Raffaele D'Ippolito

Eosinophils, mast cells, and basophils in induced sputum from patients with seasonal allergic rhinitis and perennial asthma: Relationship to methacholine responsiveness☆☆☆★

OBJECTIVES We attempted to determine whether inflammation is present in induced sputum of patients with seasonal allergic rhinitis (AR) as compared with those with perennial asthma (AS) and examined its relationship with bronchial responsiveness to methacholine. METHODS Sputum was induced in 30 patients with seasonal rhinitis in response to grass pollens only and in 15 patients with stable, asymptomatic asthma. The AR group was divided according to methacholine PD20 value: the AR- group (n = 15) had a methacholine PD20 greater than 24 micromol; the AR+ group (n = 15) had a methacholine PD20 ranging between 2.2 and 19.6 micromol. In the AS group, methacholine PD20 ranged between 0.42 and 2.6 micromol. The percentage of eosinophils and metachromatic cells (alcian blue-positive) was assessed in sputum by light microscopy. Tryptase-positive cells and EG2+ cells were identified by immunocytochemistry with the mouse anti-human mast cell-tryptase monoclonal antibody and the monoclonal anti-eosinophil cationic protein antibody. RESULTS We found that the number of eosinophils in the AS group was greater than that in the AR+ group (p < 0.05) and in the AR- group (p < 0.01). Moreover, the eosinophil count was lower in the AR- group compared with the AR+ group (p < 0.05). Similarly, the number of EG2+ cells was greater in the AS group than in the AR group (p < 0.02) and the AR- group (p < 0.05). Moreover, the EG2+ cell count was lower in the AR- group than in the AR+ group (p < 0.05). The number of mast cells and basophils in the AS group was greater than that in the AR group (p < 0.05 and p < 0.01, respectively). Mast cells in sputum were tryptase-positive. Basophils were present in sputum from 23% of patients with AR and 53% of patients with asthma. There was a significant correlation between methacholine PD20 and eosinophils (p < 0.005) and mast cells (p < 0.02) but not with basophils in those patients showing a measurable methacholine PD20 (AR+ and AS groups). CONCLUSIONS Inflammatory cells are present not only in the airways of patients with asthma but also in airways of patients with seasonal AR, even outside natural exposure. Moreover, we provide evidence for the presence of basophils in sputum of patients with asthma even during clinical remission. The presence of bronchial responsiveness is associated with an increase in the number of eosinophils and metachromatic cells. Our findings are consistent with the hypothesis that eosinophils, as well as mast cells, contribute to bronchial responsiveness not only in AS but also in seasonal AR.

Vascular endothelial growth factor up‐regulation and bronchial wall remodelling in asthma

Background There is increasing in vitro evidence to support a role for vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, as a mediator of fibrosis associated with neovascularization.

PTPN22 R620W polymorphism in the ANCA-associated vasculitides

OBJECTIVES PTPN22 is involved in T-cell activation and its R620W single-nucleotide polymorphism (SNP) has been shown to predispose to different autoimmune diseases. The aims of this study were to investigate the role of the PTPN22 R620W SNP in conferring susceptibility to the ANCA-associated vasculitides (AAVs), and to explore potential associations between the PTPN22 genotype and the disease manifestations. METHODS PTPN22 R620W SNP was genotyped in a cohort of 344 AAV patients [143 with granulomatosis with polyangiitis (Wegeners) (GPA), 102 with microscopic polyangiitis (MPA) and 99 with Churg-Strauss syndrome (CSS)] and in 945 healthy controls. RESULTS The frequency of the minor allele (620W) was significantly higher in GPA patients than in controls [P = 0.005, χ(2 )= 7.858, odds ratio (OR) = 1.91], while no statistically significant association was found with MPA or CSS. Among GPA patients, the 620W allele was particularly enriched in ANCA-positive patients as compared with controls (P = 0.00012, χ(2 )= 14.73, OR = 2.31); a particularly marked association was also found with ENT involvement (P = 0.0071, χ(2 )= 7.258, OR = 1.98), lung involvement (P = 0.0060, χ(2 )= 7.541, OR = 2.07) and skin manifestations of all kinds (P = 0.000047, χ(2 )= 16.567, OR = 3.73). CONCLUSION The PTPN22 620W allele confers susceptibility to the development of GPA (but not of MPA or CSS), and particularly of its ANCA-positive subset.

