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Some species have evolved the ability to use the sense of hearing to modify existing vocalizations, or even create new ones, which enlarges their repertoires and results in complex communication systems. 1 This ability corresponds to various forms of vocal production learning that are all possessed by humans and independently displayed by distantly related vertebrates. 1 , 2 , 3 , 4 , 5 , 6 , 7 Among mammals, a few species, including the Egyptian fruit bat, 8 , 9 , 10 would possess such vocal production learning abilities. 7 Yet the necessity of an intact auditory system for the development of the Egyptian fruit bat typical vocal repertoire has not been tested. Furthermore, a systematic causal examination of learned and innate aspects of the entire repertoire has never been performed in any vocal learner. Here we addressed these gaps by eliminating pups' sense of hearing at birth and assessing its effects on vocal production in adulthood. The deafening treatment enabled us to both causally test these bats' vocal learning ability and discern learned from innate aspects of their vocalizations. Leveraging wireless individual audio recordings from freely interacting adults, we show that a subset of the Egyptian fruit bat vocal repertoire necessitates auditory feedback. Intriguingly, these affected vocalizations belong to different acoustic groups in the vocal repertoire of males and females. These findings open the possibilities for targeted studies of the mammalian neural circuits that enable sexually dimorphic forms of vocal learning., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Flat epithelium (FE) is a condition characterized by the loss of both hair cells (HCs) and supporting cells and the transformation of the organ of Corti into a simple flat or cuboidal epithelium, which can occur after severe cochlear insults. The transcription factors Gfi1, Atoh1, Pou4f3, and Six1 (GAPS) play key roles in HC differentiation and survival in normal ears. Previous work using a single transcription factor, Atoh1, to induce HC regeneration in mature ears in vivo usually produced very few cells and failed to produce HCs in severely damaged organs of Corti, especially those with FE. Studies in vitro suggested combinations of transcription factors may be more effective than any single factor, thus the current study aims to examine the effect of co-overexpressing GAPS genes in deafened mature guinea pig cochleae with FE. Deafening was achieved through the infusion of neomycin into the perilymph, leading to the formation of FE and substantial degeneration of nerve fibers. Seven days post neomycin treatment, adenovirus vectors carrying GAPS were injected into the scala media and successfully expressed in the FE. One or two months following GAPS inoculation, cells expressing Myosin VIIa were observed in regions under the FE (located at the scala tympani side of the basilar membrane), rather than within the FE. The number of cells, which we define as induced HCs (iHCs), was not significantly different between one and two months, but the larger N at two months made it more apparent that there were significantly more iHCs in GAPS treated animals than in controls. Additionally, qualitative observations indicated that ears with GAPS gene expression in the FE had more nerve fibers than FE without the treatment. In summary, our results showed that co-overexpression of GAPS enhances the potential for HC regeneration in a severe lesion model of FE., (Copyright © 2023 Elsevier B.V. All rights reserved.)

CHARGE syndrome is a multiple anomaly developmental disorder characterized by a variety of sensory deficits, including sensorineural hearing loss of unknown etiology. Most cases of CHARGE are caused by heterozygous pathogenic variants in CHD7, the gene encoding Chromodomain DNA-binding Protein 7 (CHD7), a chromatin remodeler important for the development of neurons and glial cells. Previous studies in the Chd7 Gt/+ mouse model of CHARGE syndrome showed substantial neuron loss in the early stages of the developing inner ear that are compensated for by mid-gestation. In this study, we sought to determine if early developmental delays caused by Chd7 haploinsufficiency affect neurons, glial cells, and inner hair cell innervation in the mature cochlea. Analysis of auditory brainstem response recordings in Chd7 Gt/+ adult animals showed elevated thresholds at 4 kHz and 16 kHz, but no differences in ABR Wave I peak latency or amplitude compared to wild type controls. Proportions of neurons in the Chd7 Gt/+ adult spiral ganglion and densities of nerve projections from the spiral ganglion to the organ of Corti were not significantly different from wild type controls. Inner hair cell synapse formation also appeared unaffected in mature Chd7 Gt/+ cochleae. However, histological analysis of adult Chd7 Gt/+ cochleae revealed diminished satellite glial cells and hypermyelinated Type I spiral ganglion axons. We characterized the expression of CHD7 in developing inner ear glia and found CHD7 to be expressed during a tight window of inner ear development at the Schwann cell precursor stage at E9.5. While cochlear neurons appear to differentiate normally in the setting of Chd7 haploinsufficiency, our results suggest an important role for CHD7 in glial cells in the inner ear. This study highlights the dynamic nature of CHD7 activity during inner ear development in mice and contributes to understanding CHARGE syndrome pathology., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Pathogenic variants in GJB2 , the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2 -related deafness are not well understood, and cures are absent. Humans with GJB2 -related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCre ERT2 -mediated loss of Gjb2 . Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCre ERT2 ;Gjb2 flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2 -related deafness., Competing Interests: The authors declare no competing interest., (© 2021 The Author(s).)

