NOD-Like Receptor Signaling in Cholesteatoma.

Background. Cholesteatoma is a destructive process of the middle ear resulting in erosion of the surrounding bony structures with consequent hearing loss, vestibular dysfunction, facial paralysis, or intracranial complications. The etiopathogenesis of cholesteatoma is controversial but is associated with recurrent ear infections. The role of intracellular innate immune receptors, the NOD-like receptors, and their associated signaling networks was investigated in cholesteatoma, since mutations in NOD-like receptor-related genes have been implicated in other chronic inflammatory disorders. Results. The expression of NOD2 mRNA and protein was significantly induced in cholesteatoma compared to the external auditory canal skin, mainly located in the epithelial layer of cholesteatoma. Microarray analysis showed significant upregulation for NOD2, not for NOD1, TLR2, or TLR4 in cholesteatoma. Moreover, regulation of genes in an interaction network of the NOD-adaptor molecule RIPK2 was detected. In addition to NOD2, NLRC4, and PYCARD, the downstream molecules IRAK1 and antiapoptotic regulator CFLAR showed significant upregulation, whereas SMAD3, a proapoptotic inducer, was significantly downregulated. Finally, altered regulation of inflammatory target genes of NOD signaling was detected. Conclusions. These results indicate that the interaction of innate immune signaling mediated by NLRs and their downstream target molecules is involved in the etiopathogenesis and growth of cholesteatoma.

TNFA deletion alters apoptosis as well as caspase 3 and 4 expression during otitis media.

BACKGROUND: Tumor necrosis factor (TNFA) is the canonical member of the TNF superfamily, which plays a major role in both inflammation and apoptosis. To evaluate the role of TNFs in otitis media (OM), the most common disease of childhood, we evaluated middle ear (ME) expression of genes encoding the TNF and TNF receptor superfamilies during bacterial OM in the mouse, characterized OM in TNFA-deficient mice, and assessed apoptosis during OM in normal versus TNF-deficient MEs. RESULTS: TNFs and TNF receptors were broadly regulated during OM, with TNFA showing the highest level of up-regulation. TNF deficient mice exhibited mucosal hyperplasia even in the absence of infection and exuberant growth of the mucosa during OM, including the formation of mucosal polyps. Mucosal recovery during OM was also delayed, in parallel with a delay in mucosal apoptosis and reduced caspase gene expression. CONCLUSIONS: The TNF and TNF receptor superfamilies mediate both inflammation and apoptosis during OM. TNF appears to be critical for the maintenance of mucosal architecture in both the normal and infected ME, since excessive accumulation of mucosal tissue is seen in TNFA-/- MEs both before and after bacterial inoculation of the ME. TNFA is also required for appropriate regulation of caspase genes.

CC chemokine ligand 3 overcomes the bacteriocidal and phagocytic defect of macrophages and hastens recovery from experimental otitis media in TNF-/- mice.

Innate immune mechanisms are crucial in defense against bacterial illnesses in humans, as evidenced by abnormal antibacterial responses due to defects in TLR signaling, seen in children with MyD88 or IL-1R-associated kinase 4 deficiency. Otitis media (OM) is the most common disease of childhood, and the role of innate immune molecules in this disorder remains unclear. In a murine model of OM, we show that, in the absence of TNF, a key effector of innate immunity, this disease is prolonged after middle ear infection with nontypeable Haemophilus influenzae (NTHi). In the absence of TNF, mice fail to upregulate both TLRs and downstream genes and proteins, such as CCL3, resulting in defects in both inflammatory cell recruitment and macrophage function. Peritoneal macrophages of mice lacking TNF have a diminished ability to phagocytose and kill NTHi, and this defect is partially corrected in vitro by exogenous rTNF. Addition of rCCL3 alone or in combination with rTNF restores phagocytosis and killing by TNF-deficient macrophages to that of unstimulated wild-type macrophages. In vivo administration of rCCL3 to animals deficient in TNF fully restores the ability to control OM due to NTHi, whereas a CCL3-blocking Ab impaired the ability of wild-type mice to recover from OM. Thus, CCL3 is a potent downstream effector of TNF-mediated inflammation in vitro and in vivo. Manipulation of CCL3 and/or TNF may prove to be effective therapeutic approaches in OM or other conditions associated with defective TNF generation.

Histologic and morphologic evaluation of explanted bone anchors from bone-anchored hearing aids.

Bone-anchored hearing aids are a standard option in rehabilitation of patients with conductive or mixed hearing loss, and also CROS fitting. However, the skin-penetrating bone anchor repeatedly gives reason for discussion about the risk of infection of surrounding tissues as a major cause of malfunction. In the present study, explanted bone anchors with surrounding bone and soft tissue were examined and compared with the morphology of lost implants. The anchors originated from five patients. Two needed explantation due to deafness with the need of cochlea implantation. A third patient underwent explantation due to meningeal irritation by the bone anchor. Another patient lost the implant due to mechanical stress shortly after implantation. The last implant was lost in a child without apparent reason. All implants were clinically free of infection and had been stable for a median implantation period of 12 months. During the explantation procedure, the fixtures were recovered together with the attached soft tissue and bone. The specimens were examined by light microscopy or scanning electron microscopy (SEM). Sectioning for light microscopy was performed with a diamond-coated saw microtome. Histopathologic examination of the surrounding skin and subcutaneous soft tissue showed slight inflammation in one case only. The bone was regularly vital, presenting no signs of inflammation. The threads of the fixtures were filled with bone, with particularly strong attachment to the flank of traction. The SEM investigation exposed the ultrastructural interaction of bone with the implant surface. Filiform- and podocyte-like processes of osteocytes attach to the implant; lost implants did not reflect these features. Implant integration involves both osseointegration as well as soft tissue integration. Titanium oxide as the active implant surface promotes this integration even in unstable implants. The morphologic analysis exposed structural areas of the implant with weak bone-to-metal contact. Optimized implant design with modified surface and threads may additionally improve osseointegration of hearing aid bone anchors.

