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Follicular neoplasms of the thyroid include follicular thyroid carcinoma (FTC) and follicular thyroid adenoma (FTA). However, the differences in cytological findings between FTC and FTA remain undetermined. Here, we aimed to evaluate the accumulation of lipid droplets (LDs) and the expression of adipophilin (perilipin 2/ADRP/ADFP), a known LD marker, in cultured FTC cells. We also immunohistochemically compared adipophilin expression in the FTC and FTA of resected human thyroid tissues. Cultured FTC (FTC-133 and RO82W-1) possessed increased populations of LDs compared to thyroid follicular epithelial (Nthy-ori 3-1) cells. In vitro treatment with phosphatidylinositol-3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling inhibitors (LY294002, MK2206, and rapamycin) in FTC-133 cells downregulated the PI3K/Akt/mTOR/sterol regulatory element-binding protein 1 (SREBP1) signaling pathway, resulting in a significant reduction in LD accumulation. SREBP1 is a master transcription factor that controls lipid metabolism. Fluorescence immunocytochemistry revealed adipophilin expression in the LDs of FTC-133 cells. Immunohistochemical analysis of surgically resected human thyroid tissues revealed significantly increased expression of adipophilin in FTC compared with FTA and adjacent non-tumorous thyroid epithelia. Taken together, LDs and adipophilin were abundant in cultured FTC; the evaluation of adipophilin expression can help distinguish FTC from FTA in surgical specimens. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Objective: Involvement in the tracheostomy procedure for COVID-19 patients can lead to a feeling of fear in medical staff. To address concerns over infection, we gathered and analyzed experiences with tracheostomy in the COVID-19 patient population from all over Japan. Methods: The data for health-care workers involved in tracheostomies for COVID-19-infected patients were gathered from academic medical centers or their affiliated hospitals from all over Japan. Results: Tracheostomies have been performed in 35 COVID-19 patients with a total of 91 surgeons, 49 anesthesiologists, and 49 surgical staff members involved. Twenty-eight (80%) patients underwent surgery more than 22 days after the development of COVID-19-related symptoms (11: 22-28 days and 17: >= 29 days). Thirty (85.7%) patients underwent surgery >= 15 days after intubation (14: 15-21 days, 6: 22-28 days, and 10: >= 29 days). Among the total of 189 health-care workers involved in the tracheostomy procedures, 25 used a powered air-purifying respirator (PAPR) and 164 used a N95 mask and eye protection. As a result, no transmission to staff occurred during the 2 weeks of follow-up after surgery. Conclusion: No one involved in tracheostomy procedures were found to have been infected with COVID-19 in this Japanese study. The reason is thought to be that the timing of the surgery was quite late after the infections, and the surgery was performed using appropriate PPE and surgical procedure. The indications for and timing of tracheostomy for severe COVID-19 patients should be decided through multidisciplinary discussion. (c) 2021 Oto-Rhino-Laryngological Society of Japan Inc. Published by Elsevier B.V. All rights reserved.

Reactive oxygen species (ROS) produced by NADPH oxidases (Nox) contribute to the development of different types of sensorineural hearing loss (SNHL), a common impairment in humans with no established treatment. Although the essential role of Nox3 in otoconia biosynthesis and its possible involvement in hearing have been reported in rodents, immunohistological methods targeted at detecting Nox3 expression in inner ear cells reveal ambiguous results. Therefore, the mechanism underlying Nox3-dependent SNHL remains unclear and warrants further investigation. We generatedNox3-Creknock-in mice, in which Nox3 was replaced withCre recombinase(Cre). UsingNox3-Cre;tdTomatomice of either sex, in which tdTomato is expressed under the control of theNox3promoter, we determined Nox3-expressing regions and cell types in the inner ear. Nox3-expressing cells in the cochlea included various types of supporting cells, outer hair cells, inner hair cells, and spiral ganglion neurons. Nox3 expression increased with cisplatin, age, and noise insults. Moreover, increased Nox3 expression in supporting cells and outer hair cells, especially at the basal turn of the cochlea, played essential roles in ROS-related SNHL. The extent of Nox3 involvement in SNHL follows the following order: cisplatin-induced hearing loss > age-related hearing loss > noise-induced hearing loss. Here, on the basis ofNox3-Cre;tdTomato, which can be used as a reporter system (Nox3-Cre+/−;tdTomato+/+andNox3-Cre+/+;tdTomato+/+), andNox3-KO (Nox3-Cre+/+;tdTomato+/+) mice, we demonstrate that Nox3 inhibition in the cochlea is a promising strategy for ROS-related SNHL, such as cisplatin-induced HL, age-related HL, and noise-induced HL.SIGNIFICANCE STATEMENTWe found Nox3-expressing regions and cell types in the inner ear, especially in the cochlea, usingNox3-Cre;tdTomatomice, a reporter system generated in this study. Nox3 expression increased with cisplatin, age, and noise insults in specific cell types in the cochlea and resulted in the loss (apoptosis) of outer hair cells. Thus, Nox3 might serve as a molecular target for the development of therapeutics for sensorineural hearing loss, particularly cisplatin-induced, age-related, and noise-induced hearing loss.