Epigenetic regulation of gene transcription by chromatin remodeling proteins has recently emerged as an important contributing factor in inner ear development. Pathogenic variants in CHD7, the gene encoding Chromodomain Helicase DNA binding protein 7, cause CHARGE syndrome, which presents with malformations in the developing ear. Chd7 is broadly expressed in the developing mouse otocyst and mature auditory epithelium, yet the pathogenic effects of Chd7 loss in the cochlea are not well understood. Here we characterized cochlear epithelial phenotypes in mice with deletion of Chd7 throughout the otocyst (using Foxg1 Cre/+ and Pax2Cre), in the otic mesenchyme (using TCre), in hair cells (using Atoh1Cre), in developing neuroblasts (using NgnCre), or in spiral ganglion neurons (using Shh Cre/+ ). Pan-otic deletion of Chd7 resulted in shortened cochleae with aberrant projections and axonal looping, disorganized, supernumerary hair cells at the apical turn and a narrowed epithelium with missing hair cells in the middle region. Deletion of Chd7 in the otic mesenchyme had no effect on overall cochlear morphology. Loss of Chd7 in hair cells did not disrupt their formation or organization of the auditory epithelium. Similarly, absence of Chd7 in spiral ganglion neurons had no effect on axonal projections. In contrast, deletion of Chd7 in developing neuroblasts led to smaller spiral ganglia and disorganized cochlear neurites. Together, these observations reveal dosage-, tissue-, and time-sensitive cell autonomous roles for Chd7 in cochlear elongation and cochlear neuron organization, with minimal functions for Chd7 in hair cells. These studies provide novel information about roles for Chd7 in development of auditory neurons., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mice with chronic cochlear implants can significantly contribute to our understanding of the relationship between cochlear health and implant function because of the availability of molecular tools for controlling conditions in the cochlea and transgenic lines modeling human disease. To date, research in implanted mice has mainly consisted of short-term studies, but since there are large changes in implant function following implant insertion trauma, and subsequent recovery in many cases, longer-term studies are needed to evaluate function and perception under stable conditions. Because frequent anesthetic administration can be especially problematic in mice, a chronic model that can be tested in the awake condition is desirable. Electrically-evoked compound action potentials (ECAPs) recorded with multichannel cochlear implants are useful functional measures because they can be obtained daily without anesthesia. In this study, we assessed changes and stability of ECAPs, electrically-evoked auditory brainstem responses (EABRs), ensemble spontaneous activity (ESA), and impedance data over time after implanting mice with multichannel implants. We then compared these data to histological findings in these implanted cochleae, and compared results from this chronic mouse model to data previously obtained in a well-established chronically-implanted guinea pig model. We determined that mice can be chronically implanted with cochlear implants, and ECAP recordings can be obtained frequently in an awake state for up to at least 42 days after implantation. These recordings can effectively monitor changes or stability in cochlear function over time. ECAP and EABR amplitude-growth functions (AGFs), AGF slopes, ESA levels and impedances in mice with multichannel implants appear similar to those found in guinea pigs with long-term multichannel implants. Animals with better survival of spiral ganglion neurons (SGNs) and inner hair cells (IHCs) have steeper AGF slopes, and larger ESA responses. The time course of post-surgical ear recovery may be quicker in mice and can show different patterns of recovery which seem to be dependent on the degree of insertion trauma and subsequent histological conditions. Histology showed varying degrees of cochlear damage with fibrosis present in all implanted mouse ears and small amounts of new bone in a few ears. Impedance changes over time varied within and across animals and may represent changes over time in multiple variables in the cochlear environment post-implantation. Due to the small size of the mouse, susceptibility to stress, and the higher potential for implant failure, chronic implantation in mice can be challenging, but overall is feasible and useful for cochlear implant research., Competing Interests: Declaration of Competing Interest The authors declare no COI., (Copyright © 2021. Published by Elsevier B.V.)

The auditory sensory epithelium of the mammalian inner ear is a highly organized structure that contains sensory hair cells (HCs) and non-sensory supporting cells (SCs). Following the partial loss of HCs after cochlear insults such as overstimulation or ototoxic drugs, SCs seal the luminal epithelial surface (reticular lamina) and reorganize its cellular pattern. Here we investigated the changes in the sensory epithelium following a rapid and severe cochlear insult in the diphtheria toxin receptor (DTR) mouse, where diphtheria toxin (DT) injection leads to a HC-specific lesion resulting in a complete HC loss. We found that DT-induced selective HC ablation could lead to a pattern of scar formation and apical cell-cell adherens and tight junction reorganization similar to that occurring after other types of cochlear insult. Prestin, an outer HC-specific protein, was present in amorphous clumps at the sites where SCs had expanded to fill the spaces vacated by the dead HCs for up to 2 months after the DT induced lesion. Many of the prestin clumps appeared to occupy spaces within SCs, suggesting that SCs participate in the removal process of HC corpses in the DTR deafness model. Prestin clumps could be seen in different areas all along the length of the SCs, and appeared to be inside the SCs as well as in the inter-cellular spaces between SCs. The findings suggest that HC elimination in the DTR deafness model follows a mechanism similar to that in overstimulation or ototoxicity models, making the DTR mouse useful for understanding the process underlying HC elimination and the role of SCs in this process., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3 DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Methyl tert-butyl ether (MTBE) was added to gasoline in New Hampshire (NH) between 1995 and 2006 to comply with the oxygenate requirements of the 1990 Amendments to the Clean Air Act. Leaking tanks and spills released MTBE into groundwater, and as a result, MTBE has been detected in drinking water in NH. We conducted a comparative cancer risk assessment and a margin-of-safety (MOS) analysis for several constituents, including MTBE, detected in NH drinking water. Using standard risk assessment methods, we calculated cancer risks from exposure to 12 detected volatile organic compounds (VOCs), including MTBE, and to four naturally occurring compounds (i.e., arsenic, radium-226, radium-228, and radon-222) detected in NH public water supplies. We evaluated exposures to a hypothetical resident ingesting the water, dermally contacting the water while showering, and inhaling compounds volatilizing from water in the home. We then compared risk estimates for MTBE to those of the other 15 compounds. From our analysis, we concluded that the high-end cancer risk from exposure to MTBE in drinking water is lower than the risks from all the other VOCs evaluated and several thousand times lower than the risks from exposure to naturally occurring constituents, including arsenic, radium, and radon. We also conducted an MOS analysis in which we compared toxicological points of departure to the NH maximum contaminant level (MCL) of 13 µg/L. All of the MOSs were greater than or equal to 160,000, indicating a large margin of safety and demonstrating the health-protectiveness of the NH MCL for MTBE., (© 2020 Society for Risk Analysis.)

To address the safety of caffeine levels in energy drinks, we previously conducted a detailed evaluation of epidemiology studies in humans consuming coffee/caffeine, in which we assessed multiple health effects (unpublished). To further evaluate the effects of caffeine on the cardiovascular system, we turned to animal studies, which often use pure caffeine (not coffee), frequently at higher doses than those typical of human exposure. We identified key scientific studies and reviews in which effects of coffee or caffeine were evaluated in animals by conducting a comprehensive PubMed literature search and analyzing the results. We found that the human equivalent dose (HED) for the no observed adverse effect level (NOAEL) for cardiovascular effects was 260 mg caffeine (2-3 cups of coffee) for a single dose of caffeine for a 70-kg adult, while the lowest observed adverse effect level (LOAEL) was 770 mg (7-8 cups of coffee) for a 70-kg adult. Overall, the doses associated with possible adverse cardiovascular effects were more than either the amount of caffeine consumed over a 24-hour period in two regular energy shots (400 mg/day) or the amount in two extra strength energy shots (460 mg/day)., (Copyright © 2018. Published by Elsevier Ltd.)

