Human angiotensin-converting enzyme 2-specific antisense oligonucleotides reduce infection with SARS-CoV-2 variants.

BACKGROUND: The Spike protein mutation severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to decreased protective effect of various vaccines and mAbs, suggesting that blocking SARS-CoV-2 infection by targeting host factors would make the therapy more resilient against virus mutations. Angiotensin-converting enzyme 2 (ACE2) is the host receptor of SARS-CoV-2 and its variants, as well as many other coronaviruses. Downregulation of ACE2 expression in the respiratory tract may prevent viral infection. Antisense oligonucleotides (ASOs) can be rationally designed on the basis of sequence data, require no delivery system, and can be administered locally. OBJECTIVE: We sought to design ASOs that can block SARS-CoV-2 by downregulating ACE2 in human airway. METHODS: ACE2-targeting ASOs were designed using a bioinformatic method and screened in cell lines. Human primary nasal epithelial cells cultured at the air-liquid interface and humanized ACE2 mice were used to detect the ACE2 reduction levels and the safety of ASOs. ASO-pretreated nasal epithelial cells and mice were infected and then used to detect the viral infection levels. RESULTS: ASOs reduced ACE2 expression on mRNA and protein level in cell lines and in human nasal epithelial cells. Furthermore, they efficiently suppressed virus replication of 3 different SARS-CoV-2 variants in human nasal epithelial cells. In vivo, ASOs also downregulated human ACE2 in humanized ACE2 mice and thereby reduced viral load, histopathologic changes in lungs, and increased survival of mice. CONCLUSIONS: ACE2-targeting ASOs can effectively block SARS-CoV-2 infection. Our study provides a new approach for blocking SARS-CoV-2 and other ACE2-targeting virus in high-risk populations.

Screening for Psychiatric Disorders in Chronic Rhinosinusitis Patients Waiting for Surgery: A Prospective Cross-Sectional Study.

OBJECTIVE: To assess the prevalence of depression, anxiety, insomnia and somatic symptom disorder (SSD) in chronic rhinosinusitis (CRS) patients who were waiting for surgery and to predict these psychiatric disorders using the 22-item Sinonasal Outcome Test (SNOT-22). DESIGN: A prospective cross-sectional study. SETTING: The rhinology ward at our institution, a tertiary hospital. PARTICIPANTS: Adult patients (> 18 years) diagnosed with CRS who were admitted to the rhinology ward for endoscopic sinus surgery and were able to understand and complete the study questionnaires. MAIN OUTCOME MEASURES: Patient Health Questionnaire-9 (PHQ-9), Generalised Anxiety Disorder-7 (GAD-7), Insomnia Severity Index (ISI), Patient Health Questionnaire-15 (PHQ-15) and SNOT-22. RESULTS: Of the 159 participants recruited, 58 were at risk of depression (defined by PHQ-9 > 4, while 25 with PHQ-9 > 9), 49 were at risk of anxiety (defined by GAD-7 > 4, while 25 with GAD-7 > 9), 81 were at risk of insomnia (defined by ISI > 7, while 51 with ISI > 14) and 69 were at risk of SSD (defined by PHQ-15 > 4, while 24 with PHQ-15 > 9). The SNOT-22 score was closely correlated with the scores of psychometric tests and was an independent predictor of these psychiatric disorders. Patients with a high SNOT-22 score (> 30) are likely to be affected by comorbid psychiatric disorders and should be further evaluated by otolaryngologists. CONCLUSION: Depression, anxiety, insomnia and SSD are prevalent in CRS patients. Otolaryngologists should have a low threshold to ask the patient about psychiatric symptoms, especially for patients with an SNOT-22 score > 30.

Real-time detection of laryngopharyngeal cancer using an artificial intelligence-assisted system with multimodal data.

