No virologic resistance to bulevirtide monotherapy detected in patients through 24 weeks treatment in phase II and III clinical trials for chronic hepatitis delta.

BACKGROUND & AIMS: Bulevirtide (BLV) is a HDV/HBV entry inhibitor that is associated with virologic response (responders, HDV-RNA undetectable or ≥2 log10 IU/ml decrease from baseline) in >50% of patients after a 24-week treatment. However, some patients only achieve a <1 log10 IU/ml decline in HDV-RNA after the 24-week treatment (non-responders). Here, we report a viral resistance analysis in participants receiving BLV monotherapy who were non-responders or experienced virologic breakthrough (VB, i.e., two consecutive increases in HDV-RNA of ≥1 log10 IU/ml from nadir or two consecutive HDV-RNA detectable results if previously undetectable) from the phase II MYR202 and phase III MYR301 study. METHODS: Deep-sequencing of the BLV-corresponding region in HBV PreS1 and of the HDV HDAg gene, as well as in vitro phenotypic testing, were performed for the participant with VB (n = 1) and non-responders (n = 20) at baseline (BL) and Week 24 (WK24). RESULTS: No amino acid exchanges associated with reduced susceptibility to BLV within the BLV-corresponding region or within HDAg were identified in isolates from any of the 21 participants at BL or at WK24. Although variants (HBV n = 1; HDV n = 13) were detected at BL in some non-responders or in the participant with VB, none were associated with reduced sensitivity to BLV in vitro. Furthermore, the same variant was detected in virologic responders. A comprehensive phenotypic analysis demonstrated that the BLV EC50 values from 116 BL samples were similar across non-responders, partial responders (HDV RNA decline ≥1 but <2 log10 IU/ml), and responders regardless of the presence of HBV and/or HDV polymorphisms. CONCLUSIONS: No amino acid substitutions associated with reduced sensitivity to BLV monotherapy were detected at BL or WK24 in non-responders or the participant with VB after 24-week BLV treatment. IMPACT AND IMPLICATIONS: This is the first study investigating the development of resistance in patients treated with BLV. Excluding resistance to BLV as an explanation for an insufficient decrease in HDV-RNA levels during BLV therapy is an important finding for patients, clinicians, and researchers. It demonstrates that BLV has a high barrier to resistance, indicating it is safe and suitable for long-term treatment, although long-term surveillance for resistance should be performed. Our results hint at other still unknown mechanisms as an explanation for the persistence of serum HDV-RNA during inhibition of viral entry. CLINICAL TRIAL NUMBERS: NCT03546621 and NCT03852719.

Establishment and Characterization of an HBV Viral Spread and Infectious System following Long-Term Passage of an HBV Clinical Isolate in the Primary Human Hepatocyte and Fibroblast Coculture System.

The existing cell culture-based methods to study hepatitis B virus (HBV) have limitations and do not allow for viral long-term passage. The aim of this study was to develop a robust in vitro long-term viral passage system with optimized cell culture conditions and a viral isolate with the ability to spread and passage. An HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized primary human hepatocyte (PHH)/human fibroblast coculture system. The passaged UV-treated virus was sequenced and further characterized. In addition, a panel of mutant viruses containing different combinations of mutations observed in this virus was investigated. The clinical isolate was passaged for 20 rounds with 21 days per round in an optimized PHH/human fibroblast coculture system while subject to UV mutagenesis. This passaged UV-mutated isolate harbored four mutations: G225A (sR24K) in the S gene, A2062T in the core gene, and two mutations G1764A and C1766T (xV131I) in the basal core promoter (BCP) region. In vitro characterization of the four mutations suggested that the two BCP mutations G1764A and C1766T contributed to the increased viral replication and viral infectivity. A robust in vitro long-term HBV viral passage system has been established by passaging a UV-treated clinical isolate in an optimized PHH/fibroblast coculture system. The two BCP mutations played a key role in the virus's ability to passage. This passage system can be used for studying the entire life cycle of HBV and has the potential for in vitro drug-resistance selection upon further optimization. IMPORTANCE The existing cell culture-based methods to study HBV have limitations and do not allow for viral long-term passage. In this study, an HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized PHH/human fibroblast coculture system. This passaged UV-mutated isolate carried four mutations across the HBV genome, and in vitro characterization of the four mutations suggested that the two basal core promoter (BCP) mutations G1764A and C1766T played a key role in the virus's ability to passage. In summary, we have developed a robust in vitro long-term HBV viral passage system by passaging an UV-treated HBV genotype A clinical isolate in an optimized PHH/human fibroblast coculture system. This passage system can be used for studying the entire life cycle of HBV and has the potential for in vitro drug-resistance selection upon further optimization.

