Aggregated eosinophils and neutrophils characterize the properties of mucus in chronic rhinosinusitis.

BACKGROUND: Airway obstruction caused by viscous mucus is an important pathophysiologic characteristic of persistent inflammation, which can result in organ damage. OBJECTIVE: We investigated the hypothesis that the biophysical characteristics of accumulating granulocytes affect the clinical properties of mucus. METHODS: Surgically acquired nasal mucus samples from patients with eosinophilic chronic rhinosinusitis and neutrophil-dominant, noneosinophilic chronic rhinosinusitis were evaluated in terms of computed tomography density, viscosity, water content, wettability, and protein composition. Isolated human eosinophils and neutrophils were stimulated to induce the formation of extracellular traps, followed by the formation of aggregates. The biophysical properties of the aggregated cells were also examined. RESULTS: Mucus from patients with eosinophilic chronic rhinosinusitis had significantly higher computed tomography density, viscosity, dry weight, and hydrophobicity compared to mucus from patients with noneosinophilic chronic rhinosinusitis. The levels of eosinophil-specific proteins in mucus correlated with its physical properties. Eosinophil and neutrophil aggregates showed physical and pathologic characteristics resembling those of mucus. Cotreatment with deoxyribonuclease and heparin, which slenderizes the structure of eosinophil extracellular traps, efficiently induced reductions in the viscosity and hydrophobicity of both eosinophil aggregates and eosinophilic mucus. CONCLUSIONS: The present study elucidated the pathogenesis of mucus stasis in infiltrated granulocyte aggregates from a novel perspective. These findings may contribute to the development of treatment strategies for eosinophilic airway diseases.

Effects of polymorphisms in pregnane X receptor and ABC transporters on afatinib in Japanese patients with non-small cell lung cancer: pharmacogenomic-pharmacokinetic and exposure-response analysis.

PURPOSE: Because of the large interindividual variability of afatinib pharmacokinetics and adverse events, we evaluated the effects of polymorphisms in pregnane X receptor (NR1I2) and ABC transporters (ABCB1, ABCG2, and ABCC2) on the pharmacokinetics of afatinib. METHODS: The steady-state area under the concentration-time curve (AUC)0-24 of afatinib was analyzed using blood sampling just prior to and at 1, 2, 4, 6, 8, 12, and 24 h on day 15 after administration. RESULTS: The median oral clearance (CL/F) of afatinib in patients with the NR1I2 7635A allele was significantly lower than those in patients with the 7635G/G genotype (42.0 and 60.0 L/h, respectively, P = 0.025). There were no significant differences in afatinib CL/F between genotypes for NR1I2 8055C > T, -25385C > T, ABCB1, ABCG2, and ABCC2 polymorphisms. Based on the area under the receiver-operating characteristic curve, the threshold afatinib AUC0-24 value for prediction of dose reduction or withdrawal was 689 ng·h/mL at the best sensitivity (81.0%) and specificity (72.7%). In multivariate logistic regression analysis, an afatinib AUC0-24 above 689 ng·h/mL was independently associated with increased risk of dose reduction or withdrawal (OR: 11.66, P = 0.012). CONCLUSIONS: The NR1I2 7635A allele was related to a lower afatinib CL/F. Based on the AUC of 689 ng h/mL and CL/F, the optimal doses for patients with the NR1I2 7635G/G genotype and 7635A allele were recommended to be set at 40 and 30 mg/day, respectively, and subsequent adjustment of the maintenance dose based on the plasma concentrations of afatinib may be necessary to avoid afatinib-related adverse events.

Impact of the COVID-19 pandemic on the primary lung cancer screening program in Akita Prefecture, a region with the fastest aging population in Japan.

