Protein Tyrosine Phosphatase Non-Receptor 11 (PTPN11/Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of KRAS and MEK. PTPN11/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated PTPN11 in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, PTPN11 mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. PTPN11 constructs harbouring PTPN11 E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. PTPN11/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a PTPN11-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic PTPN11 hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 PTPN11 hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant PTPN11 significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype PTPN11 (p < 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant PTPN11 exhibited increased PTPN11/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype PTPN11. The transduction of the PTPN11 inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the PTPN11-mutated NCI-H661 cell line. NCI-H661 cells (PTPN11-mutated, KRAS-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (PTPN11/KRAS-wild type) cells (IC50: >10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (p < 0.0001). PTPN11/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.

Exhaled Breath Condensate (EBC) analysis of circulating tumour DNA (ctDNA) using a lung cancer specific UltraSEEK oncogene panel.

INTRODUCTION: Small diagnostic tissue samples can be inadequate in testing an expanding list of validated oncogenic driver alterations and fail to reflect intratumour heterogeneity (ITGH) in lung cancer. Liquid biopsies are non-invasive and may better reflect ITGH. Most liquid biopsies are performed in the context of circulating tumour DNA (ctDNA) in plasma but Exhaled Breath Condensate (EBC) shows promise as a lung-specific liquid biopsy. METHODS: In this prospective, proof-of-concept study we carried out targeted Next Generation Sequencing (NGS) on diagnostic tissue samples from 125 patients with lung cancer and compared results to plasma and EBC for 5 oncogenic driver mutations (EGFR, KRAS, PIK3CA, ERBB2, BRAF) using an ultrasensitive PCR technique (UltraSEEK™ Lung Panel on the MassARRAY® System, Agena Bioscience, San Diego, CA, USA). RESULTS: There was a significantly higher failure rate due to unamplifiable DNA in tissue NGS (57/125, 45.6%) compared to plasma (27/125, 21.6%, p < 0.001 and EBC (26/125,20.8%, p ≤ 0.001. Consequently, both plasma and EBC identified higher number of mutations compared to tissue NGS. Specifically, there were significantly higher numbers of mutations detected in EGFR, KRAS and PIK3CA in plasma (p = 9.82 × 10-3, p = 3.14 × 10-5, p = 1.95 × 10-3) and EBC (p = 2.18 × 10-3, p = 2.28 × 10-4,p = 0.016) compared to tissue NGS. There was considerable divergence in mutation profiles between plasma and EBC with 34/76 (44%) mutations detected in plasma and 37/74 (41.89%) in EBC unique to their respective liquid biopsy. CONCLUSIONS: The results suggest that EBC is effective in identifying clinically relevant alterations in patients with lung cancer using UltraSEEK™ and has a potential role as an adjunct to plasma testing.

Tumour Genome Characterization of a Rare Case of Pulmonary Enteric Adenocarcinoma and Prior Colon Adenocarcinoma.

Pulmonary enteric adenocarcinoma (PEAC) is a rare variant of lung adenocarcinoma first described in the early 1990s in a lung tumour with overlapping lung and small intestine features. It is a rare tumour with fewer than 300 cases described in the published literature and was only formally classified in 2011. Given these characteristics the diagnosis is challenging, but even more so in a patient with prior gastrointestinal malignancy. A 68-year-old Caucasian female presented with a cough and was found to have a right upper lobe mass. Her history was significant for a pT3N1 colon adenocarcinoma. The resected lung tumour showed invasive lung adenocarcinoma but also features of colorectal origin. Immuno-stains were strongly and diffusely positive for lung and enteric markers. Multi-region, whole-exome sequencing of the mass and archival tissue from the prior colorectal cancer showed distinct genomic signatures with higher mutational burden in the PEAC and very minimal overlap in mutations between the two tumours. This case highlights the challenge of diagnosing rare lung tumours, but more specifically PEAC in a patient with prior gastro-intestinal cancer. Our use of multi-region, next-generation sequencing revealed distinct genomic signatures between the two tumours further supporting our diagnosis, and evidence of PEAC intra-tumour heterogeneity.

Use of exhaled breath condensate (EBC) in the diagnosis of SARS-COV-2 (COVID-19).

False negatives from nasopharyngeal swabs (NPS) using reverse transcriptase PCR (RT-PCR) in SARS-CoV-2 are high. Exhaled breath condensate (EBC) contains lower respiratory droplets that may improve detection. We performed EBC RT-PCR for SARS-CoV-2 genes (E, S, N, ORF1ab) on NPS-positive (n=16) and NPS-negative/clinically positive COVID-19 patients (n=15) using two commercial assays. EBC detected SARS-CoV-2 in 93.5% (29/31) using the four genes. Pre-SARS-CoV-2 era controls (n=14) were negative. EBC was positive in NPS negative/clinically positive patients in 66.6% (10/15) using the identical E and S (E/S) gene assay used for NPS, 73.3% (11/15) using the N/ORF1ab assay and 14/15 (93.3%) combined.

An integrated multidisciplinary model of COVID-19 recovery care.

BACKGROUND: In January 2020, the WHO declared the SARS-CoV-2 outbreak a public health emergency; by March 11, a pandemic was declared. To date in Ireland, over 3300 patients have been admitted to acute hospitals as a result of infection with COVID-19. AIMS: This article aims to describe the establishment of a COVID Recovery Service, a multidisciplinary service for comprehensive follow-up of patients with a hospital diagnosis of COVID-19 pneumonia. METHODS: A hybrid model of virtual and in-person clinics was established, supported by a multidisciplinary team consisting of respiratory, critical care, infectious diseases, psychiatry, and psychology services. This model identifies patients who need enhanced follow-up following COVID-19 pneumonia and aims to support patients with complications of COVID-19 and those who require integrated community care. RESULTS: We describe a post-COVID-19 service structure together with detailed protocols for multidisciplinary follow-up. One hundred seventy-four patients were discharged from Beaumont Hospital after COVID-19 pneumonia. Sixty-seven percent were male with a median age (IQR) of 66.5 (51-97). Twenty-two percent were admitted to the ICU for mechanical ventilation, 11% had non-invasive ventilation or high flow oxygen, and 67% did not have specialist respiratory support. Early data suggests that 48% of these patients will require medium to long-term specialist follow-up. CONCLUSIONS: We demonstrate the implementation of an integrated multidisciplinary approach to patients with COVID-19, identifying those with increased physical and mental healthcare needs. Our initial experience suggests that significant physical, psychological, and cognitive impairments may persist despite clinical resolution of the infection.

Characterization of the Inflammatory Response to Severe COVID-19 Illness.

Rationale: Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood.Objectives: To define the cytokine profile of COVID-19 and to identify evidence of immunometabolic alterations in those with severe illness.Methods: Levels of IL-1ß, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVIDstable patients), patients with COVID-19 requiring ICU admission (COVIDICU patients), and patients with severe community-acquired pneumonia requiring ICU support (CAPICU patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated.Measurements and Main Results: IL-1ß, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVIDICU patients could be clearly differentiated from COVIDstable patients, and demonstrated higher levels of IL-1ß, IL-6, and sTNFR1 but lower IL-10 than CAPICU patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission (P < 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution (P < 0.0001).Conclusions: The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.

Brief Report on the Detection of the EGFR T790M Mutation in Exhaled Breath Condensate from Lung Cancer Patients.

The EGFR T790M somatic mutation is the most common mechanism of resistance to tyrosine kinase inhibitors in NSCLC. Patients with advanced disease are not always amenable to repeat biopsy for further molecular analysis. Developing noninvasive methods to detect T790M in cell-free DNA in the absence of tissue is being actively investigated. Unfortunately, the low sensitivity of plasma for detection of T790M has limited its clinical use. Exhaled breath condensate (EBC) is an easily collected sample that is known to harbor cell-free DNA, including lung cancer mutations. This report details the potential utility of exhaled breath condensate in the detection of the EGFR T790M mutation.

Bronchial dentures as a cause of airway actinomycosis.

A 65-year-old man was referred to the respiratory clinic with recurrent chest infections on a background of stage 3 chronic obstructive pulmonary disease. On examination, there was wheeze bilaterally more marked on the left lower lobe. Subsequent imaging revealed an obstruction of the left main bronchus that was concerning for malignancy. Initially, on flexible bronchoscopy, a hard mass was found and multiple biopsies were positive for actinomycosis. Subsequent rigid bronchoscopy was undertaken and a set of dentures were removed from the airway.