Targeted Long-Read Sequencing Reveals Comprehensive Architecture, Burden, and Transcriptional Signatures from Hepatitis B Virus-Associated Integrations and Translocations in Hepatocellular Carcinoma Cell Lines.

Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden, and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5, and Hep3B. To generate high-coverage, long-read sequencing data, a custom panel of HBV-targeting biotinylated oligonucleotide probes was designed. Targeted long-read DNA sequencing captured entire HBV integration events within individual reads, revealing that integrations may include deletions and inversions of viral sequences. Surprisingly, all three HCC cell lines contain integrations that are associated with host chromosomal translocations. In addition, targeted long-read RNA sequencing allowed for the assignment of transcriptional activity to specific integrations and resolved the contribution of overlapping HBV transcripts. HBV transcripts chimeric with host sequences were resolved in their entirety and often included >1,000 bp of host sequence. This study provides the first comprehensive description of HBV integrations and associated transcriptional activity in three commonly utilized HCC-derived cell lines. The application of novel methods sheds new light on the complexity of these integrations, including HBV bidirectional transcription, nested transcripts, silent integrations, and host genomic rearrangements. The observation of multiple HBV-associated chromosomal translocations gives rise to the hypothesis that HBV is a driver of genetic instability and provides a potential new mechanism for HCC development. IMPORTANCE HCC-derived cell lines have served as practical models to study HBV biology for decades. These cell lines harbor multiple HBV integrations and express only HBV surface antigen (HBsAg). To date, an accurate description of the integration burden, architecture, and transcriptional profile of these cell lines has been limited due to technical constraints. We have developed a targeted long-read sequencing assay that reveals the entire architecture of integrations in these cell lines. In addition, we identified five chromosomal translocations with integrated HBV DNA at the interchromosomal junctions. Incorporation of long-read transcriptome sequencing (RNA-Seq) data indicated that many integrations and translocations were transcriptionally silent. The observation of multiple HBV-associated translocations has strong implications regarding the potential mechanisms for the development of HBV-associated HCC.

Developing a sensitive HBV genotyping assay for HBV DNA suppressed patients using both DNA and RNA sequencing.

Hepatitis B virus (HBV) genotypes impact treatment outcomes and disease progression. The current genotyping methods have limitations in patients with low HBV viral load. In this study, a more sensitive assay has been developed for determining the HBV genotype in HBV DNA suppressed patients. Fifty-five serum samples from 55 chronic hepatitis B patients (HBeAg-, n = 20; HBeAg+, n = 35) across genotypes A to H with long-term nucleos(t)ide analogs (NAs) treatment were collected. All samples had HBV DNA less than 29 IU/mL. Total nucleic acid (viral DNA and RNA) was extracted and a 341 bp amplicon located at HBV S gene overlapping with reverse transcriptase domain of polymerase (pol/RT) was amplified via real time (RT)-nested polymerase chain reaction (PCR) followed by population sequencing. HBV genotype was determined by phylogenetic analysis. The assay successfully amplified HBV S/RT gene from 53 of 55 (96.4%) patient serum samples. Phylogenetic analysis demonstrated that the genotypes of all the 53 PCR positive samples matched the historical genotypes as determined by INNO-LiPA or RT sequence from the corresponding baseline samples. This assay was able to accurately determine HBV genotype irrespective of baseline genotype, HBeAg status, or duration of viral suppression. The ability to determine genotype in virally suppressed patients may facilitate the evaluation of novel treatment agents for HBV in this patient population.

Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice.

Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.

Broad-spectrum activity of bulevirtide against clinical isolates of HDV and recombinant pan-genotypic combinations of HBV/HDV.

Background & Aims: Bulevirtide (BLV) is a small lipopeptide agent that specifically binds to the sodium taurocholate cotransporting polypeptide (NTCP) bile salt transporter and HBV/HDV receptor on the surface of human hepatocytes and inhibits HDV and HBV entry. As a satellite virus of HBV, HDV virions are formed after assembly of HDV RNA with the HBV envelope proteins (HBsAg). Because both viruses exist as eight different genotypes, this creates a potential for high diversity in the HBV/HDV combinations. To investigate the sensitivity of various combinations of HBV/HDV genotypes to BLV, clinical and laboratory strains were assessed. Methods: For the laboratory strains, the different envelopes from HBV genotypes A through H were combined with HDV genotypes 1-8 in cotransfection assays. Clinical plasma isolates were obtained from clinical studies and academic collaborations to maximise the diversity of HBV/HDV genotypes tested. Results: The mean BLV EC50 against HDV laboratory strains ranged from 0.44 to 0.64 nM. Regardless of HBV and HDV genotypes, the clinical isolates showed similar sensitivities to BLV with mean values that ranged from 0.2 to 0.73 nM. Conclusions: These data support the use of BLV in patients infected with any HBV/HDV genotypes. Impact and implications: This study describes the potent activity of BLV against multiple laboratory strains spanning all HBV/HDV A-H/1-8 genotype combinations and the most diverse collection of HDV clinical samples tested to date, including HBV/HDV genotype combinations less frequently observed in the clinic. Overall, all isolates and laboratory strains displayed similar in vitro nanomolar sensitivity to BLV. This broad-spectrum antiviral activity of BLV has direct implications on potential simplified treatment for any patient infected with HDV, regardless of genotype, and supports the new 2023 EASL Clinical Practice Guidelines on HDV that recommend antiviral treatment for all patients with CHD.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.

Background & Aims: HBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms. Methods: DNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline. Results: HBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations. Conclusions: Targeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC. Lay summary: Fresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

A genetic model of chronic rhinosinusitis-associated olfactory inflammation reveals reversible functional impairment and dramatic neuroepithelial reorganization.

Inflammatory sinus and nasal disease is a common cause of human olfactory loss. To explore the mechanisms underlying rhinosinusitis-associated olfactory loss, we have generated a transgenic mouse model of olfactory inflammation, in which tumor necrosis factor alpha (TNF-alpha) expression is induced in a temporally controlled manner specifically within the olfactory epithelium (OE). Like the human disease, TNF-alpha expression leads to a progressive infiltration of inflammatory cells into the OE. Using this model, we have defined specific phases of the pathologic process. An initial loss of sensation without significant disruption is observed, followed by a striking reorganization of the sensory neuroepithelium. An inflamed and disrupted state is sustained chronically by continued induction of cytokine expression. After prolonged maintenance in a deficient state, there is a dramatic recovery of function and a normal histologic appearance when TNF-alpha expression is extinguished. Although obstruction of airflow is also a contributing factor in human rhinosinusitis, this in vivo model demonstrates for the first time that direct effects of inflammation on OE structure and function are important mechanisms of olfactory dysfunction. These features mimic essential aspects of chronic rhinosinusitis-associated olfactory loss, and illuminate underlying cellular and molecular aspects of the disease. This manipulable model also serves as a platform for developing novel therapeutic interventions.

Cell-Specific Transcriptional Responses to Heat Shock in the Mouse Utricle Epithelium.

