Time-series transcriptome analysis of peripheral blood mononuclear cells obtained from individuals who received the SARS-CoV-2 mRNA vaccine.

Messenger ribonucleic acid (mRNA) vaccination against coronavirus disease 2019 (COVID-19) is an effective prevention strategy, despite a limited understanding of the molecular mechanisms underlying the host immune system and individual heterogeneity of the variable effects of mRNA vaccination. We assessed the time-series changes in the comprehensive gene expression profiles of 200 vaccinated healthcare workers by performing bulk transcriptome and bioinformatics analyses, including dimensionality reduction utilizing the uniform manifold approximation and projection (UMAP) technique. For these analyses, blood samples, including peripheral blood mononuclear cells (PBMCs), were collected from 214 vaccine recipients before vaccination (T1) and on Days 22 (T2, after second dose), 90, 180 (T3, before a booster dose), and 360 (T4, after a booster dose) after receiving the first dose of BNT162b2 vaccine (UMIN000043851). UMAP successfully visualized the main cluster of gene expression at each time point in PBMC samples (T1-T4). Through differentially expressed gene (DEG) analysis, we identified genes that showed fluctuating expression levels and gradual increases in expression levels from T1 to T4, as well as genes with increased expression levels at T4 alone. We also succeeded in dividing these cases into five types based on the changes in gene expression levels. High-throughput and temporal bulk RNA-based transcriptome analysis is a useful approach for inclusive, diverse, and cost-effective large-scale clinical studies.

Heterogeneity assessment of vaccine-induced effects using point-of-care surrogate neutralization test for severe acute respiratory syndrome coronavirus 2.

INTRODUCTION: Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic even after vaccination. We aimed to identify immunological heterogeneity over time in vaccinated healthcare workers using neutralization antibodies and neutralizing activity tests. METHODS: Serum samples were collected from 214 healthcare workers before vaccination (pre) and on days 22, 90, and 180 after receiving the first dose of BNT162b2 vaccine (day 0). Neutralization antibody (NAb, SARS-CoV-2 S-RBD IgM/IgG) titers and two kinds of surrogate virus neutralization tests (sVNTs) were analyzed (UMIN000043851). RESULTS: The NAb (SARS-CoV-2 S-RBD IgG) titer peaked on day 90 after vaccination (30,808.0 µg/ml ± 35,211; p < 0.0001) and declined on day 180 (11,678.0 µg/ml ± 33,770.0; p < 0.0001). The neutralizing activity also peaked on day 90 and declined with larger individual differences than those of IgG titer on day 180 (88.9% ± 15.0%, 64.8% ± 23.7%, p < 0.0001). We also found that the results of POCT-sVNT (immunochromatography) were highly correlated with those of conventional sVNT (ELISA). CONCLUSIONS: Neutralizing activity is the gold standard for vaccine efficacy evaluation. Our results using conventional sVNT showed large individual differences in neutralizing activity reduction on day 180 (64.8% ± 23.7%), suggesting an association with the difference in vaccine efficacy. POCT-sVNT is rapid and user-friendly; it might be used for triage in homes, isolation facilities, and event venues without restrictions on the medical testing environment.

Mouthwash-Based Highly Sensitive Pyro-Genotyping for Nine Sexually Transmitted Human Papilloma Virus Genotypes.

Human papillomavirus (HPV) is a common sexually transmitted infection worldwide, which spreads via contact with infected genital, anal, and oral/pharyngeal areas (oral sex) owing to diverse manners of sexual intercourse. In this study, we devised an oral HPV detection method using mouthwash waste fluids that causes less psychological resistance to visiting the outpatient otolaryngology departments. We successfully detected only the specific unique reverse sequencing probe (using pyro-genotyping) and identified the nine genotypes of HPV targeted for vaccination by pyrosequencing the mouthwash waste fluids of non-head and neck cancer patient volunteers (n = 52). A relatively large number (11/52) of mouthwash waste fluids tested positive for HPV (21.2%; genotype 6, n = 1; 11, n = 1; 16, n = 1; and 18, n = 8). These results surpassed the sensitivity observed testing the same specimens using the conventional method (1/52, 1.9%). Our method (pyro-genotyping) was developed using nine HPV genotypes targeted for vaccination and the results were highly sensitive compared to those of the conventional method. This less expensive, high-throughput, and simple method can be used for detecting oral HPV infection with fewer socio-psychological barriers.

