Frances A. Hausman

Simultaneous measurement of multiple ear proteins with multiplex ELISA assays.

A recent advancement in enzyme-linked immunosorbent assay (ELISA) technology is the multiplex antibody array that measures multiple proteins simultaneously within a single sample. This allows reduction in sample volume, time, labor, and material costs, while increasing sensitivity over single ELISA. Current multiplex platforms include planar-based systems using microplates or slides, or bead-based suspension assay with microspheres. To determine the applicability of this technology for ear research, we used 3 different multiplex ELISA-based immunoassay arrays from 4 different companies to measure cytokine levels in mouse middle and inner ear tissue lysate extracts 24 h following transtympanic Haemophilus influenzae inoculation. Middle and inner ear tissue lysates were analyzed using testing services from Quansys Biosciences, Aushon Biosystems SearchLight (both microplate-based), MILLIPLEX MAP Sample (bead-based), and a RayBiotech, Inc (slide-based) kit. Samples were assayed in duplicate or triplicate. Results were compared to determine their relative sensitivity and reliability for measures of cytokines related to inflammation. The cytokine pg/ml amounts varied among the multiplex assays, so a comparison also was made of the mean fold increase in cytokines from untreated controls. Several cytokines and chemokines were elevated, the extent dependent upon the assay sensitivity. Those most significantly elevated were IL-1α, IL-1β, IL-6, TNFα, VEGF, and IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1β, IL-6) to assess sensitivity over the traditional method. Overall, the Quansys Biosciences and SearchLight arrays showed the greatest sensitivity, both employing the same multiplex methodology of a spotted array within a microplate well with chemiluminescent detection. They also were more sensitive than the traditional single ELISA performed with commercial kits and matched gene expression changes determined by quantitative RT-PCR. The Quansys array showed a limit of detection for ear IL-6 down to 2-4 pg/ml, indicating it is sufficiently sensitive to detect ear proteins present in low concentrations. Thus, the multiplex ELISA procedures appear suitable and reliable for the study of hearing related proteins, providing accurate, quantitative, reproducible results with considerable improvement in sensitivity and economy.

Murine middle ear inflammation and ion homeostasis gene expression

Hypothesis: Ion homeostasis genes are responsible for the movement of ions and water in the epithelium of the middle ear. Background: It is not well known to what extent disruption of ion homeostasis is a factor in the accumulation of middle ear fluid during otitis media. Methods: Balb/c mice were transtympanically injected with heat-killed Hemophilus influenza bacteria. Untreated and saline-injected mice were used as controls. Mice were euthanized at 6, 24, and 72 hours and 1 week after injection, the bullae harvested, and total ribonucleic acid isolated from the middle ear tissues. Ion homeostasis genes were analyzed with real-time quantitative reverse transcription-polymerase chain reaction from the following gene families: Na+,K+-ATPase, claudins, K+ transport channels, epithelial Na+ channels, gap junctions, and aquaporins. Inflammatory genes also were analyzed to document inflammation. Results: All inflammatory genes analyzed were significantly upregulated, more at 6 hours than at 24 hours, with the exception of vascular endothelial growth factor and Mapk8. Most middle ear ion homeostasis genes experienced downregulation because of inflammation. This was most prominent in the aquaporin and Na+,K+-ATPase genes. Significant upregulation was seen in several genes in response to inflammation and saline independently. Conclusion: The innate immune response to bacteria in the middle ear induces expression of several inflammatory genes. Coinciding with this inflammation is the downregulation of numerous ion homeostasis genes that are involved in ion and water transport and maintenance of tight junctions. This may explain the fluid accumulation within the middle ear seen with both acute and chronic otitis media.

Autoimmune mouse antibodies recognize multiple antigens proposed in human immune-mediated hearing loss.

Hypothesis: Autoimmune diseased mice with hearing loss will have autoantibodies against the various cochlear antigens proposed in clinical autoimmune inner ear disease. Background: Serum antibodies of patients with hearing loss recognize several proteins that are proposed as possible antigenic targets in the ear. This often leads to a clinical diagnosis of autoimmune inner ear disease, although it is not clear how these antibodies cause inner ear disease. Therefore, to better understand the relationship of autoantibodies and ear disease, an examination was made of serum autoantibodies in the MRL/MpJ-Faslpr autoimmune mouse with hearing loss. Similar antibody patterns in the mouse would provide an animal model in which to investigate potential autoimmune mechanisms of this clinical ear disorder. Methods: Sera from MRL/MpJ-Faslpr autoimmune mice and normal C3H mice were tested by the enzyme-linked immunosorbent assay technique for reactivity against various reported cochlear antigens: heat shock protein 70 (bovine, human, bacterial), laminin, heparan sulfate proteoglycan, cardiolipin, and collagen types II and IV. Results: The autoimmune mouse sera showed significantly greater antibody reactivity against all of the antigens when compared with normal mouse sera. Conclusions: Serum antibodies from autoimmune mice recognized several putative autoantigens reported for patients with hearing loss, suggesting that comparable antigen–antibody mechanisms might be operating. However, the recognition of multiple antigens did not identify any one as being the specific target in autoimmune hearing loss. The correlation of antibodies in the MRL/MpJ-Faslpr autoimmune mouse and human studies indicates this animal model should aid further investigations into potential cochlear antigens in autoimmune hearing loss.

