The Larynx is Protected from Secondary and Vertical Papillomavirus Infection in Immunocompetent Mice.

OBJECTIVE: Mouse papillomavirus MmuPV1 causes both primary and secondary infections of the larynx in immunocompromised mice. Understanding lateral and vertical transmission of papillomavirus to the larynx would benefit patients with recurrent respiratory papillomatosis (RRP). To test the hypothesis that the larynx is uniquely vulnerable to papillomavirus infection, and to further develop a mouse model of RRP, we assessed whether immunocompetent mice were vulnerable to secondary or vertical laryngeal infection with MmuPV1. METHODS: Larynges were collected from 405 immunocompetent adult mice that were infected with MmuPV1 in the oropharynx, oral cavity, or anus, and 31 mouse pups born to immunocompetent females infected in the cervicovaginal tract. Larynges were analyzed via polymerase chain reaction (PCR) of lavage fluid or whole tissues for viral DNA, histopathology, and/or in situ hybridization for MmuPV1 transcripts. RESULTS: Despite some positive laryngeal lavage PCR screens, all laryngeal tissue PCR and histopathology results were negative for MmuPV1 DNA, transcripts, and disease. There was no evidence for lateral spread of MmuPV1 to the larynges of immunocompetent mice that were infected in the oral cavity, oropharynx, or anus. Pups born to infected mothers were negative for laryngeal MmuPV1 infection from birth through weaning age. CONCLUSION: Secondary and vertical laryngeal MmuPV1 infections were not found in immunocompetent mice. Further work is necessary to explore immunologic control of laryngeal papillomavirus infection in a mouse model and to improve preclinical models of RRP. LEVEL OF EVIDENCE: NA Laryngoscope, 134:2322-2330, 2024.

Preclinical Models of Laryngeal Papillomavirus Infection: A Scoping Review.

OBJECTIVE: Laryngeal human papillomavirus (HPV) infection causes recurrent respiratory papillomatosis (RRP) and accounts for up to 25% of laryngeal cancers. Lack of satisfactory preclinical models is one reason that treatments for these diseases are limited. We sought to assess the literature describing preclinical models of laryngeal papillomavirus infection. DATA SOURCES: PubMed, Web of Science, and Scopus were searched from the inception of database through October 2022. REVIEW METHODS: Studies searched were screened by two investigators. Eligible studies were peer-reviewed, published in English, presented original data, and described attempted models of laryngeal papillomavirus infection. Data examined included type of papillomavirus, infection model, and results including success rate, disease phenotype, and viral retention. RESULTS: After screening 440 citations and 138 full-text studies, 77 studies published between 1923 and 2022 were included. Models used low-risk HPV or RRP (n = 51 studies), high-risk HPV or laryngeal cancer (n = 16), both low- and high-risk HPV (n = 1), and animal papillomaviruses (n = 9). For RRP, 2D and 3D cell culture models and xenografts retained disease phenotypes and HPV DNA in the short term. Two laryngeal cancer cell lines were consistently HPV-positive in multiple studies. Animal laryngeal infections with animal papillomaviruses resulted in disease and long-term retention of viral DNA. CONCLUSIONS: Laryngeal papillomavirus infection models have been researched for 100 years and primarily involve low-risk HPV. Most models lose viral DNA after a short duration. Future work is needed to model persistent and recurrent diseases, consistent with RRP and HPV-positive laryngeal cancer. LEVEL OF EVIDENCE: NA Laryngoscope, 133:3256-3268, 2023.

Efficacy of Topically Administered Dihydroartemisinin in Treating Papillomavirus-Induced Anogenital Dysplasia in Preclinical Mouse Models.

