Increased mast cell activation in eosinophilic chronic obstructive pulmonary disease.

Objectives: A subset of chronic obstructive pulmonary disease (COPD) patients have increased numbers of airway eosinophils associated with elevated markers of T2 inflammation. This analysis focussed on mast cell counts and mast cell-related gene expression in COPD patients with higher vs lower eosinophil counts. Methods: We investigated gene expression of tryptase (TPSAB1), carboxypeptidase A3 (CPA3), chymase (CMA1) and two mast cell specific gene signatures; a bronchial biopsy signature (MCbb) and an IgE signature (MCIgE) using sputum cells and bronchial epithelial brushings. Gene expression analysis was conducted by RNA-sequencing. We also examined bronchial biopsy mast cell numbers by immunohistochemistry. Results: There was increased expression of TPSAB1, CPA3 and MCbb in eosinophilhigh than in eosinophillow COPD patients in sputum cells and bronchial epithelial brushings (fold change differences 1.21 and 1.28, respectively, P < 0.01). Mast cell gene expression was associated with markers of T2 and eosinophilic inflammation (IL13, CLCA1, CST1, CCL26, eosinophil counts in sputum and bronchial mucosa; rho = 0.4-0.8; P < 0.05). There was no difference in MCIgE gene expression between groups. There was no difference in the total number of bronchial biopsy mast cells between groups. Conclusion: These results demonstrate that eosinophilic inflammation is associated with altered mast cell characteristics in COPD patients, implicating mast cells as a component of T2 inflammation present in a subset of COPD patients.

Relationships between Airway Remodeling and Clinical Characteristics in COPD Patients.

Background: Airway remodeling is a cardinal feature of chronic obstructive pulmonary disease (COPD) pathology. However, inconsistent findings have been reported regarding the nature of proximal airway remodeling in COPD. This is likely due to the heterogeneity of COPD. This study investigated the histopathological features of airway remodeling in bronchial biopsies of COPD patients compared to smoking controls (S). We tested the hypothesis that histopathological features in bronchial biopsies relate to clinical characteristics in COPD patients, focusing on smoking status, symptom burden, lung function, exacerbation risk and inhaled corticosteroid (ICS) use. Methods: We recruited 24 COPD patients and 10 S. We focused on reticular basement membrane thickness (RBM), surface immunoglobulin A (IgA) expression, goblet cell numbers (periodic acid-Schiff [PAS]+), sub-mucosal remodeling markers including collagen 4, 6 and laminin expression, and inflammatory cell counts (CD45+). Results: RBM thickness was increased in frequent exacerbators, IgA expression was reduced in COPD patients with worse lung function, and goblet cell numbers were increased in COPD patients compared to S but with no difference between the COPD subgroups. Collagen 4 expression was associated with higher symptom burden and worse quality of life. Sub-mucosal inflammatory cell counts were increased in COPD non-inhaled corticosteroid (ICS) users compared to ICS users and S. Conclusion: We observed relationships between the histopathological features of airway remodeling and clinical characteristics in COPD patients. Our data highlight the influence of clinical heterogeneity on diverse patterns of airway remodeling in COPD patients.

Red Blood Cell-Derived Iron Alters Macrophage Function in COPD.

Lung macrophage iron levels are increased in COPD patients. Lung macrophage iron levels are thought to be increased by cigarette smoke, but the role of red blood cells (RBCs) as a source of iron has not been investigated. We investigate RBCs as a potential source of alveolar iron in COPD, and determine the effect of RBC-derived iron on macrophage function. We used lung tissue sections to assess RBC coverage of the alveolar space, iron and ferritin levels in 11 non-smokers (NS), 15 smokers (S) and 32 COPD patients. Lung macrophages were isolated from lung resections (n = 68) and treated with hemin or ferric ammonium citrate (50, 100 or 200 µM). Lung macrophage phenotype marker gene expression was measured by qPCR. The phagocytosis of Non-typeable Haemophilus influenzae (NTHi) was measured by flow cytometry. Cytokine production in response to NTHi in iron-treated macrophages was measured by ELISA. Lung macrophage iron levels were significantly correlated with RBC coverage of the alveolar space (r = 0.31, p = 0.02). Furthermore, RBC coverage and lung macrophage iron were significantly increased in COPD patients and correlated with airflow obstruction. Hemin treatment downregulated CD36, CD163, HLA-DR, CD38, TLR4, CD14 and MARCO gene expression. Hemin-treated macrophages also impaired production of pro-inflammatory cytokines in response to NTHi exposure, and decreased phagocytosis of NTHi (200 µM: 35% decrease; p = 0.03). RBCs are a plausible source of pulmonary iron overload in COPD. RBC-derived iron dysregulates macrophage phenotype and function.

The relationship between airway immunoglobulin activity and eosinophils in COPD.

In chronic obstructive pulmonary disease (COPD), the effects of inhaled corticosteroids are predicted by blood eosinophil counts. We previously briefly reported increased immunoglobulin (Ig)A and IgM levels in bronchoalveolar lavage (BAL) of COPD patients with higher (eosinophilhigh ) compared to lower (eosinophillow ) blood eosinophils (>250/µL versus < 150/µL), suggesting differences in adaptive immune function. An inverse relationship exists between eosinophil counts and airway pathogenic bacteria levels. The mechanistic reasons for these associations between eosinophils, corticosteroids and pathogenic bacteria are unclear. IgA, IgM and IgG levels were assessed in BAL, bronchial biopsies and epithelium collected from eosinophilhigh (n = 20) and eosinophillow (n = 21) patients. Bronchial B-cell numbers were measured by immunohistochemistry. B-cell activity was assessed in bronchial samples and following exposure to BAL from eosinophilhigh and eosinophillow patients. BAL levels of non-typeable Haemophilus influenza (NTHi)-specific immunoglobulins were quantified. Results showed airway expression of IgA, IgG1 and IgM were lower in eosinophillow compared to eosinophilhigh patients, with lower levels of NTHi-specific IgA and IgM. Bronchial B-cell numbers were similar in both groups, but B-cell activity was lower in eosinophillow patients. In conclusion, COPD eosinophillow patients show differences in adaptive immune function compared to COPD eosinophilhigh patients. These differences may cause different microbiomes in these COPD phenotypes.

High frequency of infection of lung cancer patients with the parasite Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii is an intracellular protozoan parasite that can cause a wide range of clinical conditions, including miscarriage and pneumonia. The global prevalence is 30% in humans, but varies by locality (e.g. in the UK it is typically 10%). The association between lung cancer and T. gondii infection was investigated by direct detection in lung tissue samples. METHODS: Lung tissue samples were taken from patients undergoing lung resection surgery (n=72) for suspected lung cancer (infection prevalence 100% (95% CI: 93.9-100%)). All 72 participants were confirmed as having lung cancer following subsequent diagnostic tests. In addition, bronchial biopsy samples were collected from non-lung cancer healthy control subjects (n=10). Samples were tested for T. gondii using PCR amplification of T. gondii specific gene markers and T. gondii specific immunohistochemistry. RESULTS: All 72 lung cancer patients were infected with T. gondii (prevalence 100% (95% CI: 93.9-100%)). Of which, 95.8% (n=69) of patients showed evidence of active parasite stages. Infection prevalence in the controls (10%) was significantly lower (p<0.0001). CONCLUSIONS: Clinicians treating lung cancer patients should be aware of the potential presence of the parasite, the potential for induction of symptomatic complications and interference with treatment success.

The effect of electronic cigarette and tobacco smoke exposure on COPD bronchial epithelial cell inflammatory responses.

Background: Electronic cigarettes (e-cigs) are used to help smoking cessation. However, these devices contain harmful chemicals, and there are safety concerns. We have investigated the effects of e-cigs on the inflammatory response and viability of COPD bronchial epithelial cells (BECs). Methods: BECs from COPD patients and controls were exposed to e-cig vapor extract (ECVE) and the levels of interleukin (IL)-6, C-X-C motif ligand 8 (CXCL8), and lactate dehydrogenase release were measured. We also examined the effect of ECVE pretreatment on polyinosinic:polycytidylic acid (poly I:C)-stimulated cytokine release from BECs. Parallel experiments using Calu-3 cells were performed. Comparisons were made with cigarette smoke extract (CSE). Results: ECVE and CSE caused an increase in the release of IL-6 and CXCL8 from Calu-3 cells. ECVE only caused toxicity in BECs and Calu-3 cells. Furthermore, ECVE and CSE dampened poly I:C-stimulated C-X-C motif ligand 10 release from both cell culture models, reaching statistical significance for CSE at an optical density of 0.3. Conclusion: ECVE caused toxicity and reduced the antiviral response to poly I:C. This raises concerns over the safety of e-cig use.

Characterisation of lung macrophage subpopulations in COPD patients and controls.

Lung macrophage subpopulations have been identified based on size. We investigated characteristics of small and large macrophages in the alveolar spaces and lung interstitium of COPD patients and controls. Alveolar and interstitial cells were isolated from lung resection tissue from 88 patients. Macrophage subpopulation cell-surface expression of immunological markers and phagocytic ability were assessed by flow cytometry. Inflammatory related gene expression was measured. Alveolar and interstitial macrophages had subpopulations of small and large macrophages based on size and granularity. Alveolar macrophages had similar numbers of small and large cells; interstitial macrophages were mainly small. Small macrophages expressed significantly higher cell surface HLA-DR, CD14, CD38 and CD36 and lower CD206 compared to large macrophages. Large alveolar macrophages showed lower marker expression in COPD current compared to ex-smokers. Small interstitial macrophages had the highest pro-inflammatory gene expression levels, while large alveolar macrophages had the lowest. Small alveolar macrophages had the highest phagocytic ability. Small alveolar macrophage CD206 expression was lower in COPD patients compared to smokers. COPD lung macrophages include distinct subpopulations; Small interstitial and small alveolar macrophages with more pro-inflammatory and phagocytic function respectively, and large alveolar macrophages with low pro-inflammatory and phagocytic ability.

HIF-1α up-regulation is associated with adverse clinicopathological and biological factors in neuroblastomas.

AIMS: To study the expression of hypoxia-inducible factor 1α (HIF-1α) in clinical neuroblastoma (NB) samples and its association with the clinicopathological features, biological features and survival of NB patients. METHODS AND RESULTS: Immunohistochemistry indicated that elevated HIF-1α expression was present in 30 of 90 (33%) NBs. This expression was correlated significantly and positively with higher clinical stage (P = 0.002), ≥18 months of age at presentation (P = 0.020), high-risk group (P = 0.005), unfavourable pathology (P = 0.002), MYCN amplification (P < 0.001), 1p deletion (P = 0.004) and 17q gain (P = 0.002). Enzyme-linked immunosorbent assays showed that total HIF-1α protein was significantly higher in NBs of patients with all examined adverse prognostic factors except for age. Univariate survival analysis revealed that higher-than-median HIF-1α total protein levels were associated significantly with a decrease in event-free survival (EFS) (P = 0.017), but not in overall survival (OS) (P = 0.12). HIF-1α immunoexpression by ≥10% of tumour cells was associated significantly with decreased OS and EFS (P = 0.002 and P = 0.004, respectively), but not in multivariate analysis after adjusting for the high-risk group (P = 0.16 and P = 0.19, respectively). CONCLUSIONS: HIF-1α was increased significantly in patients with NB associated with unfavourable characteristics. HIF-1α is a prognostic indicator of poor OS and EFS and defines subgroups of NBs with aggressive clinical behaviour.

Carbonic anhydrase IX up-regulation is associated with adverse clinicopathologic and biologic factors in neuroblastomas.