Activated eosinophils in association with enteric nerves in inflammatory bowel disease.

Enteric neural dysfunction leads to increased mucous production and dysmotility in inflammatory bowel disease (IBD). Prior studies have shown that tissue eosinophilia is related to disease activity. We hypothesized that interactions between eosinophils and nerves contribute to neural dysfunction in IBD. Tissue from patients with intractable IBD, endoscopic biopsies from patients with steroid responsive IBD, both when active and quiescent, and control tissue were studied. Immunohistochemical studies showed that eosinophils localize to nerves in the mucosal layer of patients with Crohn's disease (CD) (p<0.001) and ulcerative colitis (UC), (p<0.01). Eosinophils localized to substance P and choline acetyltransferase (ChAT) immunostained nerves. Real time PCR of laser capture micro-dissected enteric ganglia demonstrated Intercellular Adhesion Molecule 1 (ICAM-1) mRNA was increased 7-fold in UC (n = 4), (p = 0.03), and 10-fold in CD (n = 3), (p = 0.05). Compared with controls, eotaxin-3 (CCL-26) mRNA was increased 9-fold in UC (p = 0.04) and 15-fold in CD (p = 0.06). Eosinophil numbers correlated with disease activity, while deposition of major basic protein (MBP) and eosinophil Transforming Growth Factor ß-1 (TGFß-1) expression were seen in therapeutically responsive disease. These data indicate a significant localization of eosinophils to nerves in IBD, mediated through neurally expressed ICAM-1 and eotaxin-3. This cell/neural interaction may influence the function of nerves and contribute to symptoms in IBD.

Interventional chest procedures in pregnancy.

Interventional pulmonology encompasses diagnostic and therapeutic bronchoscopic procedures, and pleural interventions. In the last 10 years older techniques have been refined and exciting new technologies have extended the reach and application of the instruments used. The main areas within pulmonary medicine for which these interventions have a role are malignant and nonmalignant airway disease, pleural effusion, pneumothorax, and artificial airways. There are no data from well-designed prospective trials to guide recommendations for interventional pulmonary procedures in pregnancy. The recommendations provided in this article are based on critical review of reported case series, opinion from recognized experts, and personal observations.

Comparison of suction catheter versus forceps biopsy for sampling of solitary pulmonary nodules guided by electromagnetic navigational bronchoscopy.

BACKGROUND: Electromagnetic navigation has been approved for use as an adjunct to standard bronchoscopy. The diagnostic yield varies depending on the size of the lesion and successful navigation to the lesion. OBJECTIVES: The performance of two different biopsy tools, i.e. catheter aspiration and forceps biopsy, in the diagnosis of small pulmonary nodules (SPN) guided by electromagnetic navigational bronchoscopy (ENB) was examined. METHODS: 54 patients referred for suspected lung cancer underwent ENB and 55 SPN (<3 cm) were sampled using both techniques. Endobronchial ultrasound (EBUS) was used to verify the accuracy of target lesion localization by ENB. Primary end points of the study were successful navigation to the lesion and a positive diagnosis. Patients were followed until a definitive diagnosis was obtained. RESULTS: All 55 lesions were accessed. Two lesions were excluded from data analysis as the patients were lost to follow-up and their diagnoses could not be confirmed. Of the remaining 53 lesions, 40 samples (75.5%) were diagnostic. Compared to forceps biopsy, catheter aspiration was positively correlated with the success rate (36/40 vs. 22/40; p = 0.035). The diagnostic yield was 93% when EBUS verified the lesion location after navigation and only 48% when lesion location was not confirmed. There were no significant complications. CONCLUSIONS: ENB is a useful tool in the evaluation of SPN <3 cm in diameter. For malignant lesions, sampling by catheter aspiration is associated with a higher diagnostic yield than sampling by forceps biopsy alone, in particular when EBUS could not confirm lesion location prior to sampling.

Endobronchial ultrasound-guided miniforceps biopsy in the biopsy of subcarinal masses in patients with low likelihood of non-small cell lung cancer.

BACKGROUND: Transbronchial needle aspiration (TBNA) is used to sample mediastinal masses, but the value may be limited by the small specimen size obtained. In benign diseases and hematologic malignancies, the sample size from TBNA is often considered insufficient for diagnosis. We evaluated the safety and efficacy of obtaining histologic specimens from subcarinal masses using a 1.15-mm miniforceps under endobronchial ultrasound (EBUS) guidance and compared the diagnostic yield with TBNA alone. METHODS: Patients being evaluated for subcarinal lesions exceeding 2.5 cm (short axis) and without known or suspected non-small cell lung cancer were included. Bronchoscopy was performed, and EBUS-guided BNA of the lesion was performed first with a 22-gauge needle, followed by the 19-gauge needle. The miniforceps was then passed through the airway into the lesion (three to five passes) under real-time EBUS guidance. Three biopsy specimens were obtained. RESULTS: The study enrolled 75 patients (41 men; mean age, 51.5 years). Specimens were acquired from each patient using the three techniques and processed separately. A specific diagnosis was made in 36% of patients with the 22-gauge needle, 49% with the 19-gauge needle, and in 88% with the miniforceps. The increase in diagnostic yield with miniforceps was most significant in patients with sarcoidosis (88% vs 36% for TBNA, p = 0.001) or lymphoma (81% vs 35%, p = 0.038). No complications occurred. CONCLUSIONS: Miniforceps biopsy, performed under real-time EBUS guidance, can be used to obtain tissue specimens from subcarinal masses adjacent to the airway. The diagnostic yield for lymphoma and sarcoidosis is superior to TBNA alone, and the procedure appears safe.

Histamine 4 receptor activation induces recruitment of FoxP3+ T cells and inhibits allergic asthma in a murine model.

Histamine has an important role in regulation of immune response which is mediated by differential expression of four distinct receptors, H1R-H4R. H1R and HR2 have previously been shown to be involved with modulation of lung inflammation. H4R is also expressed on inflammatory cells; therefore, we investigated the potential role of H4R in development of allergic asthma in a murine model. We determined that the H4R agonist 4-methylhistamine when delivered intratracheally before Ag challenge mitigated airway hyperreactivity and inflammation. This was associated with an increase in IL-10 and IFN-gamma, but not TGF-beta or IL-16, as well as a decrease in IL-13 in the bronchoalveolar lavage fluid. We also observed that H4R agonist instillation resulted in accumulation of FoxP3(+) T cells suggesting a direct effect on T regulatory cell recruitment. To investigate this further, we determined the in vitro effect of H4R stimulation on human T cell migration. The H4R agonist induced a 2- to 3-fold increase in T cell migration, similar to that seen for H1R agonists. Cells transmigrating to the H4R agonist, but not H1R, were skewed toward a CD4 cell expressing CD25 and intracellular FoxP3. H4R-responsive cells suppressed proliferation of autologous T cells, an effect that was dependent on IL-10 production. We conclude that H4R stimulation enriches for a regulatory T cell with potent suppressive activity for proliferation. These findings identify a novel function for H4R and suggest a potential therapeutic approach to attenuation of asthmatic inflammation.

Diverse effects of eosinophil cationic granule proteins on IMR-32 nerve cell signaling and survival.

Activated eosinophils release potentially toxic cationic granular proteins, including the major basic proteins (MBP) and eosinophil-derived neurotoxin (EDN). However, in inflammatory conditions including asthma and inflammatory bowel disease, localization of eosinophils to nerves is associated with nerve plasticity, specifically remodeling. In previous in vitro studies, we have shown that eosinophil adhesion to IMR-32 nerve cells, via nerve cell intercellular adhesion molecule-1, results in an adhesion-dependent release of granule proteins. We hypothesized that released eosinophil granule proteins may affect nerve cell signaling and survival, leading to nerve cell remodeling. Culture in serum-deprived media induced apoptosis in IMR-32 cells that was dose-dependently abolished by inclusion of MBP1 but not by EDN. Both MBP1 and EDN induced phosphorylation of Akt, but with divergent time courses and intensities, and survival was independent of Akt. MBP1 induced activation of neural nuclear factor (NF)-kappaB, from 10 min to 12 h, declining by 24 h, whereas EDN induced a short-lived activation of NF-kappaB. MBP1-induced protection was dependent on phosphorylation of ERK 1/2 and was related to a phospho-ERK-dependent upregulation of the NF-kappaB-activated anti-apoptotic gene, Bfl-1. This signaling pathway was not activated by EDN. Thus, MBP1 released from eosinophils at inflammatory sites may regulate peripheral nerve plasticity by inhibiting apoptosis.