Analysis of anatomical variation of the inclination of lamellas attached to the skull base and its correlation with the anterior ethmoidal artery floating in the ethmoid sinus for use in endoscopic sinus surgery.

PURPOSE: Attention to the inclination of lamellas attached to the skull base, including the basal lamella of the middle turbinate, facilitates the intraoperative identification of each lamella without requiring the use of a navigation system. We classified the inclination between the lamella and the skull base in preoperative computed tomography (CT) images and examined the relationship between the lamellas attached to the skull base, including the basal lamella of the middle turbinate, and the position of the anterior ethmoidal artery (AEA). We aimed to develop a preoperative classification to help prevent intraoperative injury of the AEA. METHODS: We retrospectively investigated the paranasal sinus sagittal section CT slices of 366 sides of 183 patients to assess the inclination of lamellas attached to the skull base and the AEA location. We also reviewed the AEA position, its correlation with the supraorbital ethmoid cell, and the lateral lamella of the cribriform plate. RESULTS: We classified the lamella inclination at the skull base as the anterior direction, perpendicular direction, and posterior direction types. Lamellas containing a floating AEA inclined in the anterior direction toward the skull base were observed in 68.9% of sides, inclination in the perpendicular direction was noted in 30.5% of sides, and inclination in the posterior direction was noted in 0.5% of sides. CONCLUSION: It is easier to identify the AEA intraoperatively when the lamella inclination of the skull base attachment is recognized based on preoperative CT findings. This approach could be applied to all paranasal sinus lamellas and assist in identifying the AEA and other nearby structures.

Anatomical variation of the pneumatized superior turbinate and its impact on endoscopic sinus surgery in chronic rhinosinusitis.

PURPOSE: The posterior ethmoid sinus is adjacent to important structures, such as the orbit, optic nerve, skull base, and ostium of the sphenoid sinus. The purpose of this study was to examine the effect of pneumatization of the superior turbinate (ST) and its basal lamella, and of the position of the anterior wall of the sphenoid sinus, on opening of the posterior ethmoid and sphenoid sinuses. METHODS: On axial, coronal, and sagittal computed tomography images, 394 sinuses of 197 patients who underwent endoscopic sinus surgery at Toho University Omori Medical Center in Tokyo, Japan, were classified according to the presence or absence of pneumatization of the ST and its basal lamella. The basal lamella of the ST was classified separately into the vertical and horizontal portions. We examined whether the classification of the anterior wall of the sphenoid sinus was associated with the structure of the ST. RESULTS: Pneumatization was observed in the ST in 28 sinuses (7.1%), in the vertical portion of the basal lamella in 127 (32.2%), and in the horizontal portion of the basal lamella in 90 (22.8%). Pneumatization in the horizontal portion of the basal lamella was significantly more common in the anterior sphenoidal wall classified as optic-canal type. CONCLUSION: Consideration should be given to the pneumatization of the ST and its basal lamella and optic-canal-type anterior sphenoidal wall, because these reduce the volume of the posterior-most ethmoid cell and may increase the risk of damaging the skull base and optic nerve.

Homozygous deletions of UGT2B17 modifies effects of smoking on TP53-mutations and relapse of head and neck carcinoma.