When animals are infected with helminthic parasites, resistant hosts mount type II helper T (Th2) immune responses to expel worms. Recent studies have clearly shown that epithelial cell-derived cytokines contribute to the induction of Th2 immune responses. Here we demonstrate the role of endogenous thymic stromal lymphopoietin (TSLP) for protection against Strongyloides venezuelensis (S. venezuelensis) infection, utilizing TSLP receptor-deficient Crlf2−/− mice. The number of eggs per gram of feces (EPG) and worm burden were significantly higher in Crlf2−/− mice than in wild type (WT) mice. S. venezuelensis infection induced Tslp mRNA expression in the skin, lung, and intestine and also facilitated the accumulation of mast cells in the intestine in a TSLP-dependent manner. Furthermore, CD4+ T cells from S. venezuelensis-infected Crlf2−/− mice showed diminished capacity to produce Th2 cytokines in the early stage of infection. Finally, CD4+ cell-depleted Crlf2−/− mice still showed higher EPG counts and worm burden than CD4+ cell-depleted WT mice, indicating that TSLP contributes to protecting mice against S. venezuelensis infection in both CD4+ T cell-dependent and -independent manners.

OBJECTIVES: We aimed to examine the effect of unilateral inhibition of the medullary dorsal swallowing networks on the activities of swallowing-related cranial motor nerves and swallowing interneurons. METHODS: In 25 juvenile rats, we recorded bilateral vagal nerve activity (VNA) as well as unilateral phrenic and hypoglossal activity (HNA) during fictive swallowing elicited by electrical stimulation of the superior laryngeal nerve during control and following microinjection of the GABA agonist muscimol into the caudal dorsal medulla oblongata in a perfused brainstem preparation. In 20 animals, swallowing interneurons contralateral to the muscimol injection side were simultaneously recorded extracellularly and their firing rates were analyzed during swallowing. RESULTS: Integrated VNA and HNA to the injection side decreased to 49.0 ± 16.6% and 32.3 ± 17.9%, respectively. However, the VNA on the uninjected side showed little change after muscimol injection. Following local inhibition, 11 out of 20 contralateral swallowing interneurons showed either increased or decreased of their respective firing discharge during evoked-swallowing, while no significant changes in activity were observed in the remaining nine neurons. CONCLUSION: The neuronal networks underlying the swallowing pattern generation in the dorsal medulla mediate the ipsilateral motor outputs and modulate the contralateral activity of swallowing interneurons, suggesting that the bilateral coordination of the swallowing central pattern generator regulates the spatiotemporal organization of pharyngeal swallowing movements. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2187-2198, 2021.