Use of Sniff nasal-inspiratory force to predict survival in amyotrophic lateral sclerosis.

Respiratory muscle weakness is the usual cause of death in amyotrophic lateral sclerosis. The prognostic value of the forced vital capacity (FVC), mouth-inspiratory force, and sniff nasal-inspiratory force were established in a group of 98 patients with amyotrophic lateral sclerosis who were followed trimonthly for 3 years. Sniff nasal-inspiratory force correlated with the transdiaphragmatic pressure (r = 0.9, p < 0.01). Sniff nasal-inspiratory force was most likely to be recorded at the last visit (96% of cases), compared with either the FVC or mouth-inspiratory force (86% and 81%, respectively, p < 0.01). A sniff nasal-inspiratory force less than 40 cm H(2)O was significantly related with nocturnal hypoxemia. When sniff nasal-inspiratory force was less than 40 cm H(2)O, the hazard ratio for death was 9.1 (p = 0.001), and the median survival was 6 +/- 0.3 months. The sensitivity of FVC < 50% for predicting 6-month mortality was 58% with a specificity of 96%, whereas sniff nasal-inspiratory force less than 40 H(2)O had a sensitivity of 97% and a specificity of 79% for death within 6 months. Thus the sniff nasal-inspiratory force test is a good measure of respiratory muscle strength in amyotrophic lateral sclerosis, it can be performed by patients with advanced disease, and it gives prognostic information.

Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells.

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.

Eosinophil adhesion to cholinergic IMR-32 cells protects against induced neuronal apoptosis.

Eosinophils release a number of mediators that are potentially toxic to nerve cells. However, in a number of inflammatory conditions, such as asthma and inflammatory bowel disease, it has been shown that eosinophils localize to nerves, and this is associated with enhanced nerve activity. In in vitro studies, we have shown that eosinophil adhesion via neuronal ICAM-1 leads to activation of neuronal NF-kappaB via an ERK1/2-dependent pathway. In this study, we tested the hypothesis that eosinophil adhesion to nerves promotes neural survival by protection from inflammation-associated apoptosis. Exposure of differentiated IMR-32 cholinergic nerve cells to IL-1beta, TNF-alpha, and IFN-gamma, or culture in serum-deprived medium, induced neuronal apoptosis, as detected by annexin V staining, caspase-3 activation, and DNA laddering. Addition of human eosinophils to IMR-32 nerve cells completely prevented all these features of apoptosis. The mechanism of protection by eosinophils was by an adhesion-dependent activation of ERK1/2, which led to the induced expression of the antiapoptotic gene bfl-1. Adhesion to nerve cells did not influence the expression of the related genes bax and bad. Thus, prevention of apoptosis by eosinophils may be a mechanism by which these cells regulate neural plasticity in the peripheral nervous system.

Effect of eosinophil adhesion on intracellular signaling in cholinergic nerve cells.

Eosinophil localization to cholinergic nerves occurs in a variety of inflammatory conditions, including asthma. This localization is mediated by interactions between eosinophil integrins and neuronal vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Eosinophil-nerve cell interactions lead to generation of neuronal reactive oxygen species and release of eosinophil proteins. The effects of eosinophil adhesion on neuronal intracellular signaling pathways were investigated. Eosinophil adhesion to IMR32 cholinergic nerves led to a rapid and sustained activation of the nuclear transcription factors nuclear factor (NF)-kappaB and activator protein (AP)-1 in the nerve cells. Eosinophil binding to neuronal ICAM-1 led to a rapid activation of ERK1/2 in nerve cells. Inhibition of ERK1/2 prevented NF-kappaB activation. Eosinophil adhesion to VCAM-1 resulted in AP-1 activation, mediated partially by rapid activation of the p38 mitogen-activated protein kinase. These data show that adhesion of eosinophils induces mitogen-activated protein kinase-dependent activation of the transcription factors NF-kappaB and AP-1 in nerve cells, indicating that eosinophil adhesion may control nerve growth and phenotype.