AIM: Akita Prefecture has the largest proportion of older inhabitants and the highest cancer mortality rate in Japan. Lung cancer is one of the biggest killers, and early detection is critical. Chest X-ray examinations are the main screening method for lung cancer; however, the COVID-19 pandemic has affected the screening system. Here, we evaluate how COVID-19 has affected lung cancer screening in Akita Prefecture. METHODS: Using the Akita General Health Corporation database, the average annual number of chest X-ray screening tests, close examinations and lung cancer diagnoses (stratified by sex and age) was evaluated during 2016-2019, and compared with the 2020 values. Furthermore, data on lung cancer registrations from 2018 to 2020 were obtained from the Collaborative Akita Prefecture Hospital-Based Cancer Registration System and analyzed. RESULTS: The average annual number of screening tests, close examinations and lung cancer diagnoses declined (by >50%) between 2016 to 2019 and 2020, especially among older people (aged ≥65 years). Furthermore, by stage, the number of patients with early-stage lung cancer (stage 0-I) decreased, and the number with advanced-stage cancer (stage IV) increased. CONCLUSIONS: The COVID-19 pandemic reduced lung cancer screening participation, especially among the older adult population in Akita Prefecture, resulting in a decrease in lung cancer diagnoses through screening. This might have reduced the number of early-stage cancer registrations. It is necessary to improve health education among the public regarding the importance of chest X-ray screening. Geriatr Gerontol Int 2023; 23: 622-627.

Effects of CYP3A4/5 and ABC transporter polymorphisms on osimertinib plasma concentrations in Japanese patients with non-small cell lung cancer.

The effects of polymorphisms in CYP3A4 (20230G > A), CYP3A5 (6986A > G), ABCB1 (1236C > T, 2677G > T/A, 3435C > T), ABCG2 (421C > A), and ABCC2 (-24C > T) on the area under the concentration-time curve (AUC) of osimertinib in 23 patients with non-small cell lung cancer were investigated. Blood sampling was performed just prior to and at 1, 2, 4, 6, 8, 12, and 24 h after osimertinib administration at the steady-state on day 15 after beginning therapy. The osimertinib AUC0-24 was significantly correlated with age (P = 0.038), serum albumin (P = 0.002), and serum creatinine (P = 0.012). Additionally, there were significant differences in the AUC0-24 of osimertinib among the groups administered vonoprazan, histamine 2-receptor antagonists or esomeprazole, and no acid suppressants (P = 0.021). By contrast, there were no significant differences in the AUC0-24 of osimertinib between genotypes of CYP3A4/5 or ABC transporters. Furthermore, there were no significant differences in the AUC0-24 of osimertinib between patients with diarrhea, skin rash, or hepatotoxicity and those without these conditions. In multivariate analysis, only serum albumin value was an independent factor predicting the AUC0-24 of osimertinib. Analysis of CYP3A4/5 and ABC transporter polymorphisms before osimertinib therapy may not predict the efficacy or side effects of osimertinib. The lower serum albumin values were associated with an increase in the AUC0-24 of osimertinib; however, further studies are needed to assess the factors contributing to the interindividual variability of osimertinib pharmacokinetics.

Relationship between Plasma Concentrations of Afatinib and the Onset of Diarrhea in Patients with Non-Small Cell Lung Cancer.

We evaluated the area under the plasma concentration-time curve (AUC) of afatinib required to avoid the onset of grade 2 or higher diarrhea. The C0 and AUC0-24 of afatinib were significant higher in patients with grade 2 diarrhea than in those with grade 0-1 diarrhea. The areas under the receiver operator curves were 0.795 with the highest sensitivity (89%) and specificity (74%) at an AUC0-24 threshold of 823.5 ng·h/mL, and 0.754 with the highest sensitivity (89%) and specificity (74%) at a C0 threshold of 28.5 ng/mL. In Kaplan-Meier analysis based on these cut-off AUC0-24 and C0 values, the median time to the incidence of grade 2 diarrhea was 16 days. The predicted AUC0-24 of afatinib from the single point of C6 showed the highest correlation with the measured AUC0-24 (r2 = 0.840); however, a significant correlation between the AUC0-24 and C0 was also observed (r2 = 0.761). C0 could be used as a marker of therapeutic drug monitoring because afatinib C0 was related to AUC0-24. Therefore, afatinib C0 should be monitored on day 8 after beginning therapy, and the daily dose of afatinib should be adjusted as an index with a cut-off value of 28.5 ng/mL.

Eosinophil extracellular traps in a patient with chronic eosinophilic pneumonia.