Invasive fungal rhinosinusitis (FRS) is a rare but intractable infectious disease of the sinonasal region with destructive direct infiltration into surrounding tissues, such as the bone, orbit and brain, and potential dissemination to systemic organs. Symptomatic assessments and imaging are frequently not sufficiently diagnostic, and histopathological examination is essential for definite diagnosis of FRS. We herein report a case of chronic invasive FRS (CIFRS) in a 58-year-old Japanese male with end-stage diabetic nephropathy that required maintenance dialysis after graft rejection of living kidney transplantation. His initial main clinical presentation was sinus gangrene, which gradually progressed from the paranasal sinus to the nasal septum and oral palate, but not towards the intracranial or orbital region, for two months. The patient was first strongly suspected to have extranodal natural killer/T cell lymphoma (ENKTL), nasal type, a subtype of malignant lymphoma, based on the macroscopic appearance of the gangrene, expansion pattern and high serum soluble interleukin-2 level; however, repeated biopsies and eventual resection led to diagnosis of CIFRS due to Aspergillus niger and Mucor. The disease was improved by surgical resection in combination with antifungal pharmacologic treatment with liposomal amphotericin B and voriconazole. CIFRS typically occurs in immunocompetent patients and shows intracranial progression, but this case shows that atypical CIFRS with an uncommon expansion pattern can occur in an immunodeficient patient.

Dia1, which belongs to the diaphanous-related formin family, influences a variety of cellular processes through straight actin elongation activity. Recently, novel DIA1 mutants such as p.R1213X (p.R1204X) and p.A265S, have been reported to cause an autosomal dominant sensorineural hearing loss (DFNA1). Additionally, active DIA1 mutants induce progressive hearing loss in a gain-of-function manner. However, the subcellular localization and pathological function of DIA1(R1213X/R1204X) remains unknown. In the present study, we demonstrated the localization of endogenous Dia1 and the constitutively active DIA1 mutant in the cochlea, using transgenic mice expressing FLAG-tagged DIA1(R1204X) (DIA1-TG). Endogenous Dia1 and the DIA1 mutant were regionally expressed at the organ of Corti and the spiral ganglion from early life; alongside cochlear maturation, they became localized at the apical junctional complexes (AJCs) between hair cells (HCs) and supporting cells (SCs). To investigate HC vulnerability in the DIA1-TG mice, we exposed 4-week-old mice to moderate noise, which induced temporary threshold shifts with cochlear synaptopathy and ultrastructural changes in stereocilia 4 weeks post noise exposure. Furthermore, we established a knock-in (KI) mouse line expressing AcGFP-tagged DIA1(R1213X) (DIA1-KI) and confirmed mutant localization at AJCs and the tips of stereocilia in HCs. In MDCKAcGFP-DIA1(R1213X) cells with stable expression of AcGFP-DIA1(R1213X), AcGFP-DIA1(R1213X) revealed marked localization at microvilli on the apical surface of cells and decreased localization at cell-cell junctions. The DIA1-TG mice demonstrated hazy and ruffled circumferential actin belts at AJCs and abnormal stereocilia accompanied with HC loss at 5 months of age. In conclusion, Dia1 plays a pivotal role in the development and maintenance of AJCs and stereocilia, ensuring cochlear and HC integrity. Subclinical/latent vulnerability of HCs may be the cause of progressive hearing loss in DFNA1 patients, thus suggesting new therapeutic targets for preventing HC degeneration and progressive hearing loss associated with DFNA1.

Understanding longitudinal changes of tumor-immune microenvironment during chemo/targeted therapies contributes to the development of optimized combinations of immunotherapy with chemotherapy and targeted therapy for patients with head and neck squamous cell carcinoma (HNSCC). We previously reported a chromogenic sequential immunohistochemical (IHC) platform enabling quantitative and spatial assessment of 29+ biomarkers in a single tissue section (Tsujikawa T et al. Cell Reports 2017, Banik G et al. Methods Enzymology 2020). Using this platform, densities, phenotypes, and distributions of tumor and immune cells were evaluated, comparing baseline and post-treatment specimens from the same individual treated by paclitaxel, carboplatin, and cetuximab (PCE) for advanced HNSCC (N = 30). Immune cell density analyses based on CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer cells, macrophages, dendritic cells, mast cells, granulocytes revealed the presence of differential immune cell compositions, where immune profiles were divided into hypo-, lymphoid-, and myeloid-inflamed groups according to the same criteria as in our previous report (Tsujikawa et al. Cell Reports 2017). Lymphoid and hypo-inflamed groups exhibited significant tumor volume reduction, increased CD45+ immune cell densities and elevated combined positive scores of PD-L1 at the post-treatment status, suggesting the potential involvement of immunogenic mechanisms related to therapeutic response. On the other hand, the myeloid group exhibited no significant tumor volume reduction, together with higher expression of HIF1α and ZEB2 on tumor cells which are potentially associated with hypoxia and epithelial-mesenchymal transition. In conclusion, longitudinal tissue-based monitoring revealed the presence of differential tumor-immune complexity profiles related to therapeutic efficacy and resistance. Hypo-inflamed profiles might require upfront chemo/targeted therapy before immunotherapy, and myeloid-inflamed profiles might require myeloid cell-targeted therapies, mandating the establishment of rapid clinical assessment of tumor-immune microenvironment. Citation Format: Alisa Kimura, Takahiro Tsujikawa, Junichi Mitsuda, Aya Miyagawa-Hayashino, Hiroki Morimoto, Sumiyo Saburi, Kanako Yoshimura, Gaku Ohmura, Sigeyuki Mukudai, Hikaru Nagao, Yoichiro Sugiyama, Hiroshi Ogi, Saya Shibata, Eiichi Konishi, Kyoko Itoh, Shigeru Hirano. Tumor-immune microenvironmental profiling during chemo- and targeted therapy for head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5172.