The damaged vestibular sensory epithelium of mammals has a limited capacity for spontaneous hair cell regeneration, which largely depends on the transdifferentiation of surviving supporting cells. Little is known about the response of vestibular supporting cells to a severe insult. In the present study, we evaluated the impact of a severe ototoxic insult on the histology of utricular supporting cells and the changes in innervation that ensued. We infused a high dose of streptomycin into the mouse posterior semicircular canal to induce a severe lesion in the utricle. Both scanning electron microscopy and light microscopy of plastic sections showed replacement of the normal cytoarchitecture of the epithelial layer with a flat layer of cells in most of the samples. Immunofluorescence staining showed numerous cells in the severely damaged epithelial layer that were negative for hair cell and supporting cell markers. Nerve fibers under the flat epithelium had high density at the 1 month time point but very low density by 3 months. Similarly, the number of vestibular ganglion neurons was unchanged at 1 month after the lesion, but was significantly lower at 3 months. We therefore determined that the mouse utricular epithelium turns into a flat epithelium after a severe lesion, but the degeneration of neural components is slow, suggesting that treatments to restore balance by hair cell regeneration, stem cell therapy or vestibular prosthesis implantation will likely benefit from the short term preservation of the neural substrate., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In experimental animal models of auditory hair cell (HC) loss, insults such as noise or ototoxic drugs often lead to secondary changes or degeneration in non-sensory cells and neural components, including reduced density of spiral ganglion neurons, demyelination of auditory nerve fibers and altered cell numbers and innervation patterns in the cochlear nucleus (CN). However, it is not clear whether loss of HCs alone leads to secondary degeneration in these neural components of the auditory pathway. To elucidate this issue, we investigated changes of central components after cochlear insults specific to HCs using diphtheria toxin receptor (DTR) mice expressing DTR only in HCs and exhibiting complete HC loss when injected with diphtheria toxin (DT). We showed that DT-induced HC ablation has no significant impacts on the survival of auditory neurons, central synaptic terminals, and myelin, despite complete HC loss and profound deafness. In contrast, noise exposure induced significant changes in synapses, myelin and CN organization even without loss of inner HCs. We observed a decrease of neuronal size in the auditory pathway, including peripheral axons, spiral ganglion neurons, and CN neurons, likely due to loss of input from the cochlea. Taken together, selective HC ablation and noise exposure showed different patterns of pathology in the auditory pathway and the presence of HCs is not essential for the maintenance of central synaptic connectivity and myelination., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Synaptopathy in the cochlea occurs when the connection between inner hair cells and the auditory nerve is disrupted, leading to impaired hearing and nerve degeneration. Experiments using transgenic mice have shown that overexpression of NT3 by supporting cells repairs synaptopathy caused by overstimulation. To accomplish such therapy in the clinical setting, it would be necessary to activate the neurotrophin receptor on auditory neurons by other means. Here we test the outcome of NT3 overexpression using viral-mediated gene transfer into the perilymph versus the endolymph of the normal guinea pig cochlea. We inoculated two different Ntf3 viral vectors, adenovirus (Adv) or adeno-associated virus (AAV) into the perilymph, to facilitate transgene expression in the mesothelial cells and cochlear duct epithelium, respectively. We assessed outcomes by comparing Auditory brainstem response (ABR) thresholds prior to that at baseline to thresholds at 1 and 3 weeks after inoculation, and then performed histologic evaluation of hair cells, nerve endings, and synaptic ribbons. We observed hearing threshold shifts as well as disorganization of peripheral nerve endings and disruption of synaptic connections between inner hair cells and peripheral nerve endings with both vectors. The data suggest that elevation of NT3 levels in the cochlear fluids can disrupt innervation and degrade hearing.

Dietary supplements consisting of beta-carotene (precursor to vitamin A), vitamins C and E and the mineral magnesium (ACEMg) can be beneficial for reducing hearing loss due to aminoglycosides and overstimulation. This regimen also slowed progression of deafness for a boy with GJB2 (CONNEXIN 26) mutations. To assess the potential for treating GJB2 and other forms of hereditary hearing loss with ACEMg, we tested the influence of ACEMg on the cochlea and hearing of mouse models for two human mutations: GJB2, the leading cause of childhood deafness, and DIAPH3, a cause of auditory neuropathy. One group of mice modeling GJB2 (Gjb2-CKO) received ACEMg diet starting shortly after they were weaned (4 weeks) until 16 weeks of age. Another group of Gjb2-CKO mice received ACEMg in utero and after weaning. The ACEMg diet was given to mice modeling DIAPH3 (Diap3-Tg) after weaning (4 weeks) until 12 weeks of age. Control groups received food pellets without the ACEMg supplement. Hearing thresholds measured by auditory brainstem response were significantly better for Gjb2-CKO mice fed ACEMg than for the control diet group. In contrast, Diap3-Tg mice displayed worse thresholds than controls. These results indicate that ACEMg supplementation can influence the progression of genetic hearing loss.

Lampalizumab is an antigen-binding fragment of a humanized monoclonal antibody against complement factor D (CFD), a rate-limiting enzyme in the activation and amplification of the alternative complement pathway (ACP), which is in phase III clinical trials for the treatment of geographic atrophy. Understanding of the pharmacokinetics, pharmacodynamics, and biodistribution of lampalizumab following intravitreal administration in the ocular compartments and systemic circulation is limited but crucial for selecting doses that provide optimal efficacy and safety. Here, we sought to construct a semimechanistic and integrated ocular-systemic pharmacokinetic-pharmacodynamic model of lampalizumab in the cynomolgus monkey to provide a quantitative understanding of the ocular and systemic disposition of lampalizumab and CFD inhibition. The model takes into account target-mediated drug disposition, target turnover, and drug distribution across ocular tissues and systemic circulation. Following intravitreal administration, lampalizumab achieves rapid equilibration across ocular tissues. Lampalizumab ocular elimination is relatively slow, with a τ1/2 of approximately 3 days, whereas systemic elimination is rapid, with a τ1/2 of 0.8 hours. Target-independent linear clearance is predominant in the eye, whereas target-mediated clearance is predominant in the systemic circulation. Systemic CFD synthesis was estimated to be high (7.8 mg/day); however, the amount of CFD entering the eye due to influx from the systemic circulation was small (<10%) compared with the lampalizumab dose and is thus expected to have an insignificant impact on the clinical dose-regimen decision. Our findings support the clinical use of intravitreal lampalizumab to achieve significant ocular ACP inhibition while maintaining low systemic exposure and minimal systemic ACP inhibition.