The overexpression of carbonic anhydrase IX, a hypoxia-induced enzyme, is associated with an adverse prognosis in many cancers. However, carbonic anhydrase IX expression in neuroblastoma, the most common extracranial pediatric tumor, has not been reported. Membranous and/or strong cytoplasmic carbonic anhydrase IX expression, assessed by immunohistochemistry, was present in 21 (23%) of 91 neuroblastomas but was absent in ganglioneuromas (n = 9). The proportion of neuroblastomas showing membranous carbonic anhydrase IX expression was higher in neuroblastomas with 1p deletion and MYCN amplification. Nuclear carbonic anhydrase IX expression was seen in less than 10% of ganglion cells in all ganglioneuromas. Of 91 neuroblastomas, 16 (18%) showed nuclear carbonic anhydrase IX expression in 10% or more tumoral cells. The proportion of neuroblastomas showing nuclear carbonic anhydrase IX expression was significantly higher in patients with adverse clinical (increasing stage and high-risk group), pathologic (unfavorable group and high mitosis-karyorrhexis-index), and biologic (MYCN-amplification and 1p deletion) factors. Carbonic anhydrase IX total protein levels, quantified by enzyme-linked immunosorbent assay, were higher in neuroblastomas (n = 49; geometric mean, 0.47 pg/µg; range, 0.0-6.52 pg/µg) than in ganglioneuromas (n = 6; geometric mean, 0.20 pg/µg; range, 0.09-0.47 pg/µg) and were significantly higher in MYCN-amplified neuroblastomas. Nuclear carbonic anhydrase IX expression was associated with a poorer overall survival (P = .003) and event-free survival (P = .004), although the relationships were no longer significant when adjusted for high-risk disease. There was no significant association of membranous carbonic anhydrase IX expression or higher-than-median total protein levels with overall survival or event-free survival. Carbonic anhydrase IX is expressed at significantly higher levels in neuroblastomas from patients with adverse clinicopathologic and biologic factors indicating that it is a biomarker of aggressive disease in neuroblastomas.

Angiogenin up-regulation correlates with adverse clinicopathological and biological factors, increased microvascular density and poor patient outcome in neuroblastomas.

AIMS: As new biomarkers are urgently needed to identify children with high-risk neuroblastoma (NB), we studied the contribution of angiogenin (ANG) to angiogenesis and its association with clinicopathological and biological features and patient outcome in NB. METHODS AND RESULTS: Ninety NBs and 12 ganglioneuromas (GNs) were immunostained for ANG and CD31. ANG expression in NB tumoral cells (ANG scores) and vessels [ANG microvascular density (MVD)] and total MVD (CD31 MVD) were determined. The ANG score was significantly greater in NBs than in GNs (P = 0.015) and in NBs from children with stage 4 tumours, high-risk disease, unfavourable pathology (P < 0.001 for each), MYCN amplification (P = 0.003), and 1p deletion (P = 0.002). ANG scores correlated with ANG MVD and CD31 MVD (P < 0.001 for each). Total ANG and CD31 protein levels, measured with a sensitive enzyme-linked immunosorbent assay, were highly correlated (P = 0.003). High ANG scores were associated with decreased overall and event-free survival (log-rank test, P = 0.025 and P = 0.018, respectively). High ANG MVD was associated with decreased overall and event-free survival (log-rank test, P = 0.009 and P = 0.026, respectively). High CD31 MVD was associated with decreased event-free survival (P = 0.045). CONCLUSIONS: The strong correlation of ANG up-regulation with total MVD and adverse clinicopathological and biological factors indicates that ANG supports growth and progression in NB.

LYVE-1 upregulation and lymphatic invasion correlate with adverse prognostic factors and lymph node metastasis in neuroblastoma.