Neutrophil-to-lymphocyte ratio (NLR) in peripheral blood is an emerging biomarker candidate of immunotherapy in a wide range of cancers. However, little is known about the potential relationships between the tumor immune microenvironment and systemic inflammatory markers including NLR. Here we have explored systemic and tumor-immune microenvironmental characteristics related to treatment outcomes of immune checkpoint inhibition, based on 29 consecutive patients with recurrent/metastatic head and neck squamous cell carcinoma who received pembrolizumab between 2020 and 2021. NLR greater than 4.5 at pretreatment status significantly correlated with short overall survival (OS). Although NLR did not show a significant association with tumor volumes, high NLR exhibited significant correlations with malnutrition status characterized by CONUT (controlling nutritional status), and GNRI (geriatric nutrition risk index). Among the patients whose NLR was greater than 4.5 at pretreatment status, those whose NLR decreased to less than 4.5 at day 21 had a better OS than those whose NLR did not decrease, indicating that longitudinal changes in NLR correlate with prognosis. To investigate association with tumor-immune microenvironment, 14-marker multiplex immunohistochemistry was performed to quantitatively evaluate intratumoral CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer cells, macrophages, dendritic cells, mast cells, and granulocytes. Notably, NLR at pretreatment status significantly correlated with intratumoral immune cell densities, where high NLR correlated with low lymphoid cells and high tumor associated macrophages in tissue. NLR in peripheral blood significantly correlated with myeloid to lymphoid cell ratios in tissue, suggesting the presence of association between circulating and intratumoral immune complexity profiles. This study highlights that the association between intratumoral myeloid predominance and systemic nutritional and inflammatory status might be a possible factor for resistance to immunotherapy. Understanding immune dynamics in tissue and blood during immunotherapy potentially contributes to the establishment of predictive biomarkers and monitoring for immunotherapy. Citation Format: Hiroki Morimoto, Takahiro Tsujikawa, Aya Miyagawa-Hayashino, Alisa Kimura, Sumiyo Saburi, Junichi Mitsuda, Kanako Yoshimura, Gaku Ohmura, Shigeyuki Mukudai, Hikaru Nagao, Yoichiro Sugiyama, Shibata Saya, Hiroshi Ogi, Eiichi Konishi, Kyoko Itoh, Shigeru Hirano. Neutrophil-to-lymphocyte ratio associates with intratumoral myeloid predominance and clinical outcomes of pembrolizumab in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4453.

Past studies have found an association between cochlear hydrops or early Meniere's disease and acute low-frequency hearing loss (ALHL) without vertigo. However, its mechanism remains unclear in some ALHL cases. This report presents a case of ALHL associated with iron-deficiency anemia (IDA). The patient was a 49-year-old female who had previously been treated with betamethasone for sudden hearing loss in the right ear. Eight months later, the symptoms recurred and cochlear hydrops was diagnosed. Isosorbide and betamethasone were administered orally, and intravenous hydrocortisone tapering was added 1 week later, but these treatments were ineffective. At the same time and subsequently, iron sucrose was intravenously administered for IDA, and the patient's hearing loss gradually resolved within 2 months. In view of the increase in serum hemoglobin levels after iron therapy, this might have been the most effective treatment in this case. The hearing loss could therefore be associated with the patient's IDA.