BACKGROUND: Laryngopharyngeal cancer (LPC) includes laryngeal and hypopharyngeal cancer, whose early diagnosis can significantly improve the prognosis and quality of life of patients. Pathological biopsy of suspicious cancerous tissue under the guidance of laryngoscopy is the gold standard for diagnosing LPC. However, this subjective examination largely depends on the skills and experience of laryngologists, which increases the possibility of missed diagnoses and repeated unnecessary biopsies. We aimed to develop and validate a deep convolutional neural network-based Laryngopharyngeal Artificial Intelligence Diagnostic System (LPAIDS) for real-time automatically identifying LPC in both laryngoscopy white-light imaging (WLI) and narrow-band imaging (NBI) images to improve the diagnostic accuracy of LPC by reducing diagnostic variation among on-expert laryngologists. METHODS: All 31,543 laryngoscopic images from 2382 patients were categorised into training, verification, and test sets to develop, validate, and internal test LPAIDS. Another 25,063 images from five other hospitals were used as external tests. Overall, 551 videos were used to evaluate the real-time performance of the system, and 200 randomly selected videos were used to compare the diagnostic performance of the LPAIDS with that of laryngologists. Two deep-learning models using either WLI (model W) or NBI (model N) images were constructed to compare with LPAIDS. RESULTS: LPAIDS had a higher diagnostic performance than models W and N, with accuracies of 0·956 and 0·949 in the internal image and video tests, respectively. The robustness and stability of LPAIDS were validated in external sets with the area under the receiver operating characteristic curve values of 0·965-0·987. In the laryngologist-machine competition, LPAIDS achieved an accuracy of 0·940, which was comparable to expert laryngologists and outperformed other laryngologists with varying qualifications. CONCLUSIONS: LPAIDS provided high accuracy and stability in detecting LPC in real-time, which showed great potential for using LPAIDS to improve the diagnostic accuracy of LPC by reducing diagnostic variation among on-expert laryngologists.

Microvessel quantification by fully convolutional neural networks associated with type 2 inflammation in chronic rhinosinusitis.

BACKGROUND: The pathogenesis of chronic rhinosinusitis (CRS) is still unclear, and little is known about angiogenesis in this disease. We utilized a fully convolutional network (FCN), which has been extensively used in image processing to study angiogenesis in CRS. OBJECTIVE: To explore the tissue quantification of microvessels and their potential association with inflammation in CRS by using FCN to reflect the angiogenesis condition in CRS. METHODS: For endotyping of CRS, tissue homogenates of 79 patients with CRS who had undergone functional endoscopic sinus surgery and 17 control subjects were analyzed for interferon gamma, transforming growth factor beta, interleukin (IL)-1ß, IL-5, IL-6, IL-8, IL-10, IL-17, tumor necrosis factor alpha, eosinophilic cationic protein, immunoglobulin E, and Staphylococcus aureus-immunoglobulin E(SE-IgE). A total of 552 hematoxylin and eosin-stained images of 27 CRS tissue samples were used to develop an FCN, going through training, validation, and evaluation processes. An optimized FCN was applied to quantify the microvessels of tissue samples of all subjects. Correlation analysis between microvessel quantification with phenotype, endotype, clinical characteristics, and cytokine expression of CRS was carried out. RESULTS: Quantification of microvessels in type 2 and non-type 2 CRS demonstrated considerable differences, with a higher expression in type 2 CRS. There was a strong negative correlation between the area ratio of microvessels with tissue tumor necrosis factor alpha and transforming growth factor beta levels and a mildly positive correlation with tissue IL-5 and eosinophilic cationic protein concentration. CONCLUSION: FCN proved to facilitate the analysis of microvessels in airway tissue samples. This study elucidated the close association of angiogenesis with endotyping, suggesting that treatment aiming at antagonizing angiogenesis may assist to the therapy for the recrudescent and refractory CRS.

Endoscopic surgery compared with intensity-modulated radiotherapy in resectable locally recurrent nasopharyngeal carcinoma: a multicentre, open-label, randomised, controlled, phase 3 trial.

BACKGROUND: The role of surgery compared with reirradiation in the primary treatment of patients with resectable, locally recurrent nasopharyngeal carcinoma (NPC) who have previously received radiotherapy is a matter of debate. In this trial, we compared the efficacy and safety outcomes of salvage endoscopic surgery versus intensity-modulated radiotherapy (IMRT) in patients with resectable locally recurrent NPC. METHODS: This multicentre, open-label, randomised, controlled, phase 3 trial was done in three hospitals in southern China. We included patients aged 18-70 years with a Karnofsky Performance Status score of at least 70 who were histopathologically diagnosed with undifferentiated or differentiated, non-keratinising, locally recurrent NPC with tumours confined to the nasopharyngeal cavity, the post-naris or nasal septum, the superficial parapharyngeal space, or the base wall of the sphenoid sinus. Eligible patients were randomly assigned (1:1) to receive either endoscopic nasopharyngectomy (ENPG group) or IMRT (IMRT group). Randomisation was done manually using a computer-generated random number code and patients were stratified by treatment centre. Treatment group assignment was not masked. The primary endpoint was overall survival, compared between the groups at 3 years. Efficacy analyses were done by intention to treat. Safety analysis was done in patients who received treatment according to the treatment they actually received. This trial was prospectively registered at the Chinese Clinical Trial Registry, ChiCTR-TRC-11001573, and is currently in follow-up. FINDINGS: Between Sept 30, 2011, and Jan 16, 2017, 200 eligible patients were randomly assigned to receive either ENPG (n=100) or IMRT (n=100). At a median follow-up of 56·0 months (IQR 42·0-69·0), 74 patients had died (29 [29%] of 100 patients in the ENPG group and 45 [45%] of 100 patients in the IMRT group). The 3-year overall survival was 85·8% (95% CI 78·9-92·7) in the ENPG group and 68·0% (58·6-77·4) in the IMRT group (hazard ratio 0·47, 95% CI 0·29-0·76; p=0·0015). The most common grade 3 or worse radiation-related late adverse event was pharyngeal mucositis (in five [5%] of 99 patients who underwent ENPG and 26 [26%] of 101 patients who underwent IMRT). Five [5%] of the 99 patients who underwent ENPG and 20 [20%] of the 101 patients who underwent IMRT died due to late toxic effects specific to radiotherapy; attribution to previous radiotherapy or trial radiotherapy is unclear due to the long-term nature of radiation-related toxicity. INTERPRETATION: Endoscopic surgery significantly improved overall survival compared with IMRT in patients with resectable locally recurrent NPC. These results suggest that ENPG could be considered as the standard treatment option for this patient population, although long-term follow-up is needed to further determine the efficacy and toxicity of this strategy. FUNDING: Sun Yat-sen University Clinical Research 5010 Program.

Using RNA sequencing to identify a putative lncRNA-associated ceRNA network in laryngeal squamous cell carcinoma.

Accumulating evidence indicates that lncRNAs can interact with miRNAs to regulate target mRNAs through competitive interactions. However, this mechanism remains largely unexplored in laryngeal squamous cell carcinoma (LSCC). In this study, transcriptome-wide RNA sequencing was performed on 3 pairs of LSCC tissues and adjacent normal tissues to investigate the expression profiles of lncRNAs, miRNAs and mRNAs, with differential expression of 171 lncRNAs, 36 miRNAs and 1709 mRNAs detected. Seven lncRNAs, eight mRNAs and three miRNAs were identified to be dysregulated in patients' tissues by using qRT-PCR. GO and KEGG pathway enrichment analyses were performed to elucidate the potential functions of these differentially expressed genes in LSCC. Subsequently, a ceRNA (lncRNA-miRNA-mRNA) network including 4631 ceRNA pairs was constructed based on predicted miRNAs shared by lncRNAs and mRNAs. Cis- and transregulatory lncRNAs were analysed by bioinformatics-based methods. Importantly, mRNA-related ceRNA networks (mRCNs) were further obtained based on potential cancer-related coding genes. Coexpression between lncRNAs and downstream mRNAs was used as a criterion for the validation of mRCNs, with the ZNF561-AS1-miR217-WNT5A and SATB1-AS1-miR1299-SAV1/CCNG2/SH3 KBP1/JADE1/HIPK2 ceRNA regulatory interactions determined, followed by experimental validation after siRNA transfection. Moreover, ceRNA activity analysis revealed that different activities of ceRNA modules existing in specific pathological environments may contribute to the tumorigenesis of LSCC. Consistently, both downregulated SATB1-AS1 and ZNF561-AS1 significantly promoted laryngeal cancer cell migration and invasion, indicating their important roles in LSCC via a ceRNA regulatory mechanism. Taken together, the results of this investigation uncovered and systemically characterized a lncRNA-related ceRNA regulatory network that may be valuable for the diagnosis and treatment of LSCC.

The cost-effectiveness analysis of drug therapy versus surgery for symptomatic adenoid hypertrophy by a Markov model.

PURPOSE: Adenoid hypertrophy (AH) is common among young children. Adenoid-based surgery and drug therapy could be applied for symptomatic AH patients, yet the treatment decision is difficult to make due to the diverse cost and efficacy between these two treatments. METHODS: A Markov simulation model was designed to estimate the cost-effectiveness (CE) of the adenoid-based surgery and the drug therapy for symptomatic AH patients. Transition probabilities, costs and utilities were extracted from early researches and expert opinions. Simulations using two set of parameter inputs for China and the USA were performed. Primary outcome was cost per QALY gained over a 6-year period. Deterministic and probabilistic sensitivity analyses were also conducted. RESULTS: The utility for the surgery group and the drug group were 4.10 quality-adjusted life years (QALYs) and 3.58 QALYs, respectively. The cost of the surgery group was more than that of the drug group using model parameters specific to China ($1069.0 vs. $753.7) but was less for the USA ($1994.4 vs. $3977.7). Surgery was dominant over drug therapy when US specific parameters were used. Surgery group had an ICER of $604.0 per QALY when parameters specific to China was used. CONCLUSION: Surgery is cost-effective in the simulations for both China and the USA at WTP thresholds of $9633.1 and $62,517.5, respectively.

Stromal interleukin-33 promotes regulatory T cell-mediated immunosuppression in head and neck squamous cell carcinoma and correlates with poor prognosis.

Regulatory T cells (Tregs) mediate immunosuppressive signals that can contribute to the progression of head and neck squamous cell carcinoma (HNSCC). Interleukin-33 (IL-33) is defined as an 'alarmin', an endogenous factor that is expressed during tissue and cell damage, which has been shown to promote Treg proliferation in non-lymphoid organs. However, the interaction between IL-33 and Tregs in the HNSCC tumor microenvironment remains uncertain. In this study, we examined IL-33+ and Foxp3+ cells by immunohistochemistry in 68 laryngeal squamous cell cancer patients, followed by functional analysis of IL-33 in Tregs. In addition, the suppressive function of Tregs was assessed by cell proliferation assays. The level of stromal IL-33 was significantly upregulated in advanced versus early stage HNSCC patients and positively correlated with Foxp3+ Treg infiltration as well as a poor prognosis. ST2 is regarded as the only receptor of IL-33. Infiltrated ST2-expressing Tregs were responsive to IL-33, and the percentage of Tregs was increased upon IL-33 stimulation. Functional investigation demonstrated that IL-33 increased the proportion of Foxp3+GATA3+ Tregs and improved the suppressive functions of Tregs by inducing IL-10 and TGF-ß1 as well as decreasing the proliferation of responder T cells. Blockade of ST2 abrogated the immunosuppression caused by IL-33. Our data demonstrate that stromal IL-33 both expands the Treg population and enhances their functions in the tumor microenvironment. Furthermore, stromal IL-33 has prognostic value for tumor progression. Thus, stromal IL-33 is a potential target for future HNSCC immunotherapy.

LL-37 promotes neutrophil extracellular trap formation in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Neutrophil accumulation has been observed in chronic rhinosinusitis with nasal polyps (CRSwNP). However, the functions of neutrophils are poorly understood. Neutrophils produce neutrophil extracellular traps (NETs), which are involved in a variety of chronic inflammatory pathologies. LL-37 is the only member of the cathelicidin family in human. OBJECTIVE: Our aims were to examine the presence of NETs in CRSwNP and to investigate the regulatory effect of LL-37 on NET formation. METHODS: Nasal polyp tissues were investigated for the presence of NETs by using immunofluorescent (IF) staining. The expression and distribution of LL-37 were examined by using quantitative RT-PCR, ELISA, IF, and immunohistochemistry. Purified peripheral neutrophils were stimulated with LL-37 and stained with IF to identify NETs. NETs% was defined as percentage of NET-generating neutrophils to the total number of neutrophils. RESULTS: Neutrophil extracellular traps were located in the subepithelial layer of nasal polyps and control tissues. Nasal polyps had higher NETs% compared with that of controls (23.01% ± 3.43% vs 4.52% ± 1.33%, P < 0.0001). NET count was also increased in nasal polyps. NET count correlated with neutrophil count (r = 0.908, P < 0.001). LL-37 protein and mRNA levels were upregulated in nasal polyps. LL-37 was distributed in the epithelial and subepithelial layer and mainly expressed by neutrophils. Moreover, LL-37 promoted peripheral neutrophils to form NETs in a dose-dependent manner ex vivo. Interestingly, dexamethasone did not inhibit the effect of LL-37 on inducing NET formation. Furthermore, peripheral neutrophils from CRSwNP patients were more susceptible to LL-37-mediated NET formation, compared with neutrophils derived from control subjects. In addition, NETs released LL-37 in vivo and ex vivo. CONCLUSION: Neutrophil extracellular traps are significantly increased in nasal polyps and LL-37 induces NET formation in CRSwNP patients. These findings indicate that NETs may contribute to the pathogenesis of neutrophilic inflammation in CRSwNP.

Epigenetic Modification of Enhancer of Zeste Homolog 2 Modulates the Activation of Dendritic Cells in Allergen Immunotherapy.

BACKGROUND: Allergen immunotherapy (AIT) is the only etiological and potentially curative therapy for allergic rhinitis (AR). OBJECTIVES: We sought to investigate the role of epigenetic regulator enhancer of zeste homolog 2 (EZH2) in the activation of dendritic cells (DCs) in AIT. METHOD: In this study, EZH2 expression in circulating myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) were evaluated using flow cytometry. Clinical information from 56 AR patients receiving AIT was collected, including 30 subjects with subcutaneous immunotherapy (SCIT) and 26 subjects with sublingual immunotherapy (SLIT). In vitro, the effect of EZH2 inhibitor, 3 Deazaneplanocin A (DZNep), on the phenotypic and functional activation of monocyte-derived DCs (moDCs) was evaluated. RESULTS: EZH2 expression in circulating mDCs and pDCs were both negatively correlated to treatment time of AIT (r = -0.39, p = 0.003 and r = -0.47, p = 0.0002, respectively). Furthermore, there was a higher correlation between EZH2 expression and AIT treatment time in the SCIT group compared to that of the SLIT group in mDCs (r = -0.42, p = 0.02 vs. r = -0.23, p = 0.26)and pDCs (r = -0.52, p = 0.003 vs. r = -0.33, p = 0.10). In vitro, the co-stimulatory molecules on moDCs, such as CD80, CD86, and CD83, were significantly inhibited by DZNep in a dose-dependent manner. The -DC-driven T-cell proliferation was suppressed by DZNep (MD = 22.88, 95% CI 7.809-37.96, p < 0.05). CONCLUSIONS: Our study shows that EZH2, which is required in the activation of DCs, mediates the epigenetic modification in AIT and stresses the importance of patient adherence during AIT.

A positive-feedback loop between tumour infiltrating activated Treg cells and type 2-skewed macrophages is essential for progression of laryngeal squamous cell carcinoma.

BACKGROUND: Foxp3+ regulatory T (Treg) cells and M2 macrophages are associated with increased tumour progression. However, the interaction between Treg cells and M2 macrophages remains unclear. METHODS: The expression of FoxP3 and CD163 was detected by immunohistochemistry in 65 cases of laryngeal squamous cell carcinoma (LSCC). In vitro, the generation of activated Treg (aTreg) cells and M2 macrophages by interactions with their precursor cells were analysed by flow cytometry and ELISA. In vivo, the antitumour effects were assessed by combined targeting aTreg cells and M2 macrophages, and intratumoural immunocytes were analysed by flow cytometry. RESULTS: In LSCC tissue, accumulation of aTreg cells and M2 macrophages predicted a poor prognosis and were positively associated with each other. In vitro, aTreg cells were induced from CD4+CD25- T cells by cancer cell-activated M2-like macrophages. Consequently, these aTreg cells skewed the differentiation of monocytes towards an M2-like phenotype, thereby forming a positive-feedback loop. Combined targeting aTreg cells and M2 macrophages led to potent antitumour immunity in vivo. CONCLUSIONS: The positive-feedback loop between aTreg cells and M2 macrophages is essential to maintain or promote immunosuppression in the tumour microenvironment and may be a potential therapeutic target to inhibit tumour progression.

Emerging drugs for head and neck cancer.

INTRODUCTION: Despite improvements in treatment, survival rates of head and neck squamous cell carcinoma (HNSCC) are stagnant. The existing chemotherapeutic agents are non-selective and associated with toxicities. Combinations of the only the US FDA-approved epidermal growth factor receptor (EGFR)-targeted agent, cetuximab, with chemotherapy or radiation improves overall survival. However, the response rates to cetuximab are modest. Thus, there is an urgent need for new agents that can be safely integrated into current treatment regimens to improve outcome. AREAS COVERED: Current EGFR-targeted drugs under clinical development include mAbs and tyrosine kinase inhibitors. The modest efficacy of these drugs implicates intrinsic or acquired resistance. Novel molecular agents inhibiting alternative targets to overcome anti-EGFR resistance in HNSCC are under investigation. Gene therapy and immunotherapy are also promising strategies to improve efficacy and reduce toxicity. EXPERT OPINION: To date, only six drugs have been FDA-approved for the treatment of head and neck cancer. Cetuximab is the only approved molecular targeting agent for HNSCC and despite ubiquitous expression of EGFR in HNSCC tumors, clinical responses are limited. Genetic and epigenetic characterization of HNSCC tumors, coupled with improved preclinical models, should facilitate the development of more effective drugs.

Increased expression of aldehyde dehydrogenase 1 A1 in nasopharyngeal carcinoma is associated with enhanced invasiveness.

Increased expression of aldehyde dehydrogenase 1 (ALDH1) has recently been reported in several cancers. However, whether member A1 of aldehyde dehydrogenase 1 (ALDH1A1) is involved in the formation of nasopharyngeal carcinoma (NPC) remains unknown. To investigate the expression of ALDH1A1 in NPC and its association with the tumorigenesis of NPC, we examined the expression of ALDH1A1 in NPC specimens using immunohistochemistry (IHC), quantitative RT-PCR (qRT-PCR) and Western blot. Moreover, we sorted ALDH1A1(high) cells from NPC cell line CNE-2 by flow cytometry and examined the expression of primitive embryonic stem cell markers OCT4, SOX2 and Nanog. Finally, we investigated the capacities of growth, proliferation, colony- formation and tumorigenesis of ALDH1A1(high) cells in vitro and in vivo. We found ALDH1A1 was significantly increased in human NPC samples via IHC, qRT-PCR and Western blot (p < 0.05). ALDH1A1(high) cells sorted from NPC cell line CNE-2 by flow cytometry had higher expression of primitive embryonic stem cell markers OCT4, SOX2 and Nanog, and showed enhanced capacities of growth, proliferation, colony formation and tumorigenesis in vitro and in vivo when compared with ALDH1A1(low) cells (p < 0.05). Our findings indicated that increased expression of ALDH1A1 in NPC was associated with enhanced invasiveness.

[Efficacy of endoscopic surgical management for recurrent nasopharyngeal carcinoma].

Compared with re-radiotherapy or open surgery, endoscopic surgery for recurrent nasopharyngeal carcinoma has a better effect, which not only improves the overall survival rate, but also reduces serious complications, improves the quality of life and saves medical costs. With the advancement of modern surgical equipment and instruments, the deepening of anatomical research in the field of skull base, the improvement of surgical techniques and the development of multidisciplinary teamwork, the safety of endoscopic surgery and the rate of complete tumor resection will also increase. The indications for surgery are further expanded, and more patients with recurrent nasopharyngeal carcinoma benefit from endoscopic minimally invasive surgery. This article reviews the efficacy of endoscopic surgery for recurrent nasopharyngeal carcinoma, and then provides insights for the selection of clinical treatment strategies for recurrent nasopharyngeal carcinoma.

Amphiregulin promotes activated regulatory T cell-suppressive function via the AREG/EGFR pathway in laryngeal squamous cell carcinoma.

BACKGROUND: Activated regulatory T cells (aTregs) play a vital role in promoting a tumor immunosuppressive microenvironment in laryngeal squamous cell carcinoma (LSCC). However, the regulatory factors that induce the generation of aTregs are not clear. Herein, we investigated the effect of amphiregulin (AREG) on the production of aTregs in the tumor microenvironment of LSCC. METHODS: Immunohistochemical (IHC) analysis was conducted to examine the expression of AREG and FOXP3, and their association with clinical parameters and patient outcomes was demonstrated. The expression level of EGFRs in three functional subsets of Tregs was assessed, and the induction of CD4+ T cells into aTregs in the presence or absence of AREG or Gefitinib was analyzed using flow cytometry. RESULTS: Our results showed a higher expression level of AREG was significantly related to advanced clinical stage and worse survival, particularly with increased infiltration of Tregs in LSCC tumor tissue. The in vitro study showed that AREG significantly promoted the differentiation of aTregs, and enhanced the inhibitory effect of Tregs on T cell proliferation, which could be reversed by epidermal growth factor receptor (EGFR) inhibitors. In addition, we found that EGFR was highly expressed in aTregs, but not in other subsets of Tregs. It is suggested that AREG might induce aTregs, and enhance the immunosuppressive function of Tregs via the AREG/EGFR signal pathway. CONCLUSIONS: Collectively, this study revealed the role and mechanism of AREG in negative immune regulation, and targeting AREG might be a novel immunotherapy for LSCC.

Intranasal PAMAM-G3 scavenges cell-free DNA attenuating the allergic airway inflammation.

Allergic airway inflammation (AAI), including allergic rhinitis (AR) and allergic asthma, is driven by epithelial barrier dysfunction and type 2 inflammation. However, the underlying mechanism remains uncertain and available treatments are constrained. Consequently, we aim to explore the role of cell-free DNA (cfDNA) in AAI and assess the potential alleviating effects of cationic polymers (CPs) through cfDNA elimination. Levels of cfDNA were evaluated in AR patients, allergen-stimulated human bronchial epithelium (BEAS-2B cells) and primary human nasal epithelium from both AR and healthy control (HC), and AAI murine model. Polyamidoamine dendrimers-generation 3 (PAMAM-G3), a classic type of cationic polymers, were applied to investigate whether the clearance of cfDNA could ameliorate airway epithelial dysfunction and inhibit AAI. The levels of cfDNA in the plasma and nasal secretion from AR were higher than those from HC (P < 0.05). Additionally, cfDNA levels in the exhaled breath condensate (EBC) were positively correlated with Interleukin (IL)-5 levels in EBC (R = 0.4191, P = 0.0001). Plasma cfDNA levels negatively correlated with the duration of allergen immunotherapy treatment (R = -0.4297, P = 0.006). Allergen stimulated cfDNA secretion in vitro (P < 0.001) and in vivo (P < 0.0001), which could be effectively scavenged with PAMAM-G3. The application of PAMAM-G3 inhibited epithelial barrier dysfunction in vitro and attenuated the development of AAI in vivo. This study elucidates that cfDNA, a promising biomarker for monitoring disease severity, aggravates AAI and the application of intranasal PAMAM-G3 could potentially be a novel therapeutic intervention for AAI. Allergen stimulates the secretion of cell-free DNA (cfDNA) in both human and mouse airway. Intranasal polyamidoamine dendrimers-generation 3 (PAMAM-G3) scavenges cfDNA and alleviates allergic airway inflammation.

Tackling Severe Neutrophilic Inflammation in Airway Disorders with Functionalized Nanosheets.