BACKGROUND: Smoking induces oncogenic TP53-mutations in head and neck squamous cell carcinomas (HNSCCs). Disruptive mutations of TP53-gene and expression of p16 protein [p16 (+)] in tumor tissue associate with worse and better prognosis, respectively. UDP-glucuronosyltransferase 2 family, polypeptide B17 (UGT2B17) detoxifies smoking-related metabolites. Differences among ethnic groups in UGT2B17 are extremely high. Homozygous deletions of UGT2B17 gene (UGT2B17-deletion) are a common copy number variant (CNV) among Japanese, but not a common CNV among Africans and Europeans. Thus, we examined Japanese patients with HNSCC to explore if UGT2B17-deletion and/or p16 (+) modify effects of smoking on TP53-mutations and affect relapse. METHODS: We conducted a posthoc analysis of a prospective cohort. Polymerase chain reaction, immunohistochemistry, and direct sequencing were used to determine UGT2B17-deletion, p16 (+), and detailed TP53-mutations, respectively. RESULTS: UGT2B17-deletion was observed in 80% of this study population. For this 80%, TP53-mutations were significantly more common among smokers than non-smokers (P = 0.0016), but this difference between smokers and nonsmokers was not significant for the 20% with UGT2B17. In patients with UGT2B17-deletion and p16 (+), simultaneously, TP53-mutations were much more common among smokers than among non-smokers (81% versus 17%; P = 0.0050). Patients with both UGT2B17-deletion and disruptive TP53-mutations had higher relapse rates than other patients (hazard ratio, 2.22; 95% confidence interval, 1.30 to 3.80, P = 0.004) in a stepwise method. CONCLUSIONS: These results suggest that UGT2B17-deletion interacting with p16 (+) may modify effects of smoking on TP53-mutations and may further interact with the disruptive TP53-mutations to raise relapse rates among Japanese patients with HNSCC.

'Precursory symptoms, awareness or progression of facial palsy' are more useful than 'forehead wrinkling ability' in differentiating central facial palsy examined in the emergency department.

BACKGROUND: Forehead wrinkling ability has been considered to be the sign of the central facial palsy (CFP). AIMS/OBJECTIVES: To identify characteristics of peripheral FP (PFP) patients in the emergency room (ER), differentiate PFP from central FP (CFP), and assess the utility of forehead wrinkling for this purpose. MATERIALS AND METHODS: ER patients with FP were clinically split into PFP (72 patients) and CFP (161 patients) groups. Factors like age, sex, medical history, time from onset to consultation, symptom awareness or progression, precursory symptoms, forehead wrinkling, and imaging history were compared. Multivariate analysis differentiated PFP from CFP, examining misdiagnosis risks based on forehead wrinkling. RESULTS: Precursory symptoms and symptom awareness or progression had the highest odds ratios. Some PFP patients could wrinkle their foreheads, typically examined within 1 day of symptoms. PFP patients had more same-day imaging than those assessed a day later. CONCLUSIONS AND SIGNIFICANCE: Forehead wrinkling, a traditional CFP sign, is also common in early-stage PFP, decreasing its diagnostic reliability. Patients with solely CFP unable to wrinkle the forehead are very rare at a single institution. Evaluating precursors symptoms, and FP awareness and progression is crucial for differentiation.

Bilateral Nontuberculous Mycobacterial Otitis Media: A Case Report.

Established treatment strategies for nontuberculous mycobacterial (NTM) infections are currently lacking, and whether surgical treatment should be applied in combination with antibiotic therapy remains debatable. Here, we report a case of bilateral otitis media caused by Mycobacterium abscessusa, a highly antibiotic-resistant bacterium. Many reported cases of NTM otitis media are unilateral, in which hearing of the contralateral ear is preserved. In the present case, strategies to improve hearing outcomes were considered, as both ears were affected. A 27-year-old woman presented with bilateral otorrhea that had lasted for the past 9 months. Bacterial culture showed M. abscessus in both ears. Based on drug sensitivity tests, clarithromycin, amikacin, and imipenem were administered. Three days after treatment initiation, diseased tissues were removed from the right middle ear, which had impaired hearing. On day 38, otorrhea stopped in both ears, and the hearing improved. Computed tomography revealed air in both middle ears. No apparent recurrence was detected. Under the same antibiotic therapy, resolution of diseased tissues and improvement in hearing were similar between the ears with and without surgery, suggesting that surgery is not always necessary. This finding may be incorporated into the treatment guidelines for NTM infections in the future.