Spatial profiles of the tumor-immune microenvironment are associated with disease progression and clinicopathological factors in various cancers. Follicular thyroid carcinoma (FTC) is the second most common thyroid cancer, where the presence of capsular invasion or angioinvasion determines the pathological diagnosis; however, little is known about the immune microenvironment profiles associated with the acquisition of invasive potential of FTC. In this study, we focused on FTC with minimal capsular invasion, and the spatially resolved immune microenvironment of FTC was studied in the discovery (n = 13) and validation cohorts (n = 40). CD8

Small inhaled particles, which are entrapped by the mucous layer that is maintained by mucous secretion via mucin exocytosis and fluid secretion, are removed from the nasal cavity by beating cilia. The functional activities of beating cilia are assessed by their frequency and the amplitude. Nasal ciliary beating is controlled by intracellular ions (Ca2+, H+ and Cl−), and is enhanced by a decreased concentration of intracellular Cl− ([Cl−]i) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which increases the ciliary beat amplitude. A novel method to measure both ciliary beat frequency (CBF) and ciliary beat distance (CBD, an index of ciliary beat amplitude) in cHNECs has been developed using high-speed video microscopy, which revealed that a decrease in [Cl−]i increased CBD, but not CBF, and an increase in [Cl−]i decreased both CBD and CBF. Thus, [Cl−]i inhibits ciliary beating in cHNECs, suggesting that axonemal structures controlling CBD and CBF may have Cl− sensors and be regulated by [Cl−]i. These observations indicate that the activation of Cl− secretion stimulates ciliary beating (increased CBD) mediated via a decrease in [Cl−]i in cHNECs. Thus, [Cl−]i is critical for controlling ciliary beating in cHNECs. This review introduces the concept of Cl− regulation of ciliary beating in cHNECs.

Background/aim Radiotherapy is widely accepted as the treatment of choice for early glottic squamous cell carcinoma (EGSCC), although it varies greatly with respect to dose, dose per fraction, and treatment techniques. The study aim was to evaluate the use of accelerated fractionation strategy (AFS) for EGSCC in standard clinical practice. Patients and methods Patients treated with definitive radiotherapy for EGSCC between 2008 and 2019 were retrospectively identified and received either conventional fractionation, hypofractionation, or hyperfractionation. Results One hundred six patients were analyzed, and 19, 71, and 16 patients underwent conventional fractionation, hypofractionation, and hyperfractionation, respectively. The median follow-up was 56 months. The 5-year local control and overall survival rates were 79% and 83%; 78% and 79%; and 87% and 77%, respectively, and no significant difference was observed between the fractionation schedules. Conclusion Our findings confirmed the utility of AFS in standard clinical practice and support its use for patients with EGSCC.

Small inhaled particles, which are entrapped by the mucous layer that is maintained by mucous secretion via mucin exocytosis and fluid secretion, are removed from the nasal cavity by beating cilia. The functional activities of beating cilia are assessed by their frequency and the amplitude. Nasal ciliary beating is controlled by intracellular ions (Ca2+, H+ and Cl−), and is enhanced by a decreased concentration of intracellular Cl− ([Cl−]i) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which increases the ciliary beat amplitude. A novel method to measure both ciliary beat frequency (CBF) and ciliary beat distance (CBD, an index of ciliary beat amplitude) in cHNECs has been developed using high-speed video microscopy, which revealed that a decrease in [Cl−]i increased CBD, but not CBF, and an increase in [Cl−]i decreased both CBD and CBF. Thus, [Cl−]i inhibits ciliary beating in cHNECs, suggesting that axonemal structures controlling CBD and CBF may have Cl− sensors and be regulated by [Cl−]i. These observations indicate that the activation of Cl− secretion stimulates ciliary beating (increased CBD) mediated via a decrease in [Cl−]i in cHNECs. Thus, [Cl−]i is critical for controlling ciliary beating in cHNECs. This review introduces the concept of Cl− regulation of ciliary beating in cHNECs.