The artemisinin family of compounds is cytopathic in certain cancer cell lines that are positive for human papillomaviruses (HPV) and can potentially drive the regression of dysplastic lesions. We evaluated the efficacy of topical dihydroartemisinin (DHA) on cervical dysplasia and anal dysplasia in two papillomavirus mouse models: K14E6/E7 transgenic mice, which express HPV16 oncogenes; and immunodeficient NOD/SCID gamma (NSG) mice infected with Mus musculus papillomavirus (MmuPV1). Mice started treatment with DHA at 25 weeks of age (K14E6/E7) or 20 weeks post infection (MmuPV1-infected), when the majority of mice are known to have papillomavirus-induced low- to high-grade dysplasia. Mice were treated with or without topical DHA at the cervix or anus and with or without topical treatment with the chemical carcinogen 7,12 dimethylbenz(a)anthracene (DMBA) at the anus of in transgenic mice to induce neoplastic progression. Mice were monitored for overt tumor growth, and tissue was harvested after 20 weeks of treatment and scored for severity of histological disease. For MmuPV1-infected mice, anogenital lavages were taken to monitor for viral clearance. Tissues were also evaluated for viral gene expression at the RNA and/or protein levels. Treatment with topical DHA did not reduce dysplasia in the anogenital tract in either papillomavirus-induced mouse model and did not prevent progression to anal cancer in the DMBA-treated K14E6/E7 mice.

A Novel In Vivo Model of Laryngeal Papillomavirus-Associated Disease Using Mus musculus Papillomavirus.

Recurrent respiratory papillomatosis (RRP), caused by laryngeal infection with low-risk human papillomaviruses, has devastating effects on vocal communication and quality of life. Factors in RRP onset, other than viral presence in the airway, are poorly understood. RRP research has been stalled by limited preclinical models. The only known papillomavirus able to infect laboratory mice, Mus musculus papillomavirus (MmuPV1), induces disease in a variety of tissues. We hypothesized that MmuPV1 could infect the larynx as a foundation for a preclinical model of RRP. We further hypothesized that epithelial injury would enhance the ability of MmuPV1 to cause laryngeal disease, because injury is a potential factor in RRP and promotes MmuPV1 infection in other tissues. In this report, we infected larynges of NOD scid gamma mice with MmuPV1 with and without vocal fold abrasion and measured infection and disease pathogenesis over 12 weeks. Laryngeal disease incidence and severity increased earlier in mice that underwent injury in addition to infection. However, laryngeal disease emerged in all infected mice by week 12, with or without injury. Secondary laryngeal infections and disease arose in nude mice after MmuPV1 skin infections, confirming that experimentally induced injury is dispensable for laryngeal MmuPV1 infection and disease in immunocompromised mice. Unlike RRP, lesions were relatively flat dysplasias and they could progress to cancer. Similar to RRP, MmuPV1 transcript was detected in all laryngeal disease and in clinically normal larynges. MmuPV1 capsid protein was largely absent from the larynx, but productive infection arose in a case of squamous metaplasia at the level of the cricoid cartilage. Similar to RRP, disease spread beyond the larynx to the trachea and bronchi. This first report of laryngeal MmuPV1 infection provides a foundation for a preclinical model of RRP.

Expanded Basal Compartment and Disrupted Barrier in Vocal Fold Epithelium Infected with Mouse Papillomavirus MmuPV1.

Laryngeal infection with low-risk human papillomaviruses can cause recurrent respiratory papillomatosis (RRP), a disease with severe effects on vocal fold epithelium resulting in impaired voice function and communication. RRP research has been stymied by limited preclinical models. We recently reported a murine model of laryngeal MmuPV1 infection and disease in immunodeficient mice. In the current study, we compare quantitative and qualitative measures of epithelial proliferation, apoptosis, differentiation, and barrier between mice with MmuPV1-induced disease of the larynx and surrounding tissues and equal numbers of uninfected controls. Findings supported our hypothesis that laryngeal MmuPV1 infection recapitulates many features of RRP. Like RRP, MmuPV1 increased proliferation in infected vocal fold epithelium, expanded the basal compartment of cells, decreased differentiated cells, and altered cell-cell junctions and basement membrane. Effects of MmuPV1 on apoptosis were equivocal, as with RRP. Barrier markers resembled human neoplastic disease in severe MmuPV1-induced disease. We conclude that MmuPV1 infection of the mouse larynx provides a useful, if imperfect, preclinical model for RRP that will facilitate further study and treatment development for this intractable and devastating disease.

Voice Handicap Index Changes After Microflap Surgery for Benign Vocal Fold Lesions Are Not Associated With Recommended Absolute Voice Rest Duration.

