Adenoid reservoir for pathogenic biofilm bacteria.

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

Use of distortion product otoacoustic emissions to assess middle ear transducers in rhesus monkeys.

Distortion product otoacoustic emissions (DPOAEs) can provide an objective and noninvasive assessment of the peripheral cochlear function. Auditory brainstem responses measured from implanted rhesus monkeys have shown that middle ear transducers, coupled directly to the incus, are capable of delivering the signals to the central auditory system. The DPOAEs were used as a noninvasive method of assessing the frequency specificity of this mechanical transduction. In two rhesus monkeys implanted with the middle ear transducers, one primary stimulating tone (f1) was presented acoustically, and the other primary tone (f2) was presented by the transducer, which converted the signal into a mechanical motion of the probe tip attached to the body of the incus. The nonlinear characteristics of the cochlea produced the distortion product responses at the expected frequencies (2f1 - f2). This demonstrates the fidelity of the middle ear implant signal transduction in vivo. The DPOAEs also indicate minimal changes in the post-implant middle ear transmission. This study demonstrates that the DPOAEs can be used to assess the function of implanted middle ear transducers objectively and noninvasively.

Distortion product otoacoustic emissions in rhesus (Macaca mulatta) monkey ears: normative findings.

Distortion product otoacoustic emissions (DPOAEs) in rhesus monkeys were characterized and the optimal parameters for their generation were determined. Robust DPOAEs were readily measurable from the ear canals of six rhesus monkeys (n = 12 ears). The nonmonotonic behavior of the f2/f1 ratio functions in rhesus monkeys was found to be similar to other animals and humans. The optimal mean f2/f1 ratio of 1.21 and the effect of the primary frequency and level on the optimal f2/f1 ratios were also similar to human measurements. The contour of the rhesus monkey DPOAE 'audiograms' and their behavior were also comparable to human measurements with slight differences in peak frequencies. The rhesus monkey DPOAE input/output (I/O) functions were generally monotonic with a slope approaching unity with increasing frequency. Therefore, our study shows that many basic DPOAE characteristics are remarkably similar in the two species and emphasizes the appropriateness of the rhesus monkey as a model for DPOAE research. Detailed studies of the behavior of DPOAEs can be carried out in a model that is phylogenetically close to human both in hearing and in the gross structure and histology of the inner ear.

Ongoing investigations into an implantable electromagnetic hearing aid for moderate to severe sensorineural hearing loss.

Over 20 million Americans have a hearing loss that produces problems in communication. The majority of these hearing-impaired individuals would benefit from conventional hearing aids, yet only about 15% use amplification devices. Many of the reasons for the lack of use of conventional hearing aids can be resolved by an implantable middle ear device directly coupled to the ossicular chain. This article presents the advantages of the implantable middle ear device.

Computer-stimulated test fitting of an implantable hearing aid using implantable hearing aid using three-dimensional CT scans of the temporal bone: preliminary study.

In preparation for future implantation of the implantable middle ear transducer in patients, a method was sought for preoperatively test fitting a model of the device, using computer generated three-dimensional (3-D) temporal bone images derived from spiral computed tomography (CT) data. A 3-D model of the implantable middle ear transducer was designed using NIH Image software on a Macintosh computer. High resolution human temporal bone CT scans were obtained using a spiral CT scanner (Siemens Somatom Plus S). The 3-D transducer model was superimposed onto 3-D reconstructions of the temporal bone using ANALYZE software on a computer graphics workstation (Sun SPARCstation 10), showing the transducer "implanted" in the temporal bone. Measurements were validated using a cadaver temporal bone. This process produced images demonstrating the "fit" of the current transducer design in the mastoid region of the adult temporal bone. It permitted assessment of the proximity of surrounding structures such as the external auditory meatus, dura, or sigmoid sinus. Preliminary cadaver validation measurements confirmed the accuracy of this method. Three-dimensional CT is a feasible method for preoperative planning of the surgical implantation of devices in the temporal bone. This method of 3-D test fitting will be used in the future to determine optimum orientation and size limitations for human implantable devices.

Effect of influenza A virus on ciliary activity and dye transport function in the chinchilla eustachian tube.

Previous studies have shown that influenza A virus inoculated via a transbullar approach can cause severe disorders of the chinchilla eustachian tube. In this study, we inoculated chinchillas both intranasally and transbullarly with influenza A virus (A/Alaska/6/77) to assess the effect of this virus on both the ciliary activity of eustachian tube epithelium and eustachian tube transport function. Data indicated that while the nature of the major lesions was indistinguishable, the differing routes of inoculation did influence the time course and initial location of virus-induced histopathology. Analysis of ciliary beat frequency and dye transport function indicated a maximal decrease approximately 7 to 14 days postinoculation, with return to normal function by 28 days postinoculation. Light and transmission electron microscopic observations of the evolution of histopathology and return to normal histology correlated well with functional assessment data.

Effect of KTP laser ablation of endochondral bone on bone healing.

The advantages of using a laser to cut a hole in the ossicular chain include increased precision, decreased likelihood of disarticulation, and elimination of noise-induced threshold shift associated with conventional drills. This study was designed to determine optimal energy settings and cooling technique to minimize tissue injury. Laser ablated holes were cut in rabbit femurs with different endostat fibers at power settings of 1.6 to 5.0 watts. Comparable burr holes were cut by a high-speed drill and the animals were sacrificed at different time intervals. Specimens were evaluated microscopically for tissue injury by the amount of cell necrosis of the surrounding osteocytes, bone marrow changes, and denaturation of bone matrix. Changes in healing were assessed by the production and ossification of the granulation tissue. Laser ablated holes created with 1.6 to 2.0 watts at 2 to 7 days demonstrated thermal damage comparable to conventionally drilled holes. At 14 to 28 days these holes demonstrated advanced healing and integrated lamellar bone. In contrast, higher power (> 3.5 watts) at 2 to 7 days demonstrated Significantly more osteocyte loss, marked bone marrow fibrosis, and bone matrix denaturation. At 14 to 28 days these holes showed evidence of delayed healing and osseous plugs not integrated with the surrounding bone.