Risk factors for late postoperative bleeding after partial glossectomy for tongue cancer.

BACKGROUND: Partial glossectomy is the most common procedure for early-stage tongue cancer. Although late postoperative bleeding occasionally occurs, the associated risk factors have not been adequately identified. AIMS/OBJECTIVES: We aimed to investigate the rate and risk factors for late postoperative bleeding after transoral partial glossectomy with or without neck dissection for tongue cancer at our institution. MATERIAL AND METHODS: We analysed 211 patients who had undergone transoral partial glossectomy between January 2016 and January 2023. The potential risk factors associated with late postoperative bleeding were investigated using univariate and multivariate logistic regression analyses. RESULTS: Of the 211 patients, 40 (19%) showed late postoperative bleeding, with 19 (9%) classified as grade IIIa (Clavien-Dindo classification). Regarding all grades, late postoperative bleeding was significantly higher in patients aged <70 years and in those with polyglycolic acid (PGA) sheets (p = .046 and .030, respectively). For grade ≥ IIIa, late postoperative bleeding was significantly higher in patients with a history of anticoagulant/platelet administration, a mucosal defect covered with fibrin glue and a PGA sheet (p = .045 and .026, respectively). CONCLUSIONS AND SIGNIFICANCE: The findings of this study suggest that primary closure decreases the frequency of late postoperative bleeding.

Alternaria inhibits double-stranded RNA-induced cytokine production through Toll-like receptor 3.

BACKGROUND: Fungi may be involved in asthma and chronic rhinosinusitis (CRS). Peripheral blood mononuclear cells from CRS patients produce interleukin (IL)-5, IL-13 and interferon (IFN)-γ in the presence of Alternaria. In addition, Alternaria produces potent Th2-like adjuvant effects in the airway. Therefore, we hypothesized that Alternaria may inhibit Th1-type defense mechanisms against virus infection. METHODS: Dendritic cells (DCs) were generated from mouse bone marrow. The functional responses were assessed by expression of cell surface molecules by FACS (MHC class II, CD40, CD80, CD86 and OX40L). Production of IL-6, chemokine CXCL10 (IP-10), chemokine CXCL11 (I-TAC) and IFN-ß was measured by ELISA. Toll-like receptor 3 (TLR3) mRNA and protein expression was detected by quantitative real-time PCR and Western blot. RESULTS: Alternaria and polyinosinic-polycytidylic acid (poly I:C) enhanced cell surface expression of MHC class II, CD40, CD80, CD86 and OX40L, and IL-6 production in a concentration-dependent manner. However, Alternaria significantly inhibited production of IP-10, I-TAC and IFN-ß, induced by viral double-stranded RNA (dsRNA) mimic poly I:C. TLR3 mRNA expression and protein production by poly I:C were significantly inhibited by Alternaria. These reactions are likely caused by heat-stable factor(s) in Alternaria extract with >100 kDa molecular mass. CONCLUSION: These findings suggest that the fungus Alternaria may inhibit production of IFN-ß and other cytokines by DCs by suppressing TLR3 expression. These results indicate that Alternaria may inhibit host innate immunity against virus infection.

Increased CXCL10 expression in nasal fibroblasts from patients with refractory chronic rhinosinusitis and asthma.

