David J. Lim

Activation of NF-κB by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK–IKKα/β–IκBα and MKK3/6–p38 MAP kinase signaling pathways in epithelial cells

Nontypeable Hemophilus influenzae (NTHi) is an important human pathogen in both children and adults. In children, it causes otitis media, the most common childhood infection and the leading cause of conductive hearing loss in the United States. In adults, it causes lower respiratory tract infections in the setting of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States. The molecular mechanisms underlying the pathogenesis of NTHi-induced infections remain undefined, but they may involve activation of NF-κB, a transcriptional activator of multiple host defense genes involved in immune and inflammatory responses. Here, we show that NTHi strongly activates NF-κB in human epithelial cells via two distinct signaling pathways, NF-κB translocation-dependent and -independent pathways. The NF-κB translocation-dependent pathway involves activation of NF-κB inducing kinase (NIK)–IKKα/β complex leading to IκBα phosphorylation and degradation, whereas the NF-κB translocation-independent pathway involves activation of MKK3/6–p38 mitogen-activated protein (MAP) kinase pathway. Bifurcation of NTHi-induced NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase pathways may occur at transforming growth factor-β activated kinase 1 (TAK1). Furthermore, we show that toll-like receptor 2 (TLR2) is required for NTHi-induced NF-κB activation. In addition, several key inflammatory mediators including IL-1β, IL-8, and tumor necrosis factor-α are up-regulated by NTHi. Finally, P6, a 16-kDa lipoprotein highly conserved in the outer membrane of all NTHi and H. influenzae type b strains, appears to also activate NF-κB via similar signaling pathways. Taken together, our results demonstrate that NTHi activates NF-κB via TLR2–TAK1-dependent NIK–IKKα/β-IκBα and MKK3/6–p38 MAP kinase signaling pathways. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and open up new therapeutic targets for these diseases.

Cochlear anatomy related to cochlear micromechanics. A review

The sensory cilia of the mammalian cochlea show an orderly gradation in height along the length of the cochlea. The cilia are taller in the apex and shorter in the base. Differences in ciliary gradation also exist between the different rows of outer hair cell cilia along the length of the cochlea. There are gradations in width and thickness in the moveable portion of the tectorial membrane paralleling those in the basilar membrane. There are also gradations in the relationship between the tectorial membrane and the slope of the reticular lamina along the length of the cochlea. This suggests that there may be additional mechanical fine-tuning capability built into the organ of Corti besides the basilar membrane. The tectorial membrane is firmly attached to the outer hair cell cilia along the entire length of the cochlea in all species examined. The inner hair cell cilia do not have the same firm attachment to the tectorial membrane as outer hair cell cilia. This suggests that the modes of mechanical coupling between the tectorial membrane and the inner and outer hair cell cilia are different.

A Cochlear Cell Line as an in vitro System for Drug Ototoxicity Screening

Aminoglycoside antibiotics, loop diuretics, antineoplastic agents and other commonly used pharmacological drugs are ototoxic. Understanding of the cellular and molecular mechanisms underlying drug ototoxicity, however, has been hampered by the limited availability of inner ear tissues and drug side effects on laboratory animals. Immortalized cell lines derived from the auditory sensory organ, sensitive to ototoxic drugs and growing in environments that can be systematically manipulated, would facilitate the research directed at elucidating these mechanisms. Such immortalized cell lines could also be used to discover novel therapeutic agents for preventing drug-induced sensorineural hearing loss. Here, we report a conditionally immortalized organ of Corti-derived epithelial cell line, which shows evidence of activation of apoptosis when exposed to known ototoxic drugs. This cell line may be an excellent in vitro system to investigate the cellular and molecular mechanisms involved in ototoxicity and for screening of the potential ototoxicity or otoprotective properties of new pharmacological drugs.

Human Tympanic Membrane: An Ultrastructural Observation

Submicroscopic details of the human tympanic membrane were studied utilizing a transmission electron microscope (T. E. M.) and a scanning electron microscope (S. E. M.) Three layers were distinctly recognized; outer epidermal, middle lamina propria and inner mucous layer. In pars flaccida, the middle lamina propria is formed of loose connective tissues with abundant elastic and collagen fibers. On the other hand, in the pars tensa, the middle lamina propria is formed of outer radial and inner circular fibers. Besides these two types of fibers, parabolic fibers were observed between the radial and circular fibers. The fibrils of the para tensa fibers are rectangular in cross-sectional view and are mixed with varying amounts of collagen fibrils. As in animals, the human pars flaccida was considerably thicker than the pars tensa, contrary to the popular concept that they are thinner.

Ultrastructural pathology of the middle ear mucosa in serous otitis media.

Serous otitis media (SaM) is one of the most common childhood diseases. In spite of numerous investigations on the subject, its pathogenesis still remains obscure. Even the terminology sterile otitis, middle ear effusion, catarrhal otitis. and secretory otitis is diverse and confusing. However, it is generally agreed that the Eustachian tubal dysfunction is associated with this condition.

Effects of noise and ototoxic drugs at the cellular level in the cochlea: a review.