Tissue remodeling gene expression in a murine model of chronic rhinosinusitis.

The matrix metalloproteinase (MMP), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families regulate tissue remodeling in many normal and pathophysiologic processes. We hypothesize that induction of chronic sinonasal inflammation will be associated with changes in regulation of these tissue remodeling cytokines.

Mouse Middle Ear Ion Homeostasis Channels and Intercellular Junctions

Hypothesis The middle ear contains homeostatic mechanisms that control the movement of ions and fluids similar to those present in the inner ear, and are altered during inflammation. Background The normal middle ear cavity is fluid-free and air-filled to allow for effective sound transmission. Within the inner ear, the regulation of fluid and ion movement is essential for normal auditory and vestibular function. The same ion and fluid channels active in the inner ear may have similar roles with fluid regulation in the middle ear. Methods Middle and inner ears from BALB/c mice were processed for immunohistochemistry of 10 specific ion homeostasis factors to determine if similar transport and barrier mechanisms are present in the tympanic cavity. Examination also was made of BALB/c mice middle ears after transtympanic injection with heat-killed Haemophilus influenza to determine if these channels are impacted by inflammation. Results The most prominent ion channels in the middle ear included aquaporins 1, 4 and 5, claudin 3, ENaC and Na+,K+-ATPase. Moderate staining was found for GJB2, KCNJ10 and KCNQ1. The inflamed middle ear epithelium showed increased staining due to expected cellular hypertrophy. Localization of ion channels was preserved within the inflamed middle ear epithelium. Conclusions The middle ear epithelium is a dynamic environment with intrinsic mechanisms for the control of ion and water transport to keep the middle ear clear of fluids. Compromise of these processes during middle ear disease may underlie the accumulation of effusions and suggests they may be a therapeutic target for effusion control.

Quantification of DNA binding to cell-surfaces by flow cytometry.

DNA binding to cell-surfaces has been documented in several studies. The interaction of DNA with cells has been shown to have therapeutic potential as a non-viral form of gene delivery and DNA vaccination. Recently, bacterial DNA binding and internalization has been demonstrated in some cells to trigger secretion of cytokines and cell activation. Previous studies to quantify DNA binding to cells have used radiolabeled DNA. Here we report a non-radioactive assay for quantification of cell-surface DNA binding based on the isoparametric analysis of flow cytometric data as described by Chatelier et al., Embo J., 5 (1986) 1181. This assay has the advantage over previously used procedures in not employing radioactive material and being able to discriminate viable from non-viable cells that bind DNA. With the importance of understanding the interaction of DNA with cells, this assay may have application for the identification and characterization of reagents designed to either enhance or inhibit DNA binding to cells.

Control of Middle Ear Inflammatory and Ion Homeostasis Genes by Transtympanic Glucocorticoid and Mineralocorticoid Treatments

Hypothesis Transtympanic steroid treatment will induce changes in ion homeostasis and inflammatory gene expression to decrease middle ear inflammation due to bacterial inoculation. Background Otitis media is common, but treatment options are limited to systemic antibiotic therapy or surgical intervention. Systemic glucocorticoid treatment of mice decreases inflammation and improves fluid clearance. However, transtympanic delivery of glucocorticoids or mineralocorticoid has not been explored to determine if direct steroid application is beneficial. Methods Balb/c mice received transtympanic inoculation of heat-killed Haemophilus influenzae (H flu), followed by transtympanic treatment with either prednisolone or aldosterone. Mice given PBS instead of steroid and untreated mice were used as controls. Four hours after steroid treatment, middle ears were harvested for mRNA extraction and 24 hours after inoculation middle ears were harvested and examined for measures of inflammation. Results H flu inoculation caused the increased expression of nearly all inflammatory cytokine genes and induced changes in expression of several genes related to cellular junctions and transport channels. Both steroids generally reversed the expression of inflammatory genes and caused ion and water regulatory genes to return to normal or near normal levels. Histologic evaluation of middle ears showed improved fluid and inflammatory cell clearance. Conclusion Improvement in middle ear inflammation was noted with both the glucocorticoid prednisolone and the mineralocorticoid aldosterone. This was due to reversal of inflammation-induced changes in middle ear cytokine genes, as well as those involved in ion and water homeostasis. Because glucocorticoids bind to the mineralocorticoid receptor, but not the reverse, it is concluded that much of the reduction of fluid and other inflammation measures was due to these steroids impact on ion and water transport channels. Further research is necessary to determine if this alternative mineralocorticoid treatment for otitis media will be clinically effective with fewer side effects than glucocorticoids.

Otitis Media: Molecular Impact of Inflammation in the Middle and Inner Ear: Cytokines, Steroids, and Ion Homeostasis

An overview of recent topics under investigation in our laboratory is presented. Topics include the cytokine and ion homeostasis gene response to steroids in the middle ear in a murine model of otitis media and gene chip analysis of inner ear genes affected by trans-tympanic and systemic steroid administration to the mouse.

Correction: Mouse Middle Ear Ion Homeostasis Channels and Intercellular Junctions

The version of Figure 1 that exists in the article is incorrect. The correct version can be found here: