Single cell RNA sequencing of human eosinophils from nasal polyps reveals eosinophil heterogeneity in chronic rhinosinusitis tissue.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation in the United States, but the actual roles that eosinophils play in CRSwNP remain largely unclear. OBJECTIVE: To reveal the roles and heterogeneity of eosinophils in nasal polyp (NP) tissue, we performed single cell RNA sequencing (scRNA-Seq) analysis of NP tissue. METHODS: Sinonasal tissues (NP and control sinus tissue) and patient matched peripheral blood (PB) samples were obtained from 5 control patients and 5 patients with CRSwNP. Eosinophils were enriched before processing for scRNA-Seq. The gene expression profiles in eosinophils were determined by microwell-based scRNA-Seq technology (BD Rhapsody platform). We predicted the overall function of NP eosinophils by Gene Ontology (geneontology.org) enrichment and pathway analyses and confirmed expression of selected genes by flow cytometry. RESULTS: After filtering out contaminating cells, we detected 5,542 eosinophils from control PB, 3,883 eosinophils from CRSwNP PB, 101 eosinophils from control sinus tissues (not included in further analyses), and 9,727 eosinophils from NPs by scRNA-Seq. We found that 204 genes were downregulated and 354 genes upregulated in NP eosinophils compared to all PB eosinophils (>1.5-fold, Padj < .05). Upregulated genes in NP eosinophils were associated with activation, cytokine-mediated signaling, growth factor activity, NF-κB signaling, and antiapoptotic molecules. NP eosinophils displayed 4 clusters revealing potential heterogeneity of eosinophils in NP tissue. CONCLUSIONS: Elevated eosinophils in NP tissue appear to exist in several subtypes that may play important pathogenic roles in CRSwNP, in part by controlling inflammation and hyperproliferation of other cells.

Anesthesia management in a child with mucopolysaccharidosis and toxic epidermal necrolysis: A case report.

Toxic epidermal necrolysis and mucopolysaccharidosis are both rare diseases that pose significant airway maintenance challenges to anesthesiologists. In this report, we describe the anesthesia management in a 4-year-old male with mucopolysaccharidosis type II who developed toxic epidermal necrolysis and presented for ophthalmic surgical procedures. Combined use of propofol and ketamine with the support of high-flow nasal oxygen enabled adequate analgesia and sedation while maintaining spontaneous ventilation and airway patency. The strategy presented in this report may contribute to enhancing the safety of sedation in spontaneously breathing children with abnormal airways.

[A MULTICENTER CLINICAL SURVEY ABOUT THE PREVALENCE OF JAPANESE CYPRESS POLLINOSIS AND THE EFFICACY OF SUBLINGUAL IMMUNOTHERAPY WITH JAPANESE CEDAR POLLEN EXTRACT DURING JAPANESE CYPRESS POLLEN DISPERSAL PERIOD].

BACKGROUND: Little is known whether sublingual immunotherapy using Japanese cedar pollen extract (cedar SLIT) is effective for not only Japanese cedar pollinosis but also Japanese cypress pollinosis. We investigated the prevalence rate of Japanese cypress pollinosis, efficacy of cedar SLIT on cypress pollinosis and patients' wish to receive cypress SLIT. METHODS: We investigated a multi-center (31 institutions), cross-sectional survey using a self-administrated questionnaire with four questions for patients received cedar SLIT aged from 5 to 69 years old. RESULTS: 2523 subjects were enrolled for analysis. 83.4% of them had pollinosis symptoms during cypress season before cedar SLIT. In such patients, 37.4% experienced lessened efficacy of cedar SLIT during cypress season. Both the prevalence of cypress pollinosis and the lessened efficacy of cedar SLIT on cypress pollinosis were significantly seen in western Japan as compared to eastern Japan. 76.1% of the subject having cypress pollinosis before SLIT wished to receive cypress SLIT if it is available. CONCLUSION: A lessened efficacy of cedar SLIT during cypress season was broadly seen in Japan, and further showed a regional difference. Together with the finding of high wish by patients, these results suggest a development of cypress SLIT is desirable.

Muscle Fiber Composition Changes after Selective Nerve Innervation.

Facial nerve paralysis interferes with mimetic muscle function. To reconstruct natural facial movement, free muscle flaps are transplanted as new muscles. However, it is difficult to maintain resting tonus. A dual innervation technique in which other nerves such as the hypoglossal nerve or contralateral facial nerve are added is often applied. Using 10-week-old rats (n = 10), the masseteric and hypoglossal nerves were cut, and the distal stump of the masseteric nerve and the proximal stump of the hypoglossal nerve were then sutured (suture group). In the other group, the masseteric nerve was cut and cauterized (cut group). Immunohistochemistry and microarray were performed on the extracted masseter muscle. The immunohistochemistry results suggested that the muscles in the suture group obtained oxidative characteristics. The microarray showed the genes involved in mitochondrial function, including Perm1. In summary, our data support the validity of the dualinnervation technique for facial paralysis treatment.

Mapping naso-ocular symptom scores to EQ-5D-5L utility values in Japanese cedar pollinosis.

BACKGROUND: The total naso-ocular symptom score (TSS) is widely used as an endpoint to evaluate the severity of seasonal allergic rhinitis. However, it is not a generic preference-based measure. We sought to develop an algorithm for mapping between the TSS and health utility in Japanese cedar pollinosis (JCP). We also performed a cost-utility analysis of sublingual immunotherapy (SLIT) for JCP by using this algorithm. METHODS: Patients with JCP filled out the TSS questionnaire and EQ-5D-5L simultaneously during the pollen season in 2019 and in 2020. We estimated a direct utility mapping model by regressing responses to individual TSS questions directly onto utility. The incremental cost-effectiveness ratio (ICER) of active SLIT to a placebo was determined by examining the drug expense and the estimated quality-adjusted life year (QALY) using a dataset from a double-blind placebo-controlled clinical trial. RESULTS: A total of 238 records were included for analysis. The estimated utility decreased with increasing severity of rhinitis. Patients with comorbid asthma showed lower utility. A negative and significant correlation was seen between the TSS and utility in both 2019 and 2020. The estimated equations were: Y(utility) = -0.0161∗X(TSS) + 1.005 in non-asthmatic JCP patients. The ICER of active SLIT to the placebo was estimated to be 4,049,720 and 6,011,218 JPY/QALY in the first and second year, respectively. CONCLUSIONS: It is possible to reasonably predict utility from the total naso-ocular symptom score by using regression models. In the estimated algorithm, pre-seasonal SLIT for JCP is cost-effective.

IgG4 Expression in Patients with Eosinophilic Otitis Media.

OBJECTIVE: Eosinophilic otitis media (EOM) is an intractable middle ear disease recognized by an eosinophil enriched middle ear effusion and mucosa. Although precise pathogenesis of EOM remains unclear, it is characterized by type 2 inflammation. Since IgG4 is an IgG subclass induced by type 2 cytokines such as IL-4 and IL-13, we sought to characterize and compare local IgG4 expression in patients with and without EOM. METHODS: Twelve patients with bilateral profound hearing loss, 9 of which underwent a cochlear implant surgery, were enrolled in this study (6 with EOM and 6 without EOM). The surgical specimens were harvested during surgery and were subjected to IgG4 immunostaining. RESULT: The middle ear mucosa showed the presence of a large number of IgG4-positive cells in patients with EOM, which was significantly higher than that in patients without EOM. CONCLUSION: Local IgG4 expression was observed in patients with EOM in comparison to those without EOM, suggesting that IgG4 contributes to EOM pathogenesis.

Serum IgG4 as a biomarker reflecting pathophysiology and post-operative recurrence in chronic rhinosinusitis.

BACKGROUND: Type 2 chronic rhinosinusitis (CRS), especially eosinophilic CRS (ECRS), is an intractable upper airway inflammatory disease. Establishment of serum biomarkers reflecting the pathophysiology of CRS is desirable in a clinical setting. As IgG4 production is regulated by type 2 cytokines, we sought to determine whether serum IgG4 levels can be used as a biomarker for CRS. METHODS: Association between the serum IgG4 levels and clinicopathological factors was analyzed in 336 CRS patients. Receiver operating characteristics (ROC) analysis was performed to determine the cut-off value of serum IgG4 levels that can be used to predict the post-operative recurrence. RESULTS: Serum IgG4 levels were significantly higher in patients with moderate to severe ECRS versus those with non to mild ECRS. The levels were also significantly higher in asthmatic patients and patients exhibiting recurrence after surgery compared to controls. ROC analysis determined that the best cut-off value for the serum IgG4 level to predict the post-operative recurrence was 95 mg/dL. The corresponding sensitivity and specificity were 39.7% and 80.5%, respectively. When we combined the two cut-off values for the serum IgG4 and periostin, patients with high serum levels of either IgG4 or periostin exhibited a high post-operative recurrence (OR: 3.95) as compared to patients having low serum levels of both IgG4 and periostin. CONCLUSIONS: The present results demonstrate that the serum IgG4 level is associated with disease severity and post-operative course in CRS. In particular, the combination of serum IgG4 and periostin could be a novel biomarker that predicts post-operative recurrence.

Impact of Preoperative Systemic Corticosteroids on the Histology and Diagnosis of Eosinophilic Chronic Rhinosinusitis.

BACKGROUND: The histological changes corticosteroids induce in nasal polyps, and whether these changes have an impact on the diagnosis of eosinophilic chronic rhinosinusitis (ECRS), currently remain unclear. OBJECTIVES: A prospective controlled multicenter trial was conducted to evaluate the efficacy of the low-dose and short-term oral prednisolone (oPSL) treatment for tissue eosinophil infiltrations in ECRS. METHODS: Subjects with ECRS diagnosed by previous biopsies received a low dose of oPSL for 3 days (PSL 3) or 7 days (PSL 7) before surgery. Changes in the tissue eosinophil count after these treatments were evaluated. Furthermore, the percent change of tissue eosinophil count from baseline and its impact on the diagnosis defined by the JESREC study were examined. RESULTS: There were 23 and 21 subjects in the PSL 3 and PSL 7 groups, respectively. Polyp scores, clinical symptom scores, and the proportion of blood eosinophils significantly decreased after the treatment, and no significant differences were observed between the groups. The entire tissue eosinophil count tended to be slightly decreased in both groups without reaching a statistically significant value. The median percent change of tissue eo-sinophil count from baseline was 83.6%, and only the posttreatment proportion of blood eosinophil showed a mild correlation with it. Seven out of 44 nasal polyp specimens collected from the superficial part of the middle meatus showed < 70 eosinophils/high-power field; therefore, the false negative rate was 15.9%, but decreased to 11.4% when other parts were included in the histological evaluation. CONCLUSIONS: Low-dose and short-term oPSL did not appear to markedly affect the tissue eosinophil count in ECRS patients; however, the potential for misdiagnoses due to the effects of oPSL cannot be rejected. The diagnosis of ECRS prior to the administration of corticosteroids or tissue evaluations using multiple tissue parts is desirable.

Role of whole saliva in the efficacy of sublingual immunotherapy in seasonal allergic rhinitis.

BACKGROUND: The development of methods to predict the clinical effectiveness of sublingual immunotherapy (SLIT) for allergic diseases is a crucial matter. We sought to determine whether whole saliva, which is the first body component that contacts allergen extracts during SLIT, is associated with the clinical effectiveness of SLIT in Japanese cedar pollinosis. METHODS: Blood monocytes or monocytic THP-1 cells were cultured in the presence or absence of either whole saliva or pure saliva with or without treatments including filtration and blockade of TLR2 and/or TLR4 signaling. IL-10 levels in the supernatants were then measured. Whole saliva-induced IL-10 production by THP-1 cells was compared between asymptomatic and disease-onset patients during peak pollen dispersal after SLIT. RESULTS: Both monocytes and THP-1 cells produced substantial amounts of IL-10 in response to whole saliva. IL-10 production was significantly reduced in response to pure saliva and 0.2 µm-filtered saliva. Simultaneous treatment with polymyxin B and TL2.1, a neutralizing antibody against TLR2, also reduced IL-10 production. IL-10 levels produced by THP-1 cells in response to whole saliva collected prior to SLIT were significantly higher in asymptomatic patients determined by symptom-medication scores than disease-onset patients following SLIT. Such differences were not seen in saliva collected 3 months after the initiation of SLIT or saliva collected during peak pollen dispersal. CONCLUSIONS: Our results provide a basis for why the sublingual route is effective and preferable in allergen immunotherapy. Saliva-induced IL-10 levels produced by THP-1 cells may be a predictive marker for clinical remission after SLIT.

A Study of Cases of Brainstem/cerebellar Infarction Detected as False Negatives by Initial MRI.

Brainstem/cerebellar infarction is known to cause various cranial nerve symptoms that may require otorhinolaryngological evaluation. Acute-phase cerebellar infarction is evaluated by MRI with diffusion-weighted imaging (MRI-DWI). However, in the acute phase, MRI-DWI may show false-negative results, because of which patients are referred to the department of otolaryngology for further evaluation of the cranial nerve symptoms. We investigated 250 cases of brainstem/cerebellar infarction in 245 patients who were admitted to our hospital between 2010 and 2015. Of the 250 cases, eight cases were diagnosed at the department of otolaryngology after detailed evaluators for dizziness or dysphagia, and three of them were false negative on initial MRI-DWI. In total, we examined 16 cases detected as false negatives upon initial MRI-DWI. Of the 16 cases, 12 were brainstem infarctions, three were cerebellar infarctions, and one was infarction of the brainstem and cerebellum. All 16 cases were evaluated by initial MRI-DWI within 12 h of onset. Careful observation of the neurological findings and follow-up MRI-DWI are useful for the detailed evaluation of patients suspected to have a cerebellar infarction.

Minimally invasive lymphatic supermicrosurgery (MILS): indocyanine green lymphography-guided simultaneous multisite lymphaticovenular anastomoses via millimeter skin incisions.

Among various surgical treatments, lymphaticovenular anastomosis (LVA), which bypasses congested lymph into venous circulation, is the least invasive surgical treatment. However, it usually entails skin incisions of around 3 cm, and operation time of around 4 hours. With multiple supermicrosurgeons under guidance of indocyanine green lymphography, LVAs can be simultaneously performed under local anesthesia within approximately 2 hours via small skin incisions with length less than 1 cm, allowing minimally invasive lymphatic supermicrosurgery (MILS). We performed MILS on 11 limbs of compression-refractory peripheral lymphedema cases. Length of skin incision for LVA ranged from 1 to 9 mm. Average operation time was 1.82 hours. Of the 11 limbs, 10 showed postoperative volume reduction. Indocyanine green lymphography clearly visualizes superficial lymph flows, which helps us to decide precise skin incision sites and find lymphatic vessels in LVA surgery, shortening skin incision length and operation time. Minimally invasive lymphatic supermicrosurgery can serve as the most reasonable treatment of compression-refractory peripheral lymphedema.

Predictive lymphatic mapping: a method for mapping lymphatic channels in patients with advanced unilateral lymphedema using indocyanine green lymphography.