Connexins are components of gap junctions which facilitate transfer of small molecules between cells. One member of the connexin family, Connexin 26 (Cx26), is prevalent in gap junctions in sensory epithelia of the inner ear. Mutations of GJB2, the gene encoding Cx26, cause significant hearing loss in humans. The vestibular system, however, does not usually show significant functional deficits in humans with this mutation. Mouse models for loss of Cx26 function demonstrate hearing loss and cochlear pathology but the extent of vestibular dysfunction and organ pathology are less well characterized. To understand the vestibular effects of Cx26 mutations, we evaluated vestibular function and histology of the vestibular sensory epithelia in a conditional knockout (CKO) mouse with Cx26 loss of function. Transgenic C57BL/6 mice, in which cre-Sox10 drives excision of the Cx26 gene from non-sensory cells flanking the sensory epithelium of the inner ear (Gjb2-CKO), were compared to age-matched wild types. Animals were sacrificed at ages between 4 and 40 weeks and their cochlear and vestibular sensory organs harvested for histological examination. Cx26 immunoreactivity was prominent in the peripheral vestibular system and the cochlea of wild type mice, but absent in the Gjb2-CKO specimens. The hair cell population in the cochleae of the Gjb2-CKO mice was severely depleted but in the vestibular organs it was intact, despite absence of Cx26 expression. The vestibular organs appeared normal at the latest time point examined, 40 weeks. To determine whether compensation by another connexin explains survival of the normal vestibular sensory epithelium, we evaluated the presence of Cx30 in the Gjb2-CKO mouse. We found that Cx30 labeling was normal in the cochlea, but it was decreased or absent in the vestibular system. The vestibular phenotype of the mutants was not different from wild-types as determined by time on the rotarod, head stability tests and physiological responses to vestibular stimulation. Thus presence of Cx30 in the cochlea does not compensate for Cx26 loss, and the absence of both connexins from vestibular sensory epithelia is no more injurious than the absence of one of them. Further studies to uncover the physiological foundation for this difference between the cochlea and the vestibular organs may help in designing treatments for GJB2 mutations., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Meta-analyses offer a rigorous and transparent systematic framework for synthesizing data that can be used for a wide range of research areas, study designs, and data types. Both the outcome of meta-analyses and the meta-analysis process itself can yield useful insights for answering scientific questions and making policy decisions. Development of the National Ambient Air Quality Standards illustrates many potential applications of meta-analysis. These applications demonstrate the strengths and limitations of meta-analysis, issues that arise in various data realms, how meta-analysis design choices can influence interpretation of results, and how meta-analysis can be used to address bias and heterogeneity. Reviewing available data from a meta-analysis perspective can provide a useful framework and impetus for identifying and refining strategies for future research. Moreover, increased pervasiveness of a meta-analysis mindset-focusing on how the pieces of the research puzzle fit together-would benefit scientific research and data syntheses regardless of whether or not a quantitative meta-analysis is undertaken. While an individual meta-analysis can only synthesize studies addressing the same research question, the results of separate meta-analyses can be combined to address a question encompassing multiple data types. This observation applies to any scientific or policy area where information from a variety of disciplines must be considered to address a broader research question., (© 2015 Society for Risk Analysis.)

The inhalation unit risk (IUR) that currently exists in the United States Environmental Protection Agency's (US EPA's) Integrated Risk Information System was developed in 1984 based on studies examining the relationship between respiratory cancer and arsenic exposure in copper smelters from two US locations: the copper smelter in Anaconda, Montana, and the American Smelting And Refining COmpany (ASARCO) smelter in Tacoma, Washington. Since US EPA last conducted its assessment, additional data have become available from epidemiology and mechanistic studies. In addition, the California Air Resources Board, Texas Commission of Environmental Quality, and Dutch Expert Committee on Occupational Safety have all conducted new risk assessments. All three analyses, which calculated IURs based on respiratory/lung cancer mortality, generated IURs that are lower (i.e., less restrictive) than the current US EPA value of 4.3×10(-3) (μg/m(3))(-1). The IURs developed by these agencies, which vary more than 20-fold, are based on somewhat different studies and use different methodologies to address uncertainties in the underlying datasets. Despite these differences, all were developed based on a cumulative exposure metric assuming a low-dose linear dose-response relationship. In this paper, we contrast and compare the analyses conducted by these agencies and critically evaluate strengths and limitations inherent in the data and methodologies used to develop quantitative risk estimates. In addition, we consider how these data could be best used to assess risk at much lower levels of arsenic in air, such as those experienced by the general public. Given that the mode of action for arsenic supports a threshold effect, and epidemiological evidence suggests that the arsenic concentration in air is a reliable predictor of lung/respiratory cancer risk, we developed a quantitative cancer risk analysis using a nonlinear threshold model. Applying a nonlinear model to occupational data, we established points of departure based on both cumulative exposure (μg/m(3)-years) to arsenic and arsenic concentration (μg/m(3)) via inhalation. Using these values, one can assess the lifetime risk of respiratory cancer mortality associated with ambient air concentrations of arsenic for the general US population., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Anti-factor D (AFD; FCFD4514S, lampalizumab) is a humanized IgG Fab fragment directed against factor D (fD), a rate-limiting serine protease in the alternative complement pathway (AP). Evaluation of AFD as a potential intravitreal (IVT) therapeutic for dry age-related macular degeneration patients with geographic atrophy (GA) is ongoing. However, it is unclear whether IVT administration of AFD can affect systemic AP activation and potentially compromise host-immune responses. We characterized the pharmacologic properties of AFD and assessed the effects of AFD administered IVT (2 or 20 mg) or intravenous (0.2, 2, or 20 mg) on systemic complement activity in cynomolgus monkeys. For the IVT groups, serum AP activity was reduced for the 20 mg dose group between 2 and 6 hours postinjection. For the intravenous groups, AFD inhibited systemic AP activity for periods of time ranging from 5 minutes (0.2 mg group) to 3 hours (20 mg group). Interestingly, the concentrations of total serum fD increased up to 10-fold relative to predose levels following administration of AFD. Furthermore, AFD was found to inhibit systemic AP activity only when the molar concentration of AFD exceeded that of fD. This occurred in cynomolgus monkeys at serum AFD levels ≥2 µg/ml, a concentration 8-fold greater than the maximum serum concentration observed following a single 10 mg IVT dose in a clinical investigation in patients with GA. Based on these findings, the low levels of serum AFD resulting from IVT administration of a clinically relevant dose are not expected to appreciably affect systemic AP activity., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