BACKGROUND: Chronic rhinosinusitis (CRS) is characterized by local inflammation of the sinonasal tissues. CRS patients with nasal polyps and asthma often develop acute exacerbation of sinonasal symptoms after upper respiratory tract infections. However, the influence of concomitant asthma on the nasal immune response to viral infection remains unclear. METHODS: Specimens of nasal polyp and mucosal tissues were obtained from 3 groups of CRS patients (n = 14 per group): 1) patients without asthma (CRS group), 2) patients with aspirin-tolerant asthma (ATA group), and 3) patients with aspirin-intolerant asthma (AIA group). Nasal fibroblasts isolated from the specimens were stimulated with poly I:C. CXCL10 expression was analyzed by the quantitative real-time polymerase chain reaction and enzyme-linked immunoadsorbent assay. Biopsy specimens from CRS patients without asthma were subjected to immunohistochemistry for detection of T-bet and GATA-3 expression in CD3+ T cells by double labeling. RESULTS: Nasal fibroblasts from the ATA and AIA groups showed significantly enhanced expression of CXCL10 mRNA and protein after poly I:C stimulation compared with cells from the CRS group and the control group (normal nasal mucosa). In addition to T helper (Th)2 cells, there was more abundant infiltration of Th1 cells into tissues from the AIA and ATA groups. CONCLUSIONS: Our findings suggest that CRS associated with asthma may become intractable through the over-production of CXCL10 in response to viral infection.

Indications of Effective Hyperbaric Oxygen Therapy Combined With Steroid Therapy for Sudden Hearing Loss.

OBJECTIVE: This study evaluated the therapeutic effect of hyperbaric oxygen therapy (HBOT) combined with steroid therapy to treat sudden hearing loss and examined the index associated with excellent therapeutic effect. METHODS: We included 109 patients with sudden hearing loss. Patients were divided into the HBOT combination group (59 sides) treated with HBOT and steroid therapy and HBOT noncombination group (50 sides) involving steroid therapy only. The recovery rate of each group was compared according to the severity of hearing loss. Blood samples were evaluated and inflammatory markers, such as neutrophil-lymphocyte ratio (NLR) and platelet-lymphocyte ratio (PLR), were calculated and compared by severity. We evaluated the usefulness of inflammatory markers for predicting recovery rate, and calculated cutoff values were also evaluated. RESULTS: The HBOT combination group exhibited a higher overall recovery rate than the HBOT noncombination group, particularly in severe cases. However, there was no significant difference in the severity of hearing loss based on various inflammatory markers. NLR and PLR are useful for predicting the effect in patients treated with concomitant HBOT. By setting 2.43 and 146.67 as cutoff values for NLR and PLR, respectively, we observed that lower values resulted in better recovery rates. CONCLUSION: The use of HBOT is effective for severe cases and early blood flow disorders with low NLR and PLR and less inflammation. When determining treatment, not only should the severity of hearing loss be considered, but also the NLR and PLR should be evaluated and examined based on the cutoff values.

In situ electron tomography for the thermally activated solid reaction of anaerobic nanoparticles.

The nanoscale characterization of thermally activated solid reactions plays a pivotal role in products manufactured by nanotechnology. Recently, in situ observation in transmission electron microscopy combined with electron tomography, namely four-dimensional observation for heat treatment of nanomaterials, has attracted great interest. However, because most nanomaterials are highly reactive, i.e., oxidation during transfer and electron beam irradiation would likely cause fatal artefacts; it is challenging to perform the artifact-free four-dimensional observation. Herein, we demonstrate our development of a novel in situ three-dimensional electron microscopy technique for thermally activated solid-state reaction processes in nanoparticles (NPs). The sintering behaviour of Cu NPs was successfully visualized and analyzed in four-dimensional space-time. An advanced image processing protocol and a newly designed state-of-the-art MEMS-based heating holder enable the implementation of considerably low electron dose imaging and prevent air exposure, which is of central importance in this type of observation. The total amount of electron dose for a single set of tilt-series images was reduced to 250 e- nm-2, which is the lowest level for inorganic materials electron tomography experiments. This study evaluated the sintering behaviour of Cu NPs in terms of variations in neck growth and particle distance. A negative correlation between the two parameters is shown, except for the particle pair bound by neighbouring NPs. The nanoscale characteristic sintering behavior of neck growth was also captured in this study.