Glycoconjugates in the chinchilla tubotympanum.

Various biotinylated lectins were used to characterize and semiquantitate glycoconjugate residues in the tubotympanum. Epithelial goblet cells were stained predominantly by WGA, LFA, SNA, RCA-I, Con-A, LCA, SBA, PHA-E, and UEA; this finding suggests they contain alpha-neuraminic acid, beta-galactose, alpha-mannose, N-acetyl alpha-galactosamine, and alpha-fucose. Glandular mucous cells were stained predominantly by WGA, LFA, SNA, and RCA-I; this finding suggests that they contain alpha-neuraminic acid and beta-galactose. The glandular serous cells were stained predominantly by Con-A, WGA, and LFA; this finding suggests that they produced alpha-mannose and alpha-neuraminic acid that represented serum-type glycoprotein. The positive staining of epithelial goblet cells and glandular mucous cells with PNA after neuraminidase digestion suggests that they produced mucin-type glycoproteins. The staining of the mucous blanket by WGA, LFA, SNA, RCA-I, LCA, PNA, SBA, PHA-E, and UEA suggests the presence of alpha-neuraminic acid, beta-galactose, N-acetyl alpha-galactosamine, and alpha-fucose. The epithelial cell (nonsecretory) surface was stained largely by WGA, LFA, SNA, RCA-I, Con-A, and LCA; this finding suggests the presence of alpha-neuraminic acid, beta-galactose, and alpha-mannose.

Surface active substances in the chinchilla tubotympanum. A biochemical study.

It has been demonstrated that the eustachian tube and middle ear epithelium produce Tubal Surface Active Substances (TSAS), which facilitate the opening of the eustachian tube. In order to characterize the biochemical contents of chinchilla TSAS, the tubal washings were analyzed using 2-D thin layer chromatography. The results indicate that phosphatidylcholine was the predominant phospholipid, followed by sphingomyelin, phosphatidylinositol phosphatidylethanolamine, and phosphatidylserine. In comparison, pulmonary lavage showed phosphatidylcholine to be highest allowed by phosphatidylethanolamine and sphingomyelin. Phosphatidylcholine/phosphatidylethanolanim ratios were 5:1 in the tubal lavage, and 8:1 in the pulmonary lavage. Phosphatidylcholine/sphingomyelin ratios were 2:1 in the tubal lavage, and 67:1 in the pulmonary lavage. It is concluded that the biochemical content of TSAS is similar but not identical to that of pulmonary surfactants.

Peritoneal fluid in human acute pancreatitis blocks hepatic mitochondrial respiration.

Peritoneal fluid may be the source of multi-system failure during the initial stages of acute pancreatitis. The toxicity of human pancreatic ascitic fluid (HPAF) collected under sterile conditions from 12 patients with acute pancreatitis was investigated with the use of an in vitro preparation of mitochondria. Hepatocyte mitochondria from Sprague-Dawley rats were prepared by standard techniques. Mitochondrial oxidative activity was determined in glutamate-malate medium, with micromodification of the YSI Model 53 (Yellow Springs, Ohio) oxygen electrode system. The oxygen uptake of 150 to 200 microliter samples of mitochondria suspension (20 to 25 mg/ml) was monitored for 3 minutes, after which 3 microliter of 0.1 M adenosine diphosphate (ADP) was added to the chamber to induce state 3 respiration. Two minutes after the oxygen trace returned to state 4 respirations, 20, 40, or 80 microliter of the HPAF sample was introduced into the reaction vessel and the oxygen measurement continued for an additional 3 minutes. After this, 3 microliter of 0.1 M ADP again was added to the reaction chamber and the oxygen measurement continued. Control runs were made with reconstituted pooled human serum used in place of the HPAF samples. A significant (p less than 0.05) increase in state 4 respiration occurred in all 12 samples of HPAF. Addition of ADP showed a significant decline (p less than 0.05) in respiratory control ratio in all specimens. A dose-response curve was observed in 8 of 12 HPAF samples tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial dysfunction induced by pancreatitis-associated ascitic fluid.

Acute hemorrhagic pancreatitis (AHP) involves multiple organ failure probably caused by the toxic factor(s) released in pancreatitis-associated ascitic fluid (PAAF). We found that PAAF interferes with hepatic mitochondrial respiration resulting in severe disturbances in respiratory control (RCR) and ADP/O ratios. Pancreatitis was induced in dogs by retrograde pancreatic duct infusion and the resultant PAAF was centrifuged, filtered, and frozen until used. Two human PAAFs collected from AHP patients were treated in a similar manner. Rat liver mitochondrial oxygen uptake was measured at 30 degrees C before and after addition of ADP and PAAF. Paired control runs were made using pooled heat-inactivated dog serum. Tests with nine canine PAAFs showed a mean increase of 120% in state 4 respiration (P less than 0.0001). After exposure to PAAF, addition of ADP to previously coupled mitochondria did not induce state 3 respiration. The human PAAFs both showed significant increases in state 4 respiration (P less than 0.01) and a marked decrease in RCR. Dose-response tests with human and canine PAAFs showed a positive correlation between percentage increase in state 4 respiration and the concentration of PAAF used. These results confirm the presence in PAAF of mitotoxic substance(s) which cause irreversible mitochondrial damage. Inhibition of coupled mitochondrial respiration by PAAF with the resultant fall in ATP may be the causative agent for the tissue and organ damage observed in AHP.