Severe airway inflammatory disorders impose a significant societal burden, and the available treatments are unsatisfactory. High levels of neutrophil extracellular trap (NET) and cell-free DNA (cfDNA) were detected in the inflammatory microenvironment of these diseases, which are closely associated with persistent uncontrolled neutrophilic inflammation. Although DNase has proven to be effective in mitigating neutrophilic airway inflammation in mice by reducing cfDNA and NET levels, its clinical use is hindered by severe side effects. Here, we synthesized polyglycerol-amine (PGA) with a series of hydroxyl/amine ratios and covered them with black phosphorus (BP) nanosheets. The BP nanosheets functionalized with polyglycerol-50% amine (BP-PGA50) efficiently lowered cfDNA levels, suppressed toll-like receptor 9 (TLR9) activation and inhibited NET formation in vitro. Importantly, BP-PGA50 nanosheets demonstrated substantial accumulation in inflamed airway tissues, excellent biocompatibility, and potent inflammation modulation ability in model mice. The 2D sheet-like structure of BP-PGA50 was identified as a crucial factor for the therapeutic efficacy, and the hydroxyl/amine ratio was revealed as a significant parameter to regulate the protein resistance, cfDNA-binding efficacy, and cytotoxicity. This study shows the promise of the BP-PGA50 nanosheet for tackling uncontrolled airway inflammation, which is also significant for the treatment of other neutrophilic inflammatory diseases. In addition, our work also highlights the importance of proper surface functionalization, such as hydroxyl/amine ratio, in therapeutic nanoplatform construction for inflammation modulation.

A novel inflammatory endotype diagnostic model based on cytokines in chronic rhinosinusitis with nasal polyps.

Background: Type 2 CRSwNP is characterized by severe symptoms, multiple comorbidities, longer recovery course and high recurrence rate. A simple and cost-effective diagnostic model for CRSwNP endotype integrating clinical characteristics and histopathological features is urgently needed. Objective: To establish a clinical diagnostic model of inflammatory endotype in CRSwNP based on the clinical characteristics, pathological characteristics, and cytokines profile in the polyp tissue of patients. Methods: A total of 244 participants with CRSwNP were enrolled at 2 different centers in China and Belgium from 2018 to 2020. IL-5 level of nasal polyp tissue was used as gold standard. Clinical characteristics were used to establish diagnostic models. The area under the receiver operating curve (AUC) was used to evaluate the diagnostic performance. The study was approved by the ethics board of the First Affiliated Hospital of Sun Yat-sen University ([2020] 302), and written informed consent was obtained from all subjects before inclusion. Results: In total, 134 patients from China (training set) and 110 patients from Belgium (validation set) were included. The logistic regression (LR) model in predicting inflammatory endotype of CRSwNP showed the AUC of 83%, which was better than the diagnostic performance of machine learning models (AUC of 61.14%-82.42%), and single clinical variables. We developed a simplified scoring system based on LR model which shows similar diagnostic performance to the LR model (P = 0.6633). Conclusion: The LR model in this diagnostic study provided greater accuracy in prediction of inflammatory endotype of CRSwNP than those obtained from the machine learning model and single clinical variable. This indicates great potential for the use of diagnostic model to facilitate inflammatory endotype evaluation when tissue cytokines are unable to be measured.

Improving Survival of T3cN0M0 Glottic Squamous Cell Cancer With Elective Neck Dissection.

OBJECTIVES/HYPOTHESIS: This study aimed to elucidate the role of elective neck dissection (END) in improving the outcome of T3cN0M0 glottic squamous cell cancer (GSCC). STUDY DESIGN: Retrospective population-based database analysis. METHODS: Patients with T3cN0M0 GSCC in the Surveillance, Epidemiology, and End Results database (SEER) were extracted and stratified into END and non-END cohorts. Propensity score matching (PSM) was used to eliminate the baseline variations. The Kaplan-Meier method was performed to access the association between END and survival. RESULTS: We retrospectively analyzed 1,589 T3cN0M0 GSCC patients in the SEER database from 2004 to 2015, and found that only 22% to 58% T3cN0M0 GSCC were performed with END. After PSM, END cohort had better overall survival (OS) (median survival time: 93 vs. 55 months, respectively; P = .0047) and cancer-specific survival (CSS) (hazard ratio [HR] 0.48, 95% confidence interval [CI] 0.29-0.77, P = .003) than non-END cohort. In addition, Subgroup analysis also indicated END cohort had better OS or CSS than non-END cohort. CONCLUSION: This study demonstrated that in patients with T3cN0M0 GSCC, END significantly associated with better survival outcomes compared with non-END. LEVEL OF EVIDENCE: 3 Laryngoscope, 132:1807-1816, 2022.