Eosinophils rapidly release extracellular filamentous chromatin fibers (extracellular traps, ETs) when they are stimulated. Reticulated ETs have been recently shown to affect secretion viscosity in eosinophilic inflammatory diseases. Here we report a 43-year-old woman with infiltrative shadows in both upper lungs that did not respond well to antibiotics. She admitted to occasional coughing and sputum, but had poor viscous regulation. Bronchoalveolar lavage fluid (BALF) collected from the upper left lobe showed many eosinophils (65%). She was diagnosed with chronic eosinophilic pneumonia, per previously reported criteria, and began treatment with prednisolone. The infiltration shadow gradually improved, and she was discharged 28 days after admission. Later, we immune-stained her BALF cell components with antibodies against major basic protein, an eosinophil granule protein, which showed a large number of agglomerating eosinophils; and antibodies against citrullinated histone H3 (CitH3-a marker for ETs), which showed CitH3-positive ETs, spread in a network. These findings confirmed that some BALF eosinophils released eosinophil ETs. This case shows the existence of ETs from BALF in patients with chronic eosinophilic pneumonia. Concentration of eosinophil ETs in eosinophilic inflammatory diseases may affect secretion viscosity in sputum, and so on.

Hypereosinophilic syndrome with abundant Charcot-Leyden crystals in spleen and lymph nodes.

Hypereosinophilic syndrome, which is characterized by eosinophilia in the peripheral blood, often causes various organ disorders. Charcot-Leyden crystals are recognized features of various diseases, such as parasite infection and asthma, and are known to be classic hallmarks of eosinophilic inflammation. Our recent study revealed the mechanism of Charcot-Leyden crystal formation (i.e., galectin-10 crystallization), namely the involvement of eosinophil extracellular trap cell death, a nonapoptotic cell death. Here we report an autopsy case of a 57-year-old man who had died of hypereosinophilic syndrome. We found numerous eosinophil extracellular trap cell death-associated Charcot-Leyden crystals in the spleen and lymph nodes. Observation of abdominal lymph nodes by electron microscopy revealed eosinophil extracellular traps and free extracellular granules, which are characteristic of typical eosinophil extracellular trap cell death. In this case, we observed various sizes of Charcot-Leyden crystals that were stained with anti-galectin-10 immunofluorescent staining. Further studies are required to understand the pathophysiological roles of Charcot-Leyden crystals and these may lead to the development of novel therapeutic modalities for severe eosinophilic inflammation.

ABCG2 C421A polymorphisms affect exposure of the epidermal growth factor receptor inhibitor gefitinib.

ATP-binding castle protein G2 (ABCG2) is thought to inhibit the activities of certain gefitinib transporters, thereby affecting drug pharmacokinetics. The C421A polymorphism affects the function and expression of ABCG2 on the cell membrane. Previous studies have shown that proton-pump inhibitors (PPIs) inhibit gefitinib absorption, as well as the function of ABCG2. We evaluated the plasma concentrations of gefitinib in patients with and without the ABCG2 C421A polymorphism, who were or were not taking PPIs. In total, 61 patients with advanced epidermal-growth-factor-positive non-small-cell lung cancer were enrolled in this study. They were treated with gefitinib at a dose of 250 mg per day. Plasma gefitinib concentration and ABCG2 C421A status were determined after 2 weeks. The patients were divided into CC- and CA/AA genotype groups. We compared the trough and peak gefitinib levels and the area under the curve (AUC) values for 24-h gefitinib concentrations. We also compared these parameters among four groups distinguished according to the presence or absence of the polymorphism and PPI use. The mean trough gefitinib level and AUC value for 24-h gefitinib concentration were significantly lower in the CA/AA group compared to the CC group (mean trough level: 333.2 vs. 454.5 ng/mL, respectively, P = 0.021; AUC: 9949.9 vs. 13,085.4 ng・h/mL, respectively, P = 0.034). Among patients taking PPIs, the mean trough gefitinib level was significantly lower in the CA/AA group than the CC group (220.1 vs. 340.5 ng/mL, respectively, P = 0.033). The CA/AA-type of ABCG2 C421A polymorphism may be associated with lower gefitinib plasma concentrations.

Effects of STAT3 polymorphisms and pharmacokinetics on the clinical outcomes of gefitinib treatment in patients with EGFR-mutation positive non-small cell lung cancer.

WHAT IS KNOWN AND OBJECTIVE: We investigated the correlations among signal transducer and activator of transcription 3 (STAT3) rs4796793C >G polymorphism, gefitinib pharmacokinetics and clinical responses in Japanese patients with non-small cell lung cancer receiving gefitinib therapy. METHODS: Forty-five patients were enrolled in this study. Plasma trough concentrations (C0 ) of gefitinib at the steady-state were measured by high-performance liquid chromatography. RESULTS AND DISCUSSION: Patients having a gefitinib C0 of at least ≥200 ng/mL had significantly longer PFS than patients having a C0 of <200 ng/mL (median [95% confidence interval (CI)] PFS: 11.0 [8.2-13.7] and 5.3 [0.0-12.0] months, respectively, P = .042). There were no significant differences in PFS between patients with STAT3 rs4796793C/C and G alleles; however, patients with STAT3 rs4796793C/C having a gefitinib C0 of ≥ 200 ng/mL had significantly longer progression-free survival (PFS) and overall survival (OS) than those with a C0 of <200 ng/mL (median [95% CI] PFS: 11.4 [4.1-18.6] and 3.0 [0.0-7.0] months, respectively, P = .008; median [95% CI] OS: 20.6 [7.4-33.7] and 12.6 [10.1-15.1] months, respectively, P = .042). In patients with the STAT3 rs4796793G allele, there were no significant differences in PFS and OS between the two gefitinib C0 groups. In addition, there were no significant differences in PFS or OS according to smoking, presence of proton pump inhibitor combination, or onset of side effects. WHAT IS NEW AND CONCLUSION: Clinical outcomes of gefitinib in patients with the STAT3 rs4796793C/C genotype depended on plasma concentrations of gefitinib. In addition to information regarding EGFR mutations, the STAT3 rs4796793C >G polymorphism and gefitinib C0 may be potential predictors of clinical outcomes after beginning of gefitinib therapy.

An autopsy case of anaplastic lymphoma kinase-positive lung cancer exacerbated in a short period of time: a case report.

BACKGROUND: Anaplastic lymphoma kinase-positive lung cancer is a form of lung cancer that accounts for approximately 5% of non-small cell lung cancers. Recently, anaplastic lymphoma kinase inhibitors have been used for treatment of anaplastic lymphoma kinase-positive lung cancer, and their high clinical effect has also been demonstrated in cases of advanced stage lung cancer. Alectinib is an anaplastic lymphoma kinase inhibitor that it is recognized as a standard drug for primary therapy because of its superiority to crizotinib. CASE PRESENTATION: A 37-year-old Japanese man was admitted to our hospital due to multiple brain metastases. An autopsy report revealed that the cause of death was anaplastic lymphoma kinase-positive lung cancer, exacerbated in a short period despite treatment with alectinib. Necropsy revealed anaplastic lymphoma kinase-positive adenosquamous carcinoma of the lung, suggesting that it was involved in the prognosis of this patient. Based on the autopsy results, we reviewed the pathological tissue from transbronchial lung biopsy at the time of clinical diagnosis. The tissue specimen for clinical diagnosis in this case was a papillary adenocarcinoma. However, when this tissue was immunostained, thyroid transcription factor 1-negative and cytokeratin 5/6-positive parts were recognized. This result indicates that we could diagnose this patient as having had adenosquamous carcinoma of the lung. CONCLUSION: In cases of anaplastic lymphoma kinase-positive lung cancer poorly responsive to anaplastic lymphoma kinase inhibitors, re-examination of the tissue should be considered because there is a possibility of anaplastic lymphoma kinase-positive adenosquamous carcinoma.

Anti-drug Antibodies Against a Novel Humanized Anti-CD20 Antibody Impair Its Therapeutic Effect on Primary Biliary Cholangitis in Human CD20- and FcγR-Expressing Mice.

There is considerable interest in expanding B cell-targeted therapies in human autoimmune diseases. However, clinical trials in human primary biliary cholangitis (PBC) using a chimeric antibody against human CD20 (hCD20) have showed limited efficacy. Two potential explanations for these disappointing results are the appearance of anti-drug antibodies (ADAs) and the high frequency of patients with moderate PBC or patients who had failed ursodeoxycholic acid treatment. Here, we studied a novel humanized IgG1 antibody against hCD20 and explored its efficacy in early stage PBC using a well-defined murine model. We developed a unique murine model consisting of dnTGF-ßRII mice expressing hCD20 and human Fcγ receptors (hFcγRs). Beginning at 4-6 weeks of age, equivalent to stage I/II human PBC, female mice were given weekly injections of an anti-hCD20 antibody (TKM-011) or vehicle control, and monitored for liver histology as well as a broad panel of immunological readouts. After 16 weeks' treatment, we observed a significant reduction in portal inflammation, a decrease in liver-infiltrating mononuclear cells as well as a reduction in liver CD8+ T cells. Importantly, direct correlations between numbers of liver non-B cells and B cells (r = 0.7426, p = 0.0006) and between numbers of liver memory CD8+ T cells and B cells (r = 0.6423, p = 0.0054) were apparent. Accompanying these changes was a dramatic reduction in anti-mitochondrial antibodies (AMAs), interleukin (IL)-12p40 and IL-5, and elevated levels of the anti-inflammatory chemokine CXCL1/KC. In mice that developed ADAs, clinical improvements were less pronounced. Sustained treatment with B cell-targeted therapies may broadly inhibit effector pathways in PBC, but may need to be administered early in the natural history of PBC.

Charcot-Leyden crystal formation is closely associated with eosinophil extracellular trap cell death.

Protein crystallization in human tissue rarely occurs. Charcot-Leyden crystals (CLCs) were described in various eosinophilic diseases >150 years ago, but our understanding of CLC formation still remains limited. In this study, we demonstrate that CLCs observed in varied inflamed human tissues are closely associated with eosinophil cell-free granules and nuclear envelope/plasma membrane disintegration with release of filamentous chromatin (extracellular traps), typical morphologies of a regulated pathway of extracellular trap cell death (ETosis). During the process of eosinophil ETosis, eccentrically localized cytoplasmic and perinuclear CLC protein (galectin-10) is homogeneously redistributed in the cytoplasm. Rapid (1-2 minutes) formation of intracytoplasmic CLCs was observed using time-lapse imaging. Plasma membrane rupture enabled the release of both intracellularly formed CLCs and soluble galectin-10 that further contributed to formation of CLCs extracellularly, in parallel with the expulsion of free intact granules and extracellular traps. CLC formation and galectin-10 release were dependent on nicotinamide adenine dinucleotide phosphate oxidase activation. To our knowledge, this is the first demonstration of natural formation of CLCs in association with an active physiological process (ie, ETosis). These results indicate that dynamic changes in intracellular localization and release of galectin-10 contribute to CLC formation in vivo and suggest that CLC/galectin-10 might serve as an indicator of ETosis.

ICAM-1 upregulation is not required for retinoic acid-induced human eosinophil survival.

Active metabolites of vitamin A, retinoic acids (RAs), are known to play critical roles in mucosal immune responses and dramatically inhibit human eosinophil apoptosis, but the detailed mechanisms have not been elucidated. We previously screened for ICAM-1 (CD54) upregulation in RA-stimulated human eosinophils by gene microarray analysis. As ICAM-1 induction and activation were observed to have a role in maintenance of eosinophil survival, we tested the hypothesis that RAs prolong eosinophil survival through ICAM-1 outside-in signaling. Blood-derived isolated eosinophils cultured with 9-cis RA and all-trans RA showed significant upregulation of ICAM-1 mRNA and cell surface expression. TTNPB, a retinoic acid receptor agonist, also induced ICAM-1 expression, while HX630, a retinoid X receptor agonist, did not. Furthermore, an RAR antagonist, HX531, completely inhibited the effect of RAs. Upregulated ICAM-1 was associated with altered kinetics of Akt, ERK, and p38 MAP kinase phosphorylation through ICAM-1 cross-linking, but an ICAM-1-blocking antibody did not affect RA-mediated cell survival. These findings indicate that RAs induce functional ICAM-1 expression through RARs, but the induced ICAM-1 does not contribute to prolongation of eosinophil survival.

Eosinophils release extracellular DNA traps in response to Aspergillus fumigatus.