Currently available information concerning the cellular mechanisms involved in acoustic trauma and aminoglycoside ototoxicity is reviewed to shed some new light on the cellular events that may be related to functional impairment of the auditory organ. Based on the available data, the following postulations can be made concerning the cellular mechanisms involved. 1) The macromolecular disruption of the stereocilia and cuticular plates is the initial cellular event in acoustic trauma. This disruption would affect the micromechanics of the transduction process, leading to temporary threshold shift. Further cellular impairment would involve basic cellular functions such as the protein, lipid, and glucose synthesis needed for cell repair and survival, and such impairment would result in permanent cell injury or cell death, leading to permanent threshold shift. 2) It can be postulated that the cellular mechanisms involved in aminoglycoside ototoxicity include two events. The early event is the reversible blockage of the transduction channels from the endolymph side of the hair cells. The later event is the interference in such cellular functions as protein and/or phospholipid synthesis because of binding of aminoglycoside to the phospholipids and/or protein, leading to cell death. The latter event may be facilitated by penetration or membrane-mediated internalization of the aminoglycoside from the perilymph side of the hair cell.

Formation and Fate of the Otoconia Scanning and Transmission Electron Microscopy

Although mammalian otoconia are known to be composed of calcium carbonate in calcite form, their morphogenesis, maintenance, and fate are not well understood. More information on these problem areas would aid considerably in better understanding various clinical conditions, such as cupulolithiasis and otolith degeneration. This study was intended to clarify the fine morphology of the otolith in normal and adverse conditions in laboratory animals with the use of the scanning and transmission electron microscopes. It was confirmed by this study that the mammalian otoconium is composed of an organic matrix and minerals (calcium carbonate). When the minerals are removed by decalcification, or chelation, a well arranged organic matrix, and even a nucleus, can be found in the crystal. The matrix of the crystal is identical to the gelatin matrix of the otolithic membrane. This finding supports the possibility that a normal protein matrix is a prerequisite for normal otoconia formation, and that the exchange of calcium ions can occur without altering the crystal structure. The vestibular dark cells, which are thought to be endolymph-secreting cells, appear to be capable of removing calcium from the otoconia that are attached to the dark cell surfaces. Although this evidence is only circumstantial, its consistency is impressive. On the basis of the foregoing, it is tempting to speculate that the dark cells participate in the removal of the dislodged otoliths, but further study is required to ascertain this point.

Glucocorticoids synergistically enhance nontypeable Haemophilus influenzae-induced Toll-like receptor 2 expression via a negative cross-talk with p38 MAP kinase.

The recognition of invading microbes followed by the induction of effective innate immune response is crucial for host survival. Human surface epithelial cells are situated at host-environment boundaries and thus act as the first line of host defense against invading microbes. They recognize the microbial ligands via Toll-like receptors (TLRs) expressed on the surface of epithelial cells. TLR2 has gained importance as a major receptor for a variety of microbial ligands. In contrast to its high expression in lymphoid tissues, TLR2 is expressed at low level in epithelial cells. Thus, it remains unclear whether the low amount of TLR2 expressed in epithelial cells is sufficient for mediating bacteria-induced host defense and immune response and whether TLR2 expression can be up-regulated by bacteria during infection. Here, we show that TLR2, although expressed at very low level in unstimulated human epithelial cells, is greatly up-regulated by nontypeableHemophilus influenzae (NTHi), an important human bacterial pathogen causing otitis media and chronic obstructive pulmonary diseases. Activation of an IKKβ-IκBα-dependent NF-κB pathway is required for TLR2 induction, whereas inhibition of the MKK3/6-p38α/β pathway leads to enhancement of NTHi-induced TLR2 up-regulation. Surprisingly, glucocorticoids, well known potent anti-inflammatory agents, synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the p38 MAP kinase pathway. These studies may bring new insights into the role of bacteria and glucocorticoids in regulating host defense and immune response and lead to novel therapeutic strategies for modulating innate immune and inflammatory responses for otitis media and chronic obstructive pulmonary diseases.

Cisplatin Cytotoxicity of Auditory Cells Requires Secretions of Proinflammatory Cytokines via Activation of ERK and NF-κB

The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, through the activation of ERK and NF-κB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-α. Immunohistochemical studies revealed that TNF-α expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-κB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-α, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

Tympanic Membrane: Electron Microscopic Observation Part I: Pars Tensa

Submicroscopic morphological details of the tympanic membrane (pars tensa) of the guinea pig, cat and squirrel monkey were studied. Three layers were distinctly recognized: outer epidermal, middle lamina propria, and inner mucous. Osmiophilic dark keratin granules, which are responsible for keratinization, were found in the granular layer (stratum granulosum) in association with tonofilaments—fine cytoplasmic microfilaments attached to desmosomes (cell bridge). Subepidermal and submucosal connective tissue layers were identified on both sides of the radiate and circular collagenous layers. These loose connective tissue layers housed nerve fibers, blood vessels, fibroblasts and wandering cells. Collagenous layers were composed of a mixture of two types of fibrils: mature collagen fibrils with distinct axial bands and finer fibrils—probably protofibrils (young form of collagen), although their exact nature is yet to be determined. Major nerve fibers (myelinated and unmyelinated) were found in the subepiderm...