PURPOSE: Voice rest is frequently prescribed after phonosurgery, but optimal type and duration for voice outcomes have not been demonstrated. Studies to date have been characterized by heterogeneity in surgical procedures and laryngeal diagnoses. We sought to analyze the effect of recommended absolute voice rest duration on outcomes of microflap surgery for benign vocal fold lesions. A secondary purpose was to identify patient factors associated with postoperative voice outcomes. METHOD: Forty-three patients were included in this retrospective review of patients aged 18 years and above who underwent direct microlaryngoscopy with microflap for vocal fold polyp or cyst over a 5-year period at a multidisciplinary voice center. Duration of recommended postoperative absolute voice rest was classified as less than 7 days, 7 days, and more than 7 days. Demographic and vocal hygiene data and voice treatment history were collected. Outcome measures consisted of one pre- and two postoperative Voice Handicap Index (VHI) scores. Effects of recommended voice rest on outcomes were analyzed using mixed models for repeated measures. Effects of patient factors on outcomes were analyzed as exploratory measures. Stroboscopy ratings were analyzed descriptively. RESULTS: Thirteen patients were recommended 7 days of absolute voice rest, 15 were recommended less than 7 days, and 15 were recommended more than 7 days. Postoperatively, VHI scores significantly improved for all patients. Voice rest as a continuous variable was associated with the Functional subscale score in the short term, but there was no effect on VHI total score and no longer term effect of voice rest on any outcome. Age, sex, and preoperative voice therapy were associated with at least one VHI subscale score on at least one time point. CONCLUSION: VHI outcomes of microflap surgery for polyps and cysts do not differ by duration of recommended absolute postoperative voice rest. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.19178459.

Role of Voice Therapy in Adherence to Voice Rest After Office-Based Vocal Fold Procedures.

Purpose Patients undergoing vocal fold procedures significantly reduce but often do not cease voice use during absolute postprocedure voice rest. We hypothesized that patients who completed preprocedure voice therapy would increase adherence to postprocedure voice rest. Method Eighty-six participants completed this prospective cohort study. Patients scheduled for office-based vocal fold procedures, 1-3 days of absolute postprocedure voice rest, and preprocedure speech-language pathology (SLP) care were recruited. SLP care consisted of either (a) multiple voice therapy sessions, (b) one counseling/therapy session, or (c) voice evaluation only. Participants reported talking and other specific voice behaviors on 100-mm visual analog scales for up to 3 days pre- and postprocedure as well as changes in overall voice use at follow-up at least 1 week postprocedure. Results Talking decreased postprocedure by 63% in the therapy group and 65% in the counseling group, both significantly more than the 35% decrease measured in the evaluation group. There were group differences in talking at baseline but not during voice rest. Coughing and throat clearing were highest in the voice evaluation group and decreased less than talking during voice rest. At follow-up, 84% of participants reported that they completed voice rest for at least as long as recommended and 39.5% reported that they never used their voices during voice rest. Participants estimated a 98% overall reduction in voice use during voice rest at follow-up. Conclusions Voice use before and after vocal fold procedures varies by participation in preprocedure voice therapy. Patients significantly decrease talking during postprocedure voice rest but are not perfectly adherent. Communicative voice use decreases more than noncommunicative voice use during voice rest. Patients may overestimate adherence to voice rest at follow-up. Supplemental Material https://doi.org/10.23641/asha.16589864.

A Mouse Model of Oropharyngeal Papillomavirus-Induced Neoplasia Using Novel Tools for Infection and Nasal Anesthesia.

Human head and neck cancers that develop from the squamous cells of the oropharynx (Oropharyngeal Squamous Cell Carcinomas or OPSCC) are commonly associated with the papillomavirus infection. A papillomavirus infection-based mouse model of oropharyngeal tumorigenesis would be valuable for studying the development and treatment of these tumors. We have developed an efficient system using the mouse papillomavirus (MmuPV1) to generate dysplastic oropharyngeal lesions, including tumors, in the soft palate and the base of the tongue of two immune-deficient strains of mice. To maximize efficiency and safety during infection and endoscopy, we have designed a nose cone for isoflurane-induced anesthesia that takes advantage of a mouse's need to breathe nasally and has a large window for oral manipulations. To reach and infect the oropharynx efficiently, we have repurposed the Greer Pick allergy testing device as a virus delivery tool. We show that the Pick can be used to infect the epithelium of the soft palate and the base of the tongue of mice directly, without prior scarification. The ability to induce and track oropharyngeal papillomavirus-induced tumors in the mouse, easily and robustly, will facilitate the study of oropharyngeal tumorigenesis and potential treatments.