Sensory epithelia of the inner ear contain mechanosensory hair cells (HCs) and glia-like supporting cells (SCs), both of which are required for hearing and balance functions. Each of these cell types has unique responses to ototoxic and cytoprotective stimuli. Non-lethal heat stress in the mammalian utricle induces heat shock proteins (HSPs) and protects against ototoxic drug-induced hair cell death. Induction of HSPs in the utricle demonstrates cell-type specificity at the protein level, with HSP70 induction occurring primarily in SCs, while HSP32 (also known as heme oxygenase 1, HMOX1) is induced primarily in resident macrophages. Neither of these HSPs are robustly induced in HCs, suggesting that HCs may have little capacity for induction of stress-induced protective responses. To determine the transcriptional responses to heat shock of these different cell types, we performed cell-type-specific transcriptional profiling using the RiboTag method, which allows for immunoprecipitation (IP) of actively translating mRNAs from specific cell types. RNA-Seq differential gene expression analyses demonstrated that the RiboTag method identified known cell type-specific markers as well as new markers for HCs and SCs. Gene expression differences suggest that HCs and SCs exhibit differential transcriptional heat shock responses. The chaperonin family member Cct8 was significantly enriched only in heat-shocked HCs, while Hspa1l (HSP70 family), and Hspb1 and Cryab (HSP27 and HSP20 families, respectively) were enriched only in SCs. Together our data indicate that HCs exhibit a limited but unique heat shock response, and SCs exhibit a broader and more robust transcriptional response to protective heat stress.

Exosomes mediate sensory hair cell protection in the inner ear.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.

Reporting quality of music intervention research in healthcare: A systematic review.

INTRODUCTION: Concomitant with the growth of music intervention research, are concerns about inadequate intervention reporting and inconsistent terminology, which limits validity, replicability, and clinical application of findings. OBJECTIVE: Examine reporting quality of music intervention research, in chronic and acute medical settings, using the Checklist for Reporting Music-based Interventions. In addition, describe patient populations and primary outcomes, intervention content and corresponding interventionist qualifications, and terminology. METHODS: Searching MEDLINE, PubMed, CINAHL, HealthSTAR, and PsycINFO we identified articles meeting inclusion/exclusion criteria for a five-year period (2010-2015) and extracted relevant data. Coded material included reporting quality across seven areas (theory, content, delivery schedule, interventionist qualifications, treatment fidelity, setting, unit of delivery), author/journal information, patient population/outcomes, and terminology. RESULTS: Of 860 articles, 187 met review criteria (128 experimental; 59 quasi-experimental), with 121 publishing journals, and authors from 31 countries. Overall reporting quality was poor with <50% providing information for four of the seven checklist components (theory, interventionist qualifications, treatment fidelity, setting). Intervention content reporting was also poor with <50% providing information about the music used, decibel levels/volume controls, or materials. Credentialed music therapists and registered nurses delivered most interventions, with clear differences in content and delivery. Terminology was varied and inconsistent. CONCLUSIONS: Problems with reporting quality impedes meaningful interpretation and cross-study comparisons. Inconsistent and misapplied terminology also create barriers to interprofessional communication and translation of findings to patient care. Improved reporting quality and creation of shared language will advance scientific rigor and clinical relevance of music intervention research.

Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle.

Recruitment of endogenous progenitors is critical during tissue repair. The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here we show that HC damage in neonatal mouse utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized ß-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.

Sinonasal epithelial cells synthesize active vitamin D, augmenting host innate immune function.