Preoperative prediction of sinonasal papilloma by artificial intelligence using nasal video endoscopy: a retrospective study.

Sinonasal inverted papilloma (IP) is at risk of recurrence and malignancy, and early diagnosis using nasal endoscopy is essential. We thus developed a diagnostic system using artificial intelligence (AI) to identify nasal sinus papilloma. Endoscopic surgery videos of 53 patients undergoing endoscopic sinus surgery were edited to train and evaluate deep neural network models and then a diagnostic system was developed. The correct diagnosis rate based on visual examination by otolaryngologists was also evaluated using the same videos and compared with that of the AI diagnostic system patients. Main outcomes evaluated included the percentage of correct diagnoses compared to AI diagnosis and the correct diagnosis rate for otolaryngologists based on years of practice experience. The diagnostic system had an area under the curve of 0.874, accuracy of 0.843, false positive rate of 0.124, and false negative rate of 0.191. The average correct diagnosis rate among otolaryngologists was 69.4%, indicating that the AI was highly accurate. Evidently, although the number of cases was small, a highly accurate diagnostic system was created. Future studies with larger samples to improve the accuracy of the system and expand the range of diseases that can be detected for more clinical applications are warranted.

Alternaria fungus induces the production of GM-CSF, interleukin-6 and interleukin-8 and calcium signaling in human airway epithelium through protease-activated receptor 2.

RATIONALE: Recent studies suggest that host immune responses to environmental fungi may play an important role in the development of allergic diseases, such as human asthma. Epithelium is considered an active participant in allergic inflammation. We previously reported that aspartate protease from Alternaria induces the activation and degranulation of human eosinophils that are mediated through protease-activated receptor 2 (PAR-2). However, our current knowledge on the innate immune responses of epithelium to environmental fungi is very limited. We investigated the responses of epithelium to fungi and the mechanisms of these responses. METHODS: Human airway epithelial cell line BEAS-2B and Calu-3 (both from American Type Culture Collection) were incubated with PAR-2 peptides and extracts of various fungi. The cellular responses, including GM-CSF, interleukin (IL)-6, IL-8, eotaxin, eotaxin-2 and RANTES production as well as increases in intracellular calcium concentration ([Ca(2+)](i)), were examined. To characterize the proteases involved in these responses, protease inhibitors such as pepstatin A and alkalo-thermophilic Bacillus inhibitor (ATBI), HIV protease inhibitors and 4-amidinophenylmethanesulfonyl fluoride hydrochloride were used. To investigate the role of PAR-2, PAR-2-agonistic and PAR-2-antagonistic peptides were used. RESULTS: PAR-2-activating peptide, but not the control peptide, induced GM-CSF, IL-6 and IL-8 production; these cellular responses were accompanied by a quick and marked increase in [Ca(2+)](i). Among 7 common environmental fungi, only Alternaria induced GM-CSF, IL-6 and IL-8 production and increased [Ca(2+)](i) response. Both cytokine production and increased [Ca(2+)](i) were significantly inhibited by PAR-2 antagonist peptide and by aspartate protease inhibitors (pepstatin A, ritonavir, nelfinavir and ATBI), but not by the PAR-2 control peptide or by other protease inhibitors. CONCLUSIONS: Aspartate proteases from Alternaria induce cytokine production and calcium response in airway epithelium that is mediated through PAR-2. This protease-mediated activation of airway epithelium may be implicated in the development and exacerbation of airway allergic disease.

[Endoscopic sinus surgery complications a prospective multicenter study].

OBJECTIVE: Our objective was to determine the rate of complications in endoscopic sinus surgery (ESS) and associated risk factors. METHODS: We prospectively studied 1,382 subjects undergoing ESS for rhinosinusitis and cystic sinus disease at 16 hospitals during 2007 and 2008. Surgeons provided information on peri-and postoperative complication occurrence. RESULT: Results of complications were seen in 80 subjects (5.8%), the most frequent was perioperative lamina papyracea injury. Analysis showed the complication rate to be linked to gender, and anesthesia type, but not the grade of surgeon. CONCLUSIONS: While care should be taken to avoid them, complications should be identified and treated in a timely and accurate manner.