Biocompatibility and Viscoelastic Properties of Injectable Resilin-Like Polypeptide and Hyaluronan Hybrid Hydrogels in Rabbit Vocal Folds.

Vocal fold scar, characterized by alterations in the lamina propria extracellular matrix, disrupts normal voice quality and function. Due to a lack of satisfactory clinical treatments, there is a need for tissue engineering strategies to restore voice. Candidate biomaterials for vocal fold tissue engineering must match the unique biomechanical and viscoelastic properties of native tissue without provoking inflammation. We sought to introduce elastomeric properties to hyaluronic acid (HA)-based biomaterials by incorporating resilin-like polypeptide (RLP) into hybrid hydrogels. Physically crosslinked RLP/HA and chemically crosslinked RLP-acrylamide/thiolated HA (RLP-AM/HA-SH) hydrogels were fabricated using cytocompatible chemistries. Mechanical properties of hydrogels were assessed in vitro using oscillatory rheology. Hybrid hydrogels were injected into rabbit vocal folds and tissues were assessed using rheology and histology. A small number of animals underwent acute vocal fold injury followed by injection of RLP-AM/HA-SH hydrogel alone or as a carrier for human bone marrow mesenchymal stem cells (BM-MSCs). Rheological testing confirmed that mechanical properties of materials in vitro resembled native vocal fold tissue and that viscoelasticity of vocal fold mucosa was preserved days 5 and 21 after injection. Histological analysis revealed that hybrid hydrogels provoked only mild inflammation in vocal fold lamina propria with demonstrated safety in the airway for up to 3 weeks, confirming acute biocompatibility of crosslinking chemistries. After acute injury, RLP-AM/HA-SH gel with and without BM-MSCs did not result in adverse effects or increased inflammation. Collectively, results indicate that RLP and HA-based hybrid hydrogels are highly promising for engineering the vocal fold lamina propria.

Magnetic resonance imaging quantification of dehydration and rehydration in vocal fold tissue layers.

Clinicians commonly recommend increased hydration to patients with voice disorders. However, effects on clinical voice outcome measures have been inconsistent. Hydration-induced change within different layers of vocal fold tissue is currently unknown. Magnetic Resonance Imaging (MRI) is a promising method of noninvasively measuring water content in vocal folds. We sought to image and quantify changes in water content within vocal fold mucosa and thyroarytenoid muscle after dehydration and rehydration. Excised porcine larynges were imaged using proton density (PD) weighted MRI (1) at baseline and (2) after immersion in one of five hypertonic, isotonic, or hypotonic solutions or in dry air. Larynges dehydrated in hypertonic solutions or dry air were rehydrated and imaged a third time. Scans revealed fluid-rich vocal fold mucosa that was distinct from muscle at baseline. Baseline normalized signal intensity in mucosa and muscle varied by left vs. right vocal fold (p < 0.01) and by anterior, middle, or posterior location (p < 0.0001). Intensity changes in the middle third of vocal fold mucosa differed by solution after immersion (p < 0.01). Hypertonic solutions dehydrated the middle third of mucosa by over 30% (p < 0.001). No difference from baseline was found in anterior or posterior mucosa or in muscle after immersion. No association was found between intensity change in mucosa and muscle after immersion. After rehydration, intensity did not differ by solution in any tissue, and was not different from baseline, but post-rehydration intensity was correlated with post-immersion intensity in both mucosa and muscle (p < 0.05), suggesting that degree of change in vocal fold water content induced by hypertonic solutions ex vivo persists after rehydration. These results indicate that PD-MRI can be used to visualize large mammalian vocal fold tissue layers and to quantify changes in water content within vocal fold mucosa and thyroarytenoid muscle independently.