We present a case of primary systemic amyloidosis diagnosed by endoscopic sinus surgery. A 75-year-old woman had blurred vision in her left eye; computed tomography and magnetic resonance imaging showed shadows of the bilateral paranasal sinuses. Endoscopic sinus surgery was performed, and amyloidosis was diagnosed by histopathology. She had previously been diagnosed with amyloidosis of the stomach, and therefore, she was diagnosed with primary systemic amyloidosis. A systemic workup for additional amyloid deposits revealed no evidence of other diseases. The patient remained under follow-up without further treatment, as no further amyloid deposition or progression of the lesions was seen. Amyloidosis is a rare condition characterized by the deposition of abnormal protein filaments in the extracellular tissue. Generally, systemic amyloidosis does not involve the head and neck region, and the presence of amyloid in the nasal and paranasal sinus mucosa is more likely to be indicative of a localized process. However, in our patient, the lesions were located in both the sinonasal tract and the stomach, indicating systemic amyloidosis. To our knowledge, there have been no previous reports of systemic amyloidosis involving the sinonasal tract, and therefore, we consider this case to be extremely rare.

Immune‐based tumor characteristics in the context of tumor heterogeneity are associated with suppression as well as promotion of cancer progression in various tumor types. As immunity typically functions based on intercellular contacts and short‐distance cytokine communications, the location and spatial relationships of the tumor immune microenvironment can provide a framework to understand the biology and potential predictive biomarkers related to disease outcomes. Immune spatial analysis is a newly emerging form of cancer research based on recent methodological advances in in situ single‐cell analysis, where cell‐cell interaction and the tissue architecture can be analyzed in relation to phenotyping the tumor immune heterogeneity. Spatial characteristics of tumors can be stratified into the tissue architecture level and the single‐cell level. At the tissue architecture level, the prognostic significance of the density of immune cell lineages, particularly T cells, is leveraged by understanding longitudinal changes in cell distribution in the tissue architecture such as intra‐tumoral and peri‐tumoral regions, and invasive margins. At the single‐cell level, the proximity of the tumor to the immune cells correlates with disease aggressiveness and therapeutic resistance, providing evidence to understand biological interactions and characteristics of the tumor immune microenvironment. In this review, we summarize recent findings regarding spatial information of the tumor immune microenvironment and review advances and challenges in spatial single‐cell analysis toward developing tissue‐based biomarkers rooted in the immune spatial landscape., The tumor immune microenvironment is characterized by multiple layers of spatial information. The whole tumor is dissected by tissue segments such as intratumoral and peritumoral regions at the tissue structure level, and by cell‐cell spatial relationships at the single‐cell level.