In severe lymphedema, indocyanine green lymphography cannot be used to map lymphatic channels before lymphaticovenular anastomosis (LVA) because linear lymphatics cannot be detected in a severely affected leg. Here, we describe a new method, which we refer to as predictive lymphatic mapping, to predict the location of lymphatics for anastomosis in unilateral lymphedema, thereby improving surgical accuracy and efficiency. The approach consists of marking anatomical landmarks and joining selected landmarks with fixed lines. The distance from these fixed lines to lymphatic channels mapped by indocyanine green lymphography in the unaffected leg is then measured, scaled up based on the difference in circumference between the legs, and transposed to the affected leg. To date, we have used this method in 5 cases of unilateral or asymmetric lymphedema of the lower extremities. In no cases have we failed to find a lymphatic channel suitable for LVA within a 2-cm incision. These results suggest that predictive lymphatic mapping is a useful additional tool for surgeons performing LVA under local anesthesia, which will help to improve the accuracy of incisions and the efficiency of surgery.

[A review of the complications and candida colonization associated with voice rehabilitation using a voice prosthesis].

Between December 2004 and December 2011, we have used the indwelling voice prosthesis for voice rehabilitation in 28 patients after total laryngectomy in our department. To clarify both the complications and safety of voice reconstruction using a voice prosthesis, and the occurrence of candida colonization, we conducted a retrospective study with a review of the Japanese literature. Twenty-six patients who were observed over a period of more than six months at our hospital were enrolled in this study. We examined the interval of prosthesis replacement, types and frequency of complications, candida colonization, and clinical outcomes. The median follow-up time postoperatively was 28.8 months (range 8.1-95.7). The average interval of prosthesis replacement was 147 days (4.9 months). Complications occurred in 14 patients (54%), and 6 patients (23%) of whom were hospitalized. The main complications were periprosthetic leakage, increased granulation around the tracheoesophageal shunt and stenosis of the trachea stoma. There were no significant differences in the frequency of complications in the background factor of the patients as far as age (p = 0.495), radiation therapy (p = 0.686) or reconstruction time (p = 0.257) were concerned. Candida species was detected in 81% of the specimens which we submitted to a culture test and confirmed the pseudohyphae which comfirmed the pathogenicity from the histopathological examination. Moreover, radiation therapy was significantly associated with the detection of candida (p = 0.004). Permanent closure of the tracheoesophageal shunt for periprosthetic leakage was required in one patient, but we were able to deal with the other complications. No patient experienced any life-threatening complications and all are safely using the prostheses. It has been reported that complications will occur over the long-term, thus careful follow-up is necessary.

Pathogenesis-based application of biologics for chronic rhinosinusitis: Current and future perspectives.

Chronic rhinosinusitis (CRS) is heterogeneous and contains diverse pathogenesis including type 1, type 2, and/or type 3 inflammation. For severe type 2 CRS especially CRS with nasal polyps (CRSwNP), biologics that target inflammatory molecules have recently been applied along with further changes in the treatment algorithm for CRS. Currently, a completed phase 3 clinical trial for biologics for severe CRSwNP with inadequate response to surgery and/or intranasal corticosteroids, including omalizumab (anti-IgE), mepolizumab (anti-IL-5), benralizumab (anti-IL-5Rα), and dupilumab (anti-IL-4Rα), have all shown efficacy. Similar phase 3 clinical trials for tezepelumab (anti-TSLP) and etokimab (anti-IL-33) are now underway and completed, respectively. Further studies need to evaluate how to optimally and cost-effectively use biologics for CRS and determine if any biomarkers are indicative of which biologics should be administered. A definition of complete and/or clinical remission of CRS is also needed to determine when to reduce or discontinue biologics. In addition, more precise basic research on CRS, such as endotyping and genotyping, will need to be undertaken in order to determine novel targets for biologics.

Gene expression of cytokines and prostaglandin metabolism-related proteins in eosinophilic otitis media.

The objective of this study was to investigate the levels of gene expression in the middle ear mucosa of 2 patients diagnosed with eosinophilic otitis media. One patient with severe hearing loss showed high expression levels of genes encoding IL-5 and IL-33 receptors.