microRNAs (miRNAs) are regulators of differentiation and development of inner ear cells. Mutations in miRNAs lead to deafness in humans and mice. Among inner ear pathologies, inflammation may lead to structural and neuronal defects and eventually to hearing loss and vestibular dysfunction. While the genetic factors of these pathways have not been defined, autoimmunity participates in these processes. We report that inflammatory stimuli in the inner ear induce activation of the innate immune system via miR-224 and pentraxin 3 (Ptx3). miR-224 is a transcriptional target of nuclear factor κB, a key mediator of innate immunity. Ptx3 is a regulator of the immune response. It is released in response to inflammation and regulated by nuclear factor κB. We show that miR-224 and Ptx3 are expressed in the inner ear and we demonstrate that miR-224 targets Ptx3. As a model of the innate immune response, we injected lipopolysaccharide into the scala tympani of mouse inner ears. This resulted in changes in the levels of miR-224 and Ptx3, in addition to activation of the complement system, as measured by immune cell infiltration and activated C3. This suggests that while miR-224 regulates Ptx3 under normal conditions, upon inflammation, both are recruited to offer a front line of defense in acting as responders to inflammation in the inner ear. miR-224 diminishes the innate immune response by down-regulating Ptx3 expression, while Ptx3 stimulates the innate immune response. An understanding of the molecular components of the inflammatory pathway may help develop therapeutics for reducing inflammation associated with inner ear injury., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media.

Mutations in the connexin 26 gene (GJB2) are the most common genetic cause of deafness, leading to congenital bilateral non-syndromic sensorineural hearing loss. Here we report the generation of a mouse model for a connexin 26 (Cx26) mutation, in which cre-Sox10 drives excision of the Cx26 gene from non-sensory cells flanking the auditory epithelium. We determined that these conditional knockout mice, designated Gjb2-CKO, have a severe hearing loss. Immunocytochemistry of the auditory epithelium confirmed absence of Cx26 in the non-sensory cells. Histology of the organ of Corti and the spiral ganglion neurons (SGNs) performed at ages 1, 3, or 6 months revealed that in Gjb2-CKO mice, the organ of Corti began to degenerate in the basal cochlear turn at an early stage, and the degeneration rapidly spread to the apex. In addition, the density of SGNs in Rosenthal's canal decreased rapidly along a gradient from the base of the cochlea to the apex, where some SGNs survived until at least 6 months of age. Surviving neurons often clustered together and formed clumps of cells in the canal. We then assessed the influence of brain derived neurotrophic factor (BDNF) gene therapy on the SGNs of Gjb2-CKO mice by inoculating Adenovirus with the BDNF gene insert (Ad.BDNF) into the base of the cochlea via the scala tympani or scala media. We determined that over-expression of BDNF beginning around 1 month of age resulted in a significant rescue of neurons in Rosenthal's canal of the cochlear basal turn but not in the middle or apical portions. This data may be used to design therapies for enhancing the SGN physiological status in all GJB2 patients and especially in a sub-group of GJB2 patients where the hearing loss progresses due to ongoing degeneration of the auditory nerve, thereby improving the outcome of cochlear implant therapy in these ears., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We reported in 2003 that exposure to metals on laundered shop towels (LSTs) could exceed toxicity criteria. New data from LSTs used by workers in North America document the continued presence of metals in freshly laundered towels. We assessed potential exposure to metals based on concentrations of metals on the LSTs, estimates of LST usage by employees, and the transfer of metals from LST-to-hand, hand-to-mouth, and LST-to-lip, under average- or high-exposure scenarios. Exposure estimates were compared to toxicity criteria. Under an average-exposure scenario (excluding metals' data outliers), exceedances of the California Environmental Protection Agency, U.S. Environmental Protection Agency, and the Agency for Toxic Substances and Disease Registry toxicity criteria may occur for aluminum, cadmium, cobalt, copper, iron, and lead. Calculated intakes for these metals were up to more than 400-fold higher (lead) than their respective toxicity criterion. For the high-exposure scenario, additional exceedances may occur, and high-exposure intakes were up to 1,170-fold higher (lead) than their respective toxicity criterion. A sensitivity analysis indicated that alternate plausible assumptions could increase or decrease the magnitude of exceedances, but were unlikely to eliminate certain exceedances, particularly for lead.

Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of (125)Iodide and (111)Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.

Onartuzumab is a unique, humanized, monovalent (one-armed) monoclonal antibody (mAb) against the MET receptor. The intravenous (IV) pharmacokinetics (PK) of onartuzumab were investigated in a phase I study and a phase II study in recurrent non-small cell lung cancer (NSCLC) patients. The potential for drug-drug interaction (DDI) was assessed during co-administration of IV onartuzumab with oral erlotinib, by measuring the PK of both drugs. The concentration-time profiles of onartuzumab were adequately described using a two-compartment model with linear clearance (CL) at doses between 4 and 30 mg/kg. The estimates for CL, central compartment volume (V1 ), and median terminal half-life were 0.439 L/day, 2.77 L, and 13.4 days, respectively. Statistically significant covariates included creatinine clearance (CrCL) on clearance, weight and gender on V1 , and weight on peripheral compartment volume (V2 ), but the clinical relevance of these covariates needs to be further evaluated. The current analysis did not indicate obvious DDI between onartuzumab and erlotinib. MET diagnostic status did not impact the exposure of either agent. Despite the slightly faster clearance compared with typical bivalent mAbs, the PK of onartuzumab support dosing regimens of 15 mg/kg every 3 weeks or doses equivalent to achieve the target minimum tumoristatic concentration in patients., (© 2013, The American College of Clinical Pharmacology.)

Purpose: We characterized the pharmacokinetics of onartuzumab (MetMAb) in animals and determined a concentration-effect relationship in tumor-bearing mice to enable estimation of clinical pharmacokinetics and target doses., Experimental Design: A tumor growth inhibition model was used to estimate tumoristatic concentrations (TSC) in mice. Human pharmacokinetic parameters were projected from pharmacokinetics in cynomolgus monkeys by the species-invariant time method. Monte Carlo simulations predicted the percentage of patients achieving steady-state trough serum concentrations (Ctrough ss) ≥TSC for every 3-week (Q3W) dosing., Results: Onartuzumab clearance (CL) in the linear dose range was 21.1 and 12.2 mL/d/kg in mice and cynomolgus monkeys with elimination half-life at 6.10 and 3.37 days, respectively. The estimated TSC in KP4 pancreatic xenograft tumor-bearing mice was 15 μg/mL. Projected CL for humans in the linear dose range was 5.74 to 9.36 mL/d/kg with scaling exponents of CL at 0.75 to 0.9. Monte Carlo simulations projected a Q3W dose of 10 to 30 mg/kg to achieve Ctrough ss of 15 μg/mL in 95% or more of patients., Conclusions: Onartuzumab pharmacokinetics differed from typical bivalent glycosylated monoclonal antibodies with approximately 2-times faster CL in the linear dose range. Despite this higher CL, xenograft efficacy data supported dose flexibility with Q1W to Q3W dose regimens in the clinical setting with a TSC of 15 μg/mL as the Ctrough ss target. The projected human efficacious dose of 10 to 30 mg/kg Q3W should achieve the target TSC of 15 μg/mL. These data show effective pharmacokinetic/pharmacodynamic modeling to project doses to be tested in the clinic., (©2013 AACR.)