Background: Biomarkers predicting therapeutic response to immunotherapy have been widely explored via monitoring the liquid and tissue-derived components. Increasing treatment options for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) mandates prediction of the therapeutic response of anti-PD-1 antibody alone as well as optimization of the treatment sequence. In view of improving biomarkers predicting the efficacy of immunotherapy for R/M HNSCC, we hypothesized that biomarkers can be personalized depending on clinicopathological backgrounds and treatment sequence. Methods: In this study, we retrospectively included formalin-fixed paraffin-embedded (FFPE) samples, peripheral blood cell counts at treatment, clinicopathological information, and outcome data for patients with R/M HNSCC receiving nivolumab across 22 institutions in Japan (N = 100). FFPE samples were subjected to 14-marker multiplex immunohistochemistry (IHC) and image cytometry analysis (Tsujikawa T et al. Cell Reports, 2017) to quantitatively evaluate CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, CD66b+ granulocytes, mast cells, programmed death ligand 1 (PD-L1) and PD-1 expression in a single slide. Intratumoral and circulating immune cell frequencies were comparatively analyzed between responders (CR, n = 14; PR, n = 39) and non-responders (SD, n = 2; PD, n = 45). Results: Of 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-L1 expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. Next, focusing on the history of prior therapy, stratified analysis revealed that the frequency of NK cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Furthermore, stratified analysis by patient age revealed that nivolumab response was significantly associated with high CPS and lymphoid-inflamed profiles based on cell densities of nine immune cell lineages in the group aged 65 years or older, but not in the group under 65 years of age. On the contrary, the neutrophil/lymphocyte ratios (NLR) in peripheral blood counts at nivolumab treatment were significantly lower in responders (mean 4.96) than those in non-responders (mean 10.46) in the group under 65 years of age, but not in those over 65 years of age (7.41 versus 8.47). Conclusions: Using peripheral blood data and tumor tissue profiling stratified by patient age and prior treatment might provide better predictive biomarkers in nivolumab response to HNSCC. Further preclinical and clinical studies elucidating immune mechanisms in different patient backgrounds will be warranted. Citation Format: Takahiro Tsujikawa, Kazuchika Ohno, Sumiyo Saburi, Junichi Mitsuda, Kanako Yoshimura, Alisa Kimura, Hiroki Morimoto, Gaku Ohmura, Akihito Arai, Hiroshi Ogi, Saya Shibata, Yosuke Ariizumi, Akihisa Tasaki, Ryosuke Takahashi, Yumiko Tateishi, Hiroaki Kawabe, Sadakatsu Ikeda, Kei-ichi Morita, Tatsuhiko Tsunoda, Takumi Akashi, Morito Kurata, Issei Imoto, Yasushi Shimizu, Akihito Watanabe, Yukinori Asada, Ryuichi Hayashi, Yuki Saito, Hiroyuki Ozawa, Kiyoaki Tsukahara, Nobuhiko Oridate, Arata Horii, Takashi Maruo, Nobuhiro Hanai, Hidenori Inohara, Hiroshi Iwai, Takashi Fujii, Ken-ichi Nibu, Shigemichi Iwae, Tsutomu Ueda, Ryuji Yasumatsu, Hirohito Umeno, Muneyuki Masuda, Kyoko Itoh, Shigeru Hirano, Takahiro Asakage. Tumor immune characterization identifies age-stratified biomarkers for nivolumab in patients with head and neck squamous cell carcinoma: A nationwide collaborative study in Japan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5210.

OBJECTIVES/HYPOTHESIS We aimed to investigate and validate the cellular activity patterns and the potential topographical organization of neurons of the medullary swallowing pattern generator (Sw-CPG). We used the perfused brainstem preparation as an innovative experimental model that allows for stable neuronal recording in the brainstem. STUDY DESIGN Animal model. METHODS Experiments were conducted in 14 juvenile Wistar rats. The activities of the phrenic, vagus, and hypoglossal nerves were recorded at baseline, and fictive swallowing was elicited by stimulation of the superior laryngeal nerve. Extracellular action potentials of 72 swallowing-related neurons were recorded in the Sw-CPG of the dorsal medulla oblongata. RESULTS Neurons could be classified into three types: sensory relay, and neurons that were excited or inhibited during fictive swallowing. Approximately one-third of the neurons likely received monosynaptic input from the laryngeal afferents. One-third of neurons recorded showed respiratory-related activity, most of which exhibited inspiratory modulation. The neurons were widely distributed in the nucleus tractus solitarius and reticular formation. CONCLUSIONS The perfused brainstem preparation of rat fully preserves the Sw-CPG. The recorded cellular activities and general topographical organization of swallowing neurons are in accordance with previous in vivo studies. Thus, the perfused brainstem preparation is an ideal experimental model to advance the understanding of neuronal mechanisms underlying swallowing. LEVEL OF EVIDENCE NA Laryngoscope, 129:E72-E79, 2019.

Ambient particulate matter (PM) epidemiologically exacerbates respiratory and immune health, including allergic rhinitis (AR) and bronchial asthma (BA). Although fine and coarse particles can affect respiratory tract, the differences in their effects on the upper and lower respiratory tract and immune system, their underlying mechanism, and the components responsible for the adverse health effects have not been yet completely elucidated. In this study, ambient fine and coarse particles were collected at three different locations in Japan by cyclone technique. Both particles collected at all locations decreased the viability of nasal epithelial cells and antigen presenting cells (APCs), increased the production of IL-6, IL-8, and IL-1β from bronchial epithelial cells and APCs, and induced expression of dendritic and epithelial cell (DEC) 205 on APCs. Differences in inflammatory responses, but not in cytotoxicity, were shown between both particles, and among three locations. Some components such as Ti, Co, Zn, Pb, As, OC (organic carbon) and EC (elemental carbon) showed significant correlations to inflammatory responses or cytotoxicity. These results suggest that ambient fine and coarse particles differently affect nasal and bronchial epithelial cells and immune response, which may depend on particles size diameter, chemical composition and source related particles types.