BACKGROUND: Vitamin D, long recognized for its role in bone metabolism and calcium homeostasis, has been increasingly shown to augment innate immunity. 1-α-Hydroxylase, the enzyme responsible for the synthesis of active vitamin D, has been shown to have extrarenal expression in multiple cell types, including airway epithelial cells. The purpose of this study is to explore whether sinonasal epithelial cells (SNECs) express 1-α-hydroxylase, allowing for the local production of active vitamin D, thereby augmenting innate immune function. METHODS: Human SNECs were grown in culture and stimulated by inactive vitamin D. Expression of 1-α-hydroxylase was measured by real-time polymerase chain reaction and immunocytochemistry. Active vitamin D production was measured by enzyme-linked immunosorbent assay (ELISA). The expression of cathelicidin, an antimicrobial peptide, was measured by real-time polymerase chain reaction and immunocytochemistry. RESULTS: SNECs constitutively express the enzyme 1-α-hydroxylase resulting in active vitamin D production. SNECs exposed to inactive vitamin D had a significant 8-fold increase in cathelicidin expression when compared to controls. CONCLUSION: SNECs can generate active vitamin D, which significantly increases expression of the antimicrobial peptide cathelicidin. © 2013 ARS-AAOA, LLC.

Inner ear supporting cells protect hair cells by secreting HSP70.

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug-induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70-/- mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress.

Dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection.

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1). The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells. Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium. Supporting cells are also important mediators of hair cell survival and death. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

The role of TNF-α in inflammatory olfactory loss.

BACKGROUND: Despite the significant health impact of olfactory loss in chronic rhinosinusitis (CRS), the underlying pathophysiology is incompletely understood. A transgenic mouse model of olfactory inflammation induced by tumor necrosis factor-alpha (TNF-α) has provided new insights into the cellular and molecular basis of inflammatory olfactory loss. Here, we utilize systemic corticosteroids to suppress downstream cytokine expression, in order to study the direct role of TNF-α in CRS-associated olfactory dysfunction. METHODS: Transgenic mice were induced to express TNF-α in the olfactory epithelium for 6 weeks. In a subset of mice, 1 mg/kg prednisolone was administered concurrently to inhibit downstream inflammatory responses. The olfactory epithelium (OE) was analyzed by histology and electro-olfactogram (EOG) recordings. RESULTS: Treatment with prednisolone successfully prevented inflammatory infiltration over significant regions of the OE. In areas where significant subepithelial inflammation was present, a corresponding loss of olfactory neurons was observed. In contrast, areas without major inflammatory changes had normal olfactory neuron layers, despite chronic local expression of TNF-α. Prednisolone partially reversed the complete loss of olfaction in the mouse model, preserving odorant responses that were significantly diminished compared to controls, but not absent. CONCLUSIONS: The addition of prednisolone to the transgenic model of olfactory inflammation isolates the direct effects of induced TNF-α expression on the OE. The finding that prednisolone treatment prevents neuronal loss in some regions of the OE suggests that TNF-α does not directly cause neuronal apoptosis--rather, that subepithelial inflammation or other downstream mediators may be responsible. At the same time, EOG results imply that TNF-α directly causes physiologic dysfunction of olfactory neurons, independent of the inflammatory state. An understanding of the role of TNF-α and other inflammatory cytokines may suggest novel therapeutic strategies for CRS-associated olfactory loss.

Reversible loss of neuronal marker protein expression in a transgenic mouse model for sinusitis-associated olfactory dysfunction.

BACKGROUND: Chronic rhinosinusitis (CRS) is among the most common causes of olfactory loss. The loss of the sense of smell is thought to result from structural and functional changes occurring in the olfactory epithelium caused by inflammation. However, the cellular mechanisms underlying CRS-associated olfactory loss remain incompletely understood. METHODS: Transgenic mice expressing TNF-alpha specifically within the olfactory epithelium were used as a model for CRS-associated olfactory loss. TNF-alpha expression was induced over different time intervals, and olfactory epithelial tissue was assessed for the expression of neuronal markers by laser scanning confocal microscopy and Western blot. RESULTS: TNF-alpha expression results in an inflammatory infiltrate in the olfactory epithelium, thinning of the olfactory neuron layer, and a progressive loss of olfactory function. Reduced expression of markers for neurons and mature olfactory neurons (neural cell adhesion molecule [NCAM] and olfactory marker protein [OMP], respectively) was observed in the neuroepithelium and in the subepithelial axon bundles. Expression of growth-associated protein (GAP) 43, a marker for immature neurons, was also reduced. These alterations were reversed when TNF-alpha expression was discontinued. CONCLUSION: TNF-alpha expression in a transgenic model of CRS-associated olfactory loss results in progressive loss of olfactory neurons. Decreased GAP-43 expression suggests that TNF-alpha-associated inflammation inhibits differentiation of progenitor cells into immature olfactory neurons. Therefore, reduced regeneration of olfactory neurons may be an important mechanism underlying olfactory loss in CRS, in addition to neuronal loss or apoptosis. This mouse model represents a potential tool in the development of novel therapeutic strategies for the prevention of olfactory neuron loss in CRS.