Purpose: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities., Experimental Design: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities., Results: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12β, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats., Conclusion: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans., (©2013 AACR.)

Purpose: PRO95780, a human monoclonal antibody (mAb) against death receptor 5 (DR5/TRAIL-R2/TNFRSF10B), was developed for the treatment for cancer. Our objective was to characterize pharmacokinetics (PK) in mice, rats, and cynomolgus monkeys and concentration-effect relationships of PRO95780 in xenograft mouse models of human cancers; this would guide the selection of dose and regimen for clinical trials., Methods: The PK profiles were determined in mice, rats, and cynomolgus monkeys. Three xenograft models with a wide range of in vitro sensitivities to PRO95780 were selected for efficacy studies. Tumoristatic serum concentrations (TSCs) were determined using PK/pharmacodynamic (PD) modeling with tumor growth as a PD endpoint. A species-invariant time PK scaling method was employed to estimate disposition in humans using PK data in cynomolgus monkeys. Furthermore, the predicted human PK parameters were used to estimate dose and regimen to achieve TSC observed in mice at the steady-state trough concentrations (C trough ss) in the clinic., Results: Linear PK was observed across species. A serum concentration of 22 μg/mL was identified to be the target TSC in mice. A dose of 10 mg/kg administered once every 2 weeks (Q2W) was predicted to achieve a TSC at C trough ss in 95 % of patients., Conclusions: PRO95780 has linear PK in mice, rats, and monkeys. Estimated TSCs varied among different xenograft models. A projected target dose in humans is achievable for Q2W administration within the dose range used for other commercial mAbs.

Purpose: To characterize ranibizumab pharmacokinetics in patients with AMD., Methods: A population approach of nonlinear mixed-effect pharmacokinetic modeling based on concentration-time data from 2993 serum samples from 674 AMD patients enrolled in 5 phase 1 to 3 clinical trials of single or multiple intravitreal (ITV) doses of ranibizumab (0.3-2.0 mg/eye) administered biweekly or monthly for up to 24 months., Results: A TOTAL OF 696 CONCENTRATION-TIME RECORDS FROM 229 SUBJECTS WITH ONE OR MORE MEASURABLE TOTAL SERUM RANIBIZUMAB CONCENTRATIONS WERE ANALYZED. THE SYSTEMIC CONCENTRATION-TIME DATA FOR RANIBIZUMAB WERE BEST DESCRIBED BY A ONE-COMPARTMENT MODEL WITH FIRST-ORDER ABSORPTION INTO AND FIRST-ORDER ELIMINATION FROM THE SYSTEMIC CIRCULATION. VITREOUS ELIMINATION HALF-LIFE (T1/2) WAS CALCULATED TO BE 9 DAYS AND THE INTRINSIC SYSTEMIC ELIMINATION T1/2 WAS CALCULATED TO BE APPROXIMATELY 2 HOURS. FOLLOWING ITV ADMINISTRATION, RANIBIZUMAB EGRESSES SLOWLY INTO THE SYSTEMIC CIRCULATION, RESULTING IN AN APPARENT SERUM T1/2 OF 9 DAYS. SYSTEMIC-TO-VITREOUS EXPOSURE RATIO WAS ESTIMATED TO BE 1: 90,000. With monthly and quarterly ITV regimens, the serum concentrations of ranibizumab at steady-state for both the 0.3 and 0.5 mg/eye dose levels were estimated to be below the range needed to inhibit VEGF-A-induced endothelial cell proliferation in vitro by 50% at all times., Conclusions: Systemic exposure to ranibizumab after ITV injection was very low due to elimination on reaching systemic circulation from the vitreous. Population pharmacokinetic analysis of data from a representative sample of AMD patients did not identify clinically significant sources or correlates of variability in ranibizumab exposure. (ClinicalTrials.gov numbers, NCT00056836, NCT00056823.).

The human deafness-pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia-White (Mitf(Mi-wh) /+) mice were studied and hearing function of these mice characterized. Mitf(Mi-wh) /+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf(Mi-wh) /+ embryos have fewer melanoblasts during embryonic development than their wild-type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf(Mi-wh) /+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness-pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes., (© 2012 John Wiley & Sons A/S.)

Purpose: To compare the pharmacokinetics (PK) of MNRP1685A, a human monoclonal antibody (mAb) against neuropilin-1 (NRP1), in mice, rats, monkeys, and cancer patients from a Phase I study to model with parallel linear and nonlinear clearances., Methods: Binding characteristics of MNRP1685A in different species were evaluated using surface plasmon resonance technology. PK profiles of MNRP1685A after single and/or multiple doses in different species were analyzed using population analysis. PK parameters were compared across species., Results: MNRP1685A binds to NRP1 in all four species tested. Consistent with the wide expression of NRP1, MNRP1685A demonstrated pronounced non-linear PK over a wide dose range. PK profiles are best described by a two-compartment model with parallel linear and nonlinear clearances. Model-derived PK parameters suggest similar in-vivo target expression levels and binding affinity to target across all species tested. However, compared to typical human/humanized mAbs, non-specific clearance of MNRP1685A was faster in mice, rats, and humans (60.3, 19.4, and 8.5 ml/day/kg), but not in monkeys (3.22 ml/day/kg)., Conclusions: Monkey PK properly predicted the target-mediated clearance of MNRP1685A but underestimated its non-specific clearance in humans. This unique PK property warrants further investigation of underlying mechanisms.