Objectives/Hypothesis This study aimed to reveal the effects of adipose-derived mesenchymal stromal cells (ASCs) on prevention of vocal fold scarring by investigating how the immediate ASCs transplantation into the injured rat vocal fold affect the levels of gene transcription and translation. Study Design Prospective animal experiments with controls. Methods ASCs harvested from green fluorescent protein transgenic rat (ASCs group) or saline (sham group) were injected into the thyroarytenoid muscle of Sprague-Dawley rats immediately after stripping the vocal fold. For histological examinations, larynges were extirpated at 3, 14, and 56 days after the injection. Quantitative real-time polymerase chain reaction (PCR) analyses were performed at 3 and 14 days after the injection. Results Transplanted ASCs were detected only in larynges at day 3. At days 14 and 56, histological examination showed significantly higher amounts of hyaluronic acid and lower deposition of collagen in the ASCs group compared to the sham group. Real-time PCR revealed that the ASCs group showed low expression of procollagen (Col)1a1, Col1a3, matrix metalloproteinase (Mmp)1 and Mmp8 in each time points. The ASCs group showed high expression of fibroblast growth factor (Fgf)2 and Hepatocyte growth factor (Hgf) compared to the sham group at day 14. Conclusions ASCs increased expressions of Fgf2 and Hgf, and suppressed excessive collagen deposition during vocal fold wound healing. Given the fact that ASCs survived no more than 14 days, ASCs were thought to induce upregulations of growth factors' genes in surrounding cells. These results suggested that ASCs have potential to prevent vocal fold scarring. Level of Evidence NA. Laryngoscope, 128:E33–E40, 2018

OBJECTIVES The purpose of this study was to investigate the influence of laryngeal afferent inputs on brainstem circuits that mediate and transmit swallowing activity to the orofacial musculature. METHODS Experiments were performed on 19 arterially perfused juvenile rats. The activities of swallowing interneurons in relation to their respective motor outputs in the hypoglossal and vagus nerves were assessed during fictive swallowing with or without concurrent laryngeal sensory stimulation at intensities of 20, 40, and 60 μA. RESULTS The hypoglossal nerve activity was gradually enhanced with increasing intensity of the sensory stimulation, while the vagus nerve activity was not altered. The activities of various interneurons were modulated by the laryngeal stimulation, but more than 50% of the recorded neurons were inhibited by the stimulation. Some interneurons demonstrated no obvious change in their discharge rates with laryngeal sensory stimulation during fictive swallowing. CONCLUSION Laryngeal afferent inputs partially modulated the swallowing motor activity via enhanced or suppressed activities of the swallowing interneurons, while the essential motor pattern underlying the pharyngeal stage of swallowing remained basically unchanged. Thus, the output patterns of the complex sequential movements of swallowing could be basically predetermined and further adjusted according to sensory information related to the properties of the ingested food by a swallowing central pattern generator. LEVEL OF EVIDENCE NA Laryngoscope, 130: 1885-1893, 2020.

Objectives. Superficial-type pharyngeal squamous cell carcinoma (STPSCC) is defined as carcinoma in situ or microinvasive squamous cell carcinoma without invasion to the muscular layer. An exploration of the biological characteristics of STPSCC could uncover the invasion mechanism of this carcinoma. Phosphatidylcholine (PC) in combination with fatty acids is considered to play an important role in cell motility. Imaging mass spectrometry (IMS) is especially suitable for phospholipid analysis because this technique can distinguish even fatty acid compositions. Study Design. IMS analysis of frozen human specimens. Methods. IMS analysis was conducted to elucidate the distribution of PC species in STPSCC tissues. STPSCC tissue sections from five patients were analyzed, and we identified the signals that showed significant increases in the subepithelial invasive region relative to the superficial region. Results. Three kinds of PC species containing arachidonic acid, that is, PC (16:0/20:4), PC (18:1/20:4), and PC (18:0/20:4), were increased in the subepithelial invasive region. Conclusion. These results may be associated with the invasion mechanism of hypopharyngeal carcinoma.