs, were attached to the epithelial surface by their pili. The bacteria often formed a hollow in the epithelial cell surface. Coccus-shaped bacteria expressing positive affinity to immunogold-labelled antiserum were intermingled with bacteria, often rods, having no affinity whatsoever to the antiserum. With the immunocytological technique outlined in this study it is possible to study more closely cellular/bacterial adhesion mechanisms.

Occurrence of otitis media in an arctic region.

The occurrence of otitis media (OM) was examined retrospectively in files at Longyearbyen Hospital, Svalbard, Norway during 1991-1994. Acute OM (AOM) with and without spontaneous drum perforation, secretory OM (SOM) and chronic OM (COM) with and without suppuration were registered, as were patient's sex, age, and climatic conditions. These findings were compared with weather observations obtained from the Norwegian Meteorological Institute, Tromsø in the same period. Sixty-four percent of the OM patients registered were classified as AOM, 17% having a spontaneous drum perforation. Thirty percent suffered from SOM, whereas 6% had COM. Forty percent of the patients were younger than 6 years. The distribution of OM cases during the year showed a peak during the spring, especially in May. Another minor peak was noted in September. Average daily temperature in the period was -5.6 degrees C, range -14 degrees C to +6 degrees C. Humidity and precipitation were fairly stable throughout the observation period 74% and 240 mm/year, respectively. OM is a common disease at Svalbard and is apparently related to seasonal shifts in temperature.

Nasal septal surgery as an out-patient procedure.

Sixty consecutive patients (48 males, 12 females; age range 16 to 57, mean age 35 years) underwent nasal septal surgery at our out-patient department for symptoms of nasal obstruction. The operations were performed under local anaesthesia supplemented with 2.5-5 mg intravenous midazolam. Only one surgeon and one nurse were needed for the procedure. The total time spent at the hospital averaged three hours. The preliminary short-term results of surgery are comparable to those at our day-care unit. The complication rate was negligible. Ninety-five per cent of these patients regarded the arrangements for their operation as safe. Our results show that nasal septal surgery can be performed with good cost-effectiveness, satisfactory quality and with good safety and patient satisfaction as an out-patient procedure.

In situ localization of Streptococcus pyogenes during acute tonsillitis: an immunocytochemical study with gold markers.

Epithelial cells were harvested from the surface of the palatine tonsils of seven patients with current acute tonsillitis, proven culture-positive for Streptococcus pyogenes. The epithelial cells harboured attached bacteria, which expressed positive affinity to gold-labelled antiserum to S. pyogenes. The gold particles adhered selectively to the bacterial capsules. The microorganisms were held in place by projections protruding from the epithelial cells, which were in close contact with the pili of the bacteria. In some areas, positive immunogold-labelled bacteria intermingled with bacteria lacking such labelling. None of the culture-negative controls harboured epithelial cells with positive immunogold-labelled bacteria. Orally administered phenoxymethylpenicillin caused a significant reduction in both culture-positive S. pyogenes and bacteria displaying positive coating with specific gold-labelled antiserum to S. pyogenes.

Bacterial attachment to oropharyngeal epithelial cells in breastfed newborns.

On day 2 post partum, swab samples were obtained from the oropharyngx of 20 healthy, breast-fed babies. The samples were examined for aerobes (culture on agar plates), for bacterial coating with the immunoglobulins SIgA, IgG and IgM (immunofluorescence assay), for bacterial attachment to epithelial cells (fluorescence microscopy of acridine orange stained material, and scanning electron microscopy). alpha-Hemolytic streptococci grew almost exclusively in the oropharynx, while 32% (median value) of the epithelial cells showed bacterial attachment in abundance, viz. > 50 attached bacteria per cell, 66% (median value) of the bacteria showed positive reactivity when treated with anti-human SIgA serum. 72% (median value) of the bacteria were coated with IgG, but no IgM coating of the bacteria could be detected. Newborns do not possess IgA antibodies, as only IgG is able to pass the placental barrier. The SIgA-rich colostrum is capable of coating the oropharyngeal bacteria of breast-fed babies during their first days after birth. However, despite abundant SIgA-coating, bacteria still succeed in attaching to the epithelial cells of the oropharynx. This finding hints that factors other than SIgA impede the bacterial/cellular adhesion mechanism.

Bacterial adherence to the surface and isolated cell epithelium of the palatine tonsils.

Bacterial adherence to the oropharyngeal epithelium is a significant factor in normal microecology, etiopathogenesis of diseases (tonsillitis, gingivitis) and possibly also induction of immune response. Bacterial adhesion to human tonsillar epithelium of whole tonsils and swabs was studied by fluorescence, scanning, and transmission electron microscopy. The epithelial cell borders were well demarcated. On the apical surface of the cells there were irregular microridges. All forms of microscopy visualized epithelial cells with attached bacteria, often forming microcolonies on the free surface. Some bacteria formed excavations on the cell surface. Most attached bacteria were coccoid, but variously sized rods were also visible. In transmission electron microscopy, epithelial cells with intracellular bacteria were regularly observed.