BACKGROUND: Eosinophils mediate the immune response in different infectious conditions. The release of extracellular DNA traps (ETs) by leukocytes has been described as an innate immune response mechanism that is relevant in many disorders including fungal diseases. Different stimuli induce the release of human eosinophil ETs (EETs). Aspergillus fumigatus is an opportunistic fungus that may cause eosinophilic allergic bronchopulmonary aspergillosis (ABPA). It has been reported that eosinophils are important to the clearance of A fumigatus in infected mice lungs. However, the immunological mechanisms that underlie the molecular interactions between A fumigatus and eosinophils are poorly understood. OBJECTIVE: Here, we investigated the presence of EETs in the bronchial mucus plugs of patients with ABPA. We also determined whether A fumigatus induced the release of human eosinophil EETs in vitro. METHODS: Mucus samples of patients with ABPA were analyzed by light and confocal fluorescence microscopy. The release of EETs by human blood eosinophils was evaluated using different pharmacological tools and neutralizing antibodies by fluorescence microscopy and a fluorimetric method. RESULTS: We identified abundant nuclear histone-bearing EETs in the bronchial secretions obtained from patients with ABPA. In vitro, we demonstrated that A fumigatus induces the release of EETs through a mechanism independent of reactive oxygen species but associated with eosinophil death, histone citrullination, CD11b, and the Syk tyrosine kinase pathway. EETs lack the killing or fungistatic activities against A fumigatus. CONCLUSIONS: Our findings may contribute to the understanding of how eosinophils recognize and act as immune cells in response to A fumigatus, which may lead to novel insights regarding the treatment of patients with ABPA.

Validity of Ultrasound Lung Comets for Assessment of the Severity of Interstitial Pneumonia.

OBJECTIVES: Ultrasound (US) lung comets are often observed in patients with interstitial lung disease or congestive heart failure, but few studies have explored the clinical importance of US lung comets in patients with the former condition. We explored whether the US lung comet number could be used to assess the severity of interstitial pneumonia. METHODS: Forty stable patients with interstitial pneumonia were examined. Lung comets evident on transthoracic US imaging in 12 selected regions of the posterior chest wall were analyzed. We defined lung comets accompanied by thickened and irregular pleural lines as interstitial US lung comets; these predominated in patients with interstitial pneumonia. The total number of interstitial US lung comets was correlated with the data from chest high-resolution computed tomography, pulmonary function tests, serologic tests, and the 6-minute walk test. RESULTS: The 40 patients included 16 with idiopathic pulmonary fibrosis and 24 with nonspecific interstitial pneumonia. Thirty-four patients had interstitial US lung comets, which were more common in the lower than the upper lung area. Good correlations were evident between the lung comet number and the extent of the reticular pattern on chest high-resolution computed tomography (r = 0.710; P < .01), predicted forced vital capacity (r = -0.614; P < .01), and lung diffusion capacity for carbon monoxide (r = -0.577; P < .01). Notably, the lung comet number had a strong negative correlation with the percutaneous oxygen saturation level after the 6-minute walk test (r = -0.751; P < .01). CONCLUSIONS: The number of interstitial US lung comets evident on transthoracic US imaging may be a valuable marker of disease severity in patients with interstitial pneumonia.

Association Between Endothelial Progenitor Cells and Treatment Response in Non-Squamous Non-small Cell Lung Cancer Treated with Bevacizumab.

BACKGROUND/AIM: To investigate the association between the number of circulating endothelial progenitor cells (EPCs) in non-squamous non-small cell lung cancer (NSCLC) and disease outcome, in combination chemotherapy with and without bevacizumab. MATERIALS AND METHODS: We retrospectively identified 25 non-squamous NSCLC cases, and divided them into high-EPC and low-EPC groups. Within each group, we compared disease outcomes, with or without the administration of bevacizumab. RESULTS: In the high-EPC group, chemotherapy with bevacizumab produced a significantly higher tumor reduction rate and objective response rate, with significantly longer progression-free survival, compared to chemotherapy without bevacizumab (p<0.001, p=0.010, and p<0.001, respectively). However, in the low-EPC group, there were no significant differences in disease outcomes in groups with versus those without bevacizumab. CONCLUSION: The number of EPCs may be a useful biomarker to guide decision-making in the use of bevacizumab in non-squamous NSCLC.