Myeloid differentiation primary response gene 88 is required for the resolution of otitis media.

BACKGROUND: Signaling defects in the Toll-like receptor (TLR) pathway, such as interleukin-1 receptor-associated kinase 4 deficiency, highlight the prominence of TLR signaling in the defense against bacterial disease. Because myeloid differentiation primary response gene 88 (MyD88) can transduce signals from almost all TLRs, we studied its role in otitis media (OM), the most common upper respiratory tract bacterial infectious disease in young children. METHODS: The middle ears (MEs) of wild-type (WT) and MyD88(-/-) mice were inoculated with nontypeable Haemophilus influenzae (NTHi). ME infection and inflammation were monitored for 21 days after surgery. Bone marrow-derived macrophages from WT and MyD88(-/-) mice were infected with NTHi in vitro to assess their interaction with bacteria. RESULTS: In WT mice, MyD88 expression was detected in the ME stroma at baseline. MyD88(-/-) mice displayed prolonged ME mucosal thickening and delayed recruitment of neutrophils and macrophages. Although WT mice cleared NTHi within 5 days, viable NTHi were isolated for up to 21 days in MyD88(-/-) mice. The interaction between macrophages and NTHi was significantly altered in MyD88(-/-) mice. CONCLUSIONS: In this mouse model, MyD88-mediated signaling was important for clearance of infection and resolution of inflammation in acute OM due to NTHi. The role played by innate signaling in children susceptible to chronic or recurrent OM deserves further study.

Interaction of cochlear nucleus explants with semiconductor materials.

OBJECTIVE/HYPOTHESIS: Implantable hearing devices such as cochlear implants and auditory brainstem implants deliver auditory information through electrical stimulation of auditory neurons. The combination of microelectronic electrodes with auditory nerve cells may lead to further improvement of the hearing quality with these devices. Whereas several kinds of neurons are known to grow on semiconductor substrates, interactions of cochlear nucleus (CN) neurons with such materials have yet to be described. MATERIALS AND METHODS: To investigate survival and growth behavior of CN neurons on different semiconductor materials. CN explants from postnatal day 10 Sprague-Dawley rats were cultured for 96 hours in Neurobasal medium on polished and unpolished silicon wafers (p-type Si [100] and p-type Si3N4[100]) as well as plastic surface. These surfaces had been coated with poly-L-lysine and laminin. Neuronal outgrowth was examined using image analysis software after immunohistologic staining for neurofilament. Neurite length and directional changes were quantified. Additionally, neurite morphology and adhesion to the semiconductor material was evaluated by scanning electron microscopy. RESULTS: Although proper adhesion of CN explants was seen, no neurite growth could be detected on unpolished silicon wafers (Si and Si3N4). Compared with the other test conditions, polished, laminin-coated Si3N4 wafers showed best biocompatibility regarding neurite length and number per explant. CN explants developed a mean of eight neurons with an average length of 236 mum in 96 hours of culture on these wafers. CONCLUSION: The results of this study demonstrate the general possibility of CN neuron growth in culture on semiconductors in vitro. The differences in neuron length and number per explant indicate that the growth of CN neurons is influenced by the semiconductor substrate as well as extracellular matrix proteins, with laminin-coated p-type Si3N4[100] being a preferable material for future hybrid experiments on auditory-neuron semiconductor chips.

Deficiencies of myeloid differentiation factor 88, Toll-like receptor 2 (TLR2), or TLR4 produce specific defects in macrophage cytokine secretion induced by Helicobacter pylori.

Helicobacter pylori is a gram-negative microaerophilic bacterium that colonizes the gastric mucosa, leading to disease conditions ranging from gastritis to cancer. Toll-like receptors (TLRs) play a central role in innate immunity by their recognition of conserved molecular patterns on bacteria, fungi, and viruses. Upon recognition of microbial components, these TLRs associate with several adaptor molecules, including myeloid differentiation factor 88 (MyD88). To investigate the contribution of the innate immune system to H. pylori infection, bone marrow-derived macrophages from mice deficient in TLR2, TLR4, TLR9, and MyD88 were infected with H. pylori SS1 and SD4 for 24 or 48 h. We demonstrate that MyD88 was essential for H. pylori induction of all cytokines investigated except alpha interferon (IFN-alpha). The secretion of IFN-alpha was substantially increased from cells deficient in MyD88. H. pylori induced interleukin-12 (IL-12) and IL-10 through TLR4/MyD88 signaling. In addition, H. pylori induced less IL-6 and IL-1beta in TLR2-deleted macrophages, suggesting that the MyD88 pathway activated by TLR2 stimulation is responsible for H. pylori induction of the host proinflammatory response (IL-6 and IL-1beta). These observations are important in light of a recent report on IL-6 and IL-1beta playing a role in the development of H. pylori-related gastric cancer. In conclusion, our study demonstrates that H. pylori activates TLR2 and TLR4, leading to the secretion of distinct cytokines by macrophages.