Morphological relationships between external auditory canal and vital structures of tympanic cavity.

PURPOSE: We aimed to evaluate the morphology of the external auditory canal (EAC) using a three-dimensional (3D) reconstruction of computed tomography (CT) scans of the temporal bone to corroborate and predict important anatomical structures involved in middle ear surgery based on the EAC morphology. METHODS: Temporal bone CT from 62 patients (120 ears) was used to perform 3D reconstruction (maximum intensity projection), of which 32 patients (60 ears) had chronic otitis media and 30 patients (60 ears) had normal temporal bones. The anatomical morphology of the EAC, tympanic sinus, vertical portion of the facial nerve, and jugular bulb were measured, and the anatomical relationship between the EAC morphology and important structures of the middle ear was analyzed. RESULTS: In ears with chronic otitis media, the overhang of the inferior wall of the EAC was significantly more than that in normal ears, and the antero-posterior length of the bony tympanic ring was short. Furthermore, the tympanic sinus was shallow, and vertical portion of the facial nerve tended to run outward. The EAC morphology correlated with the tympanic sinus depth and outward orientation of the vertical portion of the facial nerve. CONCLUSION: A severe overhang of the inferior wall of the EAC and short antero-posterior length of the bony tympanic ring indicates a higher possibility of a shallow tympanic sinus and an outward orientation of the vertical portion of the facial nerve. These findings aid in predicting the difficulty of tympanic sinus operation and reducing facial nerve damage risk during EAC excision.

Human eosinophil innate response to Alternaria fungus through protease-activated receptor-2.

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of allergic diseases. An association between eosinophilic inflammation and infection or colonization by fungi has also been long recognized. However, the mechanisms underlying how eosinophils are activated and how they release proinflammatory and immunomodulatory mediators such as major basic protein (MBP) and eosinophil-derived neurotoxin remain largely unknown. We used a fungus, i.e. Alternaria, as a model microbe relevant to human asthma and chronic rhinosinusitis (CRS) to investigate the molecular mechanisms involved in the immune recognition of ubiquitous environmental allergens. Human eosinophils are activated by live Alternaria alternata organisms, release their granule proteins, and kill the fungi. Eosinophils, but not neutrophils, respond to secreted products from A. alternata. We found that eosinophils are equipped with innate cellular activation machinery that responds to an extracellular aspartate protease secreted by Alternaria. Aspartate protease activation of protease-activated receptor (PAR)-2 probably involves a novel mechanism different from that for serine protease activation of PAR-2. Thus, human eosinophils may recognize certain danger signals or virulence factors produced by fungi and provoke inflammatory responses against these organisms. Dysregulation of such an innate immune mechanism may be involved in the pathophysiology of certain human inflammatory diseases, including asthma and CRS.

Cockroach induces inflammatory responses through protease-dependent pathways.

Exposure to cockroaches is a major risk factor for asthma. Products from cockroaches may contain proteases and ligands for pattern recognition receptors. These molecules may activate airway inflammatory cells, such as eosinophils, that are involved in asthma. Among inner-city children, cockroach allergens play an especially important role in increasing asthma morbidity. The molecular mechanism for this association between cockroach exposure and asthma is not fully understood. Enzymatic activities from cockroaches activate inflammatory cells in the airways and may also exacerbate certain human airway diseases, such as asthma. We recently reported that cockroach extracts contain pepstatin A-sensitive proteases that activate PAR-2 and induce activation and degranulation of human eosinophils. This review focuses on the effects of cockroach on various inflammatory cells, including eosinophils, epithelial cells, fibroblasts, dendritic cells, and T cells, in allergic reactions.

Recognition of fungal protease activities induces cellular activation and eosinophil-derived neurotoxin release in human eosinophils.

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils' responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils' innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.