Tumor necrosis factor alpha inhibits olfactory regeneration in a transgenic model of chronic rhinosinusitis-associated olfactory loss.

BACKGROUND: Olfactory loss is a debilitating symptom of chronic rhinosinusitis (CRS). Although olfactory sensory neurons (OSNs) are normally regenerated constantly in the olfactory epithelium (OE), a transgenic model of CRS-associated olfactory loss (inducible olfactory inflammation [IOI] mouse) shows that inflammation causes widespread OSN loss without progenitor cell proliferation. In this study, we further examine whether the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) inhibits olfactory regeneration. METHODS: IOI mice underwent either unilateral bulbectomy or sham surgery and then were induced to express TNF-alpha in the OE for 1 week. After death, the mice were assessed histologically and with bromodeoxyuridine staining to determine the effect of TNF-alpha on olfactory regeneration. RESULTS: In the absence of TNF-alpha, bulbectomy was associated with death of OSNs, followed by robust proliferation of neural progenitors and regrowth of the OE. At 12 days postbulbectomy, OE thickness on the operated side had recovered to >80% of the unoperated side. In mice in which TNF-alpha expression was induced, significantly reduced proliferation was observed, associated with failure of normal reconstitution of OE thickness. CONCLUSION: The mechanism of olfactory dysfunction in CRS remains incompletely understood. Previous studies with a transgenic mouse model suggested that inflammation inhibits progenitor cell proliferation and olfactory regeneration. Here, the role of the CRS-associated cytokine TNF-alpha was investigated using surgical ablation of the olfactory bulb to stimulate synchronous OSN turnover. We find that TNF-alpha expression prevents normal OE recovery, supporting the role of suppressed olfactory regeneration in the pathophysiology of CRS-associated olfactory loss.

The role of hepatocyte growth factor/c-Met in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Hepatocyte growth factor (HGF) is a growth factor thought to attenuate Th2-driven eosinophilic airway inflammatory responses. Increased expression of HGF and its receptor c-Met in nasal polyps suggests a role in disease pathogenesis. The effect of HGF on human sinonasal epithelial cell (SNEC) responses to Th2 inflammatory cytokines in chronic rhinosinusitis with nasal polyps (CRSwNP) has not been explored. METHODS: SNECs isolated from patients with CRSwNP and control subjects were grown in cell culture at the air-liquid interface. The Th2 cytokine IL-13 was applied for 24 hours in the presence or absence of HGF. Eotaxin-3 and c-Met expression was assessed using real-time PCR, immunohistochemistry, and flow cytometry. RESULTS: SNECs obtained from both CRSwNP and control subjects showed markedly increased expression of eotaxin-3 after exposure to IL-13. HGF significantly blocked IL-13-induced expression of eotaxin-3 in control SNECs, but not in SNECs derived from CRSwNP subjects. CONCLUSION: SNECs are active participants in sinonasal mucosal immunity, expressing inflammatory mediators in response to potential pathogens and endogenous cytokines. Although Th2 cytokines can elicit expression of proeosinophilic mediators by SNECs, HGF appears to have a down-regulating effect on this response. In patients with CRSwNP, SNECs are resistant to this attenuation, showing continued IL-13-induced eotaxin-3 expression despite HGF treatment. Abnormalities in the regulation of epithelial cell responses to endogenous cytokines and growth factors may contribute to the persistent eosinophilic inflammatory state in CRSwNP.