MNRP1685A (anti-NRP1) is a fully human IgG1 monoclonal antibody against neuropilin-1 (NRP1), a protein necessary for blood vessel maturation. MNRP1685A binds to free membrane-bound NRP1 (mNRP1) and circulating NRP1 (cNRP1). Total cNRP1 increased in a dose-dependent manner following anti-NRP1 administration in mice, rats, and monkeys. The purpose of this study is to develop a mechanism-based model to simultaneously describe the kinetics of both unbound drug (MNRP1685A) and total cNRP1 in cynomolgus monkeys. Pharmacokinetic (PK) and pharmacodynamic (PD) profiles after single- or multiple-dose administrations were well described by the two-target quasi-steady-state (QSS) model. The estimated nonspecific clearance was 3.26 mL/day/kg and central compartment volume was 38.2 mL/kg. The maximum elimination rate for mNRP1-mediated disposition was 98.8 nM/day. The synthesis rate (3.8 nM/day), degradation rate constant (1.53 day(-1)), and complex elimination rate constant (0.260 day(-1)) for cNRP1 were also derived from the model. QSS constants were 6.94 nM for mNRP1 and 2.8 nM for cNRP1. The results suggest that cNRP1 has minimal effect on MNRP1685A PK while mNRP1 plays a major role in the target-mediated drug disposition. This finding is favorable as the desired pharmacological target is mNRP1, rather than cNRP1. The two-target QSS model provides mechanistic understanding of the nonlinear PK of MNRP1685A. Based on the model prediction, the free drug concentrations to maintain free mNRP1 and cNRP1 below 10% of baseline level are 10 and 20 μg/mL, respectively. This serves as a target concentration for clinical dose selection, assuming cynomolgus monkeys are predictive for humans.

Background and Objective: Dosing of therapeutic monoclonal antibodies (mAbs) is often based on body size, with the perception that body size-based dosing would reduce inter-subject variability in drug exposure. However, most mAbs are target specific with a relatively large therapeutic window and generally a small contribution of body size to pharmacokinetic variability. Therefore, the dosing paradigm for mAbs should be assessed in the context of these unique characteristics. The objective of this study was to review the current dosing strategy and to provide a scientific rationale for dosing of mAbs using a modelling and simulation approach., Methods: In this analysis, the body weight-based or body weight-independent (fixed) dosing regimens for mAbs were systematically evaluated. A generic two-compartment first-order elimination model was developed. Individual or population pharmacokinetic profiles were simulated as a function of the body weight effects on clearance (θ(BW&#95;CL)) and on the central volume of distribution (θ(BW&#95;V1)). The variability in exposure (the area under the serum concentration-time curve [AUC], trough serum concentration [C(min)] and peak serum concentration [C(max)]) was compared between body weight-based dosing and fixed dosing in the entire population. The deviation of exposure for light and heavy subjects from median body weight subjects was also measured. The simulation results were then evaluated with clinical pharmacokinetic characteristics of various mAbs that were given either by body weight-based dosing or by fixed dosing in the case study., Results: Results from this analysis demonstrated that exposure variability was dependent on the magnitude of the body weight effect on pharmacokinetics. In contrast to the conventional assumption, body weight-based dosing does not always offer advantages over fixed dosing in reducing exposure variability. In general, when the exponential functions of θ(BW&#95;CL) and θ(BW&#95;V1) in the population pharmacokinetic model are <0.5, fixed dosing results in less variability and less deviation than body weight-based dosing; when both θ(BW&#95;CL) and θ(BW&#95;V1) are >0.5, body weight-based dosing results in less variability and less deviation than fixed dosing. In the scenarios when either θ(BW&#95;CL) or θ(BW&#95;V1) is >0.5, the impact on exposure variability is different for each exposure measure. The case study demonstrated that most mAbs had little effect or a moderate body weight effect (θ(BW&#95;CL) and θ(BW&#95;V1) <0.5 or ∼0.5). The difference of variability in exposure between body weight-based and fixed dosing is generally less than 20% and the percentages of deviation for light and heavy subpopulations are less than 40%., Conclusions: The analysis provided insights into the conditions under which either fixed or body weight-based dosing would be superior in reducing pharmacokinetic variability and exposure differences between light and heavy subjects across the population. The pharmacokinetic variability introduced by either dosing regimen is moderate relative to the variability generally observed in pharmacodynamics, efficacy and safety. Therefore, mAb dosing can be flexible. Given many practical advantages, fixed dosing is recommended to be the first option in first-in-human studies with mAbs. The dosing strategy in later stages of clinical development could then be determined based on combined knowledge of the body weight effect on pharmacokinetics, safety and efficacy from the early clinical trials.

Current therapy for patients with hereditary absence of cochlear hair cells, who have severe or profound deafness, is restricted to cochlear implantation, a procedure that requires survival of the auditory nerve. Mouse mutations that serve as models for genetic deafness can be utilized for developing and enhancing therapies for hereditary deafness. A mouse with Pou4f3 loss of function has no hair cells and a subsequent, progressive degeneration of auditory neurons. Here we tested the influence of neurotrophin gene therapy on auditory nerve survival and peripheral sprouting in Pou4f3 mouse ears. BDNF gene transfer enhanced preservation of auditory neurons compared to control ears, in which nearly all neurons degenerated. Surviving neurons in treated ears exhibited pronounced sprouting of nerve fibers into the auditory epithelium, despite the absence of hair cells. This enhanced nerve survival and regenerative sprouting may improve the outcome of cochlear implant therapy in patients with hereditary deafness.

Purpose: Oxidized low-density lipoprotein (LDL) plays an essential role in the pathogenesis of atherosclerosis. The purpose of this study was to characterize the pharmacokinetics (PK) of a human recombinant IgG1 antibody to oxidized LDL (anti-oxLDL) in cynomolgus monkey. The tissue biodistribution of anti-oxLDL was also investigated using positron emission tomography (PET) imaging., Methods: Anti-oxLDL was conjugated with the N-hydroxysuccinimide ester of DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid) and radiolabeled by chelation of radioactive copper-64 ((64)Cu) for detection by PET. Anti-oxLDL was administered as a single intravenous (IV) dose of 10 mg/kg (as a mixture of radiolabeled and non-labeled material) to two male and two female cynomolgus monkeys. Serum samples were collected over 29 days. Two ELISA methods were used to measure serum concentrations of anti-oxLDL; Assay A was a ligand binding assay that measured free anti-oxLDL (unbound and partially bound forms) and Assay B measured total anti-oxLDL. The biodistribution was observed over a 48-hour period following dose administration using PET imaging., Results: Anti-oxLDL serum concentration-time profiles showed a biphasic elimination pattern that could be best described by a two-compartment elimination model. The serum concentrations obtained using the two ELISA methods were comparable. Clearance values ranged from 8 to 17 ml/day/kg, while beta half-life ranged from 8 to 12 days. The initial volume of distribution and volume of distribution at steady state were approximately 55 mL/kg and 150 mL/kg, respectively. PET imaging showed distribution predominantly to the blood pool, visible as the heart and great vessels in the trunk and limbs, plus diffuse signals in the liver, kidney, spleen, and bone marrow., Conclusions: The clearance of anti-oxLDL is slightly higher than typical IgG1 antibodies in cynomolgus monkeys. The biodistribution pattern appears to be consistent with an antibody that has no large, rapid antigen sink outside the blood space.