Neuroblastoma (NB) accounts for 15% of all childhood cancer deaths. The majority of patients have widespread lymphatic and/or haematogenous metastases at diagnosis, but lymphangiogenesis has not been well documented. Sixty-seven NBs were immunostained for the lymphatic endothelial marker, LYVE-1, and the lymphatic density (LD) and lymphatic invasion (LI), were counted in LYVE-1-expressing lymphatics. LYVE-1-stained lymphatic vessels and LI were present in 26/67 (39%) and 14/67 (21%) of the NBs, respectively. Central LD (CLD) and LI were higher in NBs from stage 4 (p = 0.012, p = 0.004, respectively), high-risk group (p = 0.030, p = 0.002), NBs with high mitosis karyorrhexis index (MKI) (p = 0.011, p = 0.005), unfavourable histology group (p = 0.040, p = 0.017) and distant lymph node metastasis (LNM) (p < 0.001 for each). Marginal LD (MLD) was higher in patients with LNM (p < 0.001). CLD and MLD correlated with LI (p < 0.001 each). Total LYVE-1 protein levels, quantified by a sensitive enzyme-linked immunosorbent assay (n = 55), were also higher in NBs from patients with stage 4 disease (p = 0.046), high-risk group (p = 0.028), MYCN-amplified NBs (p = 0.034) and LNM (p = 0.038). Kaplan-Meier analysis showed that the presence of CLD was associated with both worse OS at 5 years (77% [95% CI: 62-87%] versus 60% [95% CI: 32-80%], p = 0.062) and EFS (74% [95% CI: 58-85%] versus 43% [95% CI: 15-69%], p = 0.070) and LI with OS (71% [95% CI: 57-81%] versus 56% [95% CI: 26-78%], p = 0.055). Significant upregulation of LYVE-1 and the presence of LI in patients with stage 4 and high-risk disease, MYCN-amplification and LNM suggests that LYVE-1 may have value as predictors of outcome.

Overexpression of carbonic anhydrase and HIF-1α in Wilms tumours.

BACKGROUND: Overexpression of carbonic anhydrase (CA IX) is associated with poor survival in several adult-type cancers but its expression is undocumented in Wilms tumour (WT), the most common tumour of the paediatric kidney. METHODS: CA9 expression was measured using polymerase chain reaction (PCR) in 13 WTs and matched-paired non-neoplastic kidneys (NKs). CA IX and hypoxia-inducible factor-1 α-subunit (HIF-1α) protein were quantified in 15 matched-paired WTs and NKs using enzyme-linked immunosorbent assays. CA IX and HIF-1α were localised by immunostaining tissue sections of 70 WTs (untreated WTs, n = 22; chemotherapy-treated WTs, n = 40; relapsed/metastatic WTs, n = 8). CA IX-positive untreated WTs (n = 14) were immunostained for vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT1) and CD31. Double staining for CA IX and CD31 was performed in WTs (n = 14). RESULTS: CA9 full length (FL) was significantly up-regulated in WTs compared to NKs (p = 0.009) by real-time PCR. Conventional PCR showed expression of alternative splice variant in all NKs and WTs but FL in WTs only. WTs showed a 2-fold increase in CA IX protein over NKs (p = 0.01). HIF-1α levels were up-regulated in WTs compared to NKs, although the difference was not statistically significant (p = 0.09). CA IX and HIF-1α immunolocalisation were observed in 63% and 93% of WTs, respectively. The median fraction of cells staining positively for CA IX and HIF-1α was 5% and 22%, respectively. There was no significant association between the expression of either CA IX or HIF-1α and clinicopathological variables in WTs resected following chemotherapy. VEGF and GLUT1 immunoreactivity was seen in 94% and 100% with the median fraction of 10% and 60% respectively. Co-expression and co-localisation of all four hypoxia markers was seen in 7/14 and 6/14 cases respectively. CA IX was seen in well vascularised areas as well as in the peri-necrotic areas. CONCLUSIONS: Carbonic anhydrase 9 (mRNA and protein), and HIF-1α protein are overexpressed in a significant portion of WTs. No significant association was detected between the expression of either CA IX or HIF-1α and clinicopathological variables in WTs resected following chemotherapy. Cellular localisation studies in untreated WTs suggest that CA IX and HIF-1α are regulated by hypoxia and non-hypoxia mechanisms.