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7(Gt)(/+) mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7(Gt)(/+) mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7(Gt)(/+) mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7(Gt)(/+) mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The membrane glycoprotein CTL2/SLC44A2 is expressed by supporting cells in the inner ear and has been identified as a target of antibodies that may induce auto-immune hearing loss. To determine if CTL2/SLC44A2 also has roles in inner ear development and to distinguish between isoform-specific roles, we assessed age-related changes in expression of CTL2/SLC44A2 isoforms and protein in the developing murine inner ear. We determined that both isoform p1 and isoform p2 (named for the upstream p1 and proximal p2 promoters that control alternate exons 1a and 1b) were robustly expressed as early as E14 and persisted during embryonic development, but after birth the p1 isoform fell to barely detectable levels while isoform p2 levels were maintained. This trend continued and became even more apparent later in post-natal development and remained in mature ears until at least 6 weeks of age. In aged (18 mo old) mice, the level of isoform p1 transcripts rose again to levels similar to the p2 isoform like that seen early in development. At the earliest stage examined, CTL2/SLC44A2 protein was expressed in both immature supporting cells and immature sensory cells, but after birth expression in the sensory cells declined in both the utricle and cochlea and by day P1 expression of CTL2/SLC44A2 was restricted to supporting cells. The changes we observed in isoform distribution are indicative of differential developmental roles and age related changes between the two isoforms of CTL2/SLC44A2 in the inner ear., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The animal testing protocols used today to evaluate the carcinogenicity of chemicals are very different from those used in the earlier part of the 20th century. To explore how cancer bioassays have changed over time, we surveyed the literature discussing test design and interpretation from the 1930s to the present. We also analyzed compendia of bioassays published by the US Public Health Service (US PHS) from 1938 to 1978, and evaluated the data to understand the evolution of testing methodology (e.g., animals used, test duration) and the types of chemicals being studied. The cancer bioassay evolved in several stages. At the beginning of the 20th century, animal bioassays were primarily used to re-create known human diseases, whereas in the 1940s to 1960s, animal bioassays were largely used to evaluate the safety of chemicals in foods, drugs, and cosmetics. Beginning in the late 1960s and 1970s, chemicals primarily associated with occupational or environmental exposures were also evaluated. Testing strategies now emphasize a suite of tests including multiple in vitro tests and both short-term and long-term animal tests. The objectives of testing are broader, too, with test goals encompassing information regarding mode of action and other parameters aimed at evaluating potential species differences (e.g., in toxicokinetics) and their relevance for evaluating human risks. It is important to consider this evolution when evaluating the testing methodology and scientific conclusions in earlier eras. As toxicology continues to develop, testing methods will continue to change in concert with increased knowledge and understanding., (© 2011 Informa Healthcare USA, Inc.)

Sensory organs typically use receptor cells and afferent neurons to transduce environmental signals and transmit them to the CNS. When sensory cells are lost, nerves often regress from the sensory area. Therapeutic and regenerative approaches would benefit from the presence of nerve fibers in the tissue. In the hearing system, retraction of afferent innervation may accompany the degeneration of auditory hair cells that is associated with permanent hearing loss. The only therapy currently available for cases with severe or complete loss of hair cells is the cochlear implant auditory prosthesis. To enhance the therapeutic benefits of a cochlear implant, it is necessary to attract nerve fibers back into the cochlear epithelium. Here we show that forced expression of the neurotrophin gene BDNF in epithelial or mesothelial cells that remain in the deaf ear induces robust regrowth of nerve fibers towards the cells that secrete the neurotrophin, and results in re-innervation of the sensory area. The process of neurotrophin-induced neuronal regeneration is accompanied by significant preservation of the spiral ganglion cells. The ability to regrow nerve fibers into the basilar membrane area and protect the auditory nerve will enhance performance of cochlear implants and augment future cell replacement therapies such as stem cell implantation or induced transdifferentiation. This model also provides a general experimental stage for drawing nerve fibers into a tissue devoid of neurons, and studying the interaction between the nerve fibers and the tissue., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.

An increased Leydig cell tumor (LCT) incidence has been reported in a study of Sprague-Dawley (SD) rats exposed via gavage to 1000 (but not 250)mg/kgday MTBE; it is unclear, however, if this finding was indeed dose-related or due to the statistical analyses not having adequately accounted for the increased survival rate in the high-dose animals and/or for multiple statistical comparisons. To address this question, we conducted Hoel-Walburg and Poly-3 analyses, using p-values of 0.01 for pair-wise comparisons and 0.005 for trend tests of common tumors. We found that MTBE does not cause a statistically significant increase in LCTs in SD rats when survival is appropriately taken into account. In addition, the original study reported some overall survival data, but did not specify which rats had LCTs. This led us to conduct separate Poly-3 analyses for the most extreme scenarios of survival age and tumor incidence to provide an illustrative example of approaches for analyzing the impact of survival rates on tumor findings in the absence of animal-specific survival data. We found this method to provide results similar to analyses using the actual data, suggesting that it can be used when full survival data are not available.

CHD7 is a chromodomain gene mutated in CHARGE syndrome, a multiple anomaly condition characterized by ocular coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, and ear defects including deafness and semicircular canal dysgenesis. Mice with heterozygous Chd7 deficiency have circling behavior and semicircular canal defects and are an excellent animal model for exploring the pathogenesis of CHARGE features. Inner ear vestibular defects have been characterized in heterozygous Chd7-deficient embryos and early postnatal mice, but it is not known whether vestibular defects persist throughout adulthood in Chd7-deficient mice or whether the vestibular sensory epithelia and their associated innervation and function are intact. Here we describe a detailed analysis of inner ear vestibular structures in mature mice that are heterozygous for a Chd7-deficient, gene-trapped allele (Chd7(Gt/+)). Chd7(Gt/+) mice display variable asymmetric lateral and posterior semicircular canal malformations, as well as defects in vestibular sensory epithelial innervation despite the presence of intact hair cells in the target organs. These observations have important functional implications for understanding the clinical manifestations of CHD7 mutations in humans and for designing therapies to treat inner ear vestibular dysfunction.