Induced sputum: diagnostic value in interstitial lung disease.

Induced sputum by inhalation of hypertonic saline solution is a noninvasive technique used to collect cellular and soluble material from lung airways. During the past decade, this method has been widely used to assess airway inflammation in asthma and chronic obstructive pulmonary disease, since it produces reliable results and compares favorably to other invasive techniques, such as biopsy and bronchoalveolar lavage (BAL). Induced sputum has been recently used to study interstitial lung disease (ILD), more specifically pneumoconiosis, sarcoidosis, and nongranulomatous ILD. Moreover, results from induced sputum supplied information comparable to BAL findings for occupational lung disease and were able to distinguish sarcoidosis patients from healthy subjects and from patients with nongranulomatous ILD. Although induced sputum had previously provided promising results in assessing patients with ILD, its diagnostic role has not yet been well defined. Further studies of the evaluation by induced sputum of grading of severity, follow-up of disease, and effects of treatment are needed. Additionally, to date no specific studies have been undertaken to evaluate the safety and functional effects of sputum induction on patients with ILD. In conclusion, we think that induced sputum can be used as a complementary tool to BAL both in research and in clinical monitoring of patients with ILD.

MICROSATELLITE ANALYSIS OF INDUCED SPUTUM DNA IN PATIENTS WITH LUNG CANCER IN HEAVY SMOKERS AND IN HEALTHY SUBJECTS

Abnormality in the fragile histidine triade (FHIT), a candidate tumor suppressor gene located in chromosome region 3 (3p14.2), has been frequently found in multiple tumor types, including lung cancer. In this study, the authors assessed the consistency of DNA microsatellite analysis of induced sputum (IS), as compared to that of blood and plasma. They also evaluated the loss of heterozigosity (LOH) and microsatellite instability (MSI) in 3 different loci, D3S1300, D3S1313, and D3S1234, all internal to the FHIT gene, in IS, blood, and plasma from patients with lung cancer, smokers, and healthy subjects. Eighteen patients with lung cancer (3 females, age mean ± SD: 63 ± 7 years), 39 smokers (23 females, age mean ± SD: 57 ± 6 years and cigarette pack-years mean ± SD: 34 ± 12), and 22 healthy nonsmoking subjects (13 females, age mean ± SD: 63 ± 5 years) were studied. DNA was extracted from blood, plasma, and IS, by means of a standard method. Analysis of LOH and MSI were performed using a fluorescent polymerase chain reaction (PCR)-based approach, followed by capillary electrophoresis. The ratios between the peak heights (phs), expressed as random fluorescence units, from plasma/blood (p/b) and induced sputum/blood (is/b) in all three loci were considered. The biases (agreement limits) between the mean ph ratio from p/b and is/b of D3S1300, D3S1313, and D3S1234 were respectively 0.07 (− 0.39 to 0.53), 0.016 (− 0.32 to 0.35), − 0.10 (− 0.51 to 0.30) in the patients; − 0.04 (− 0.52 to 0.43), − 0.06 (− 0.31 to 0.18), − 0.08 (− 0.48 to 0.30) in smokers; and − 0.11 (− 0.40 to 0.17), − 0.05 (− 0.53 to 0.43), − 0.09 (− 0.51 to 0.33) in healthy subjects. LOH and MSI in at least one locus were observed in 55% of patients, in 18% of smokers, and in 4.5% of healthy subjects (P < 0.001). These results showed that IS DNA provided data that were consistent with those from blood and plasma. These findings highlight new prospects for early tumor detection by a noninvasive technique based on the analysis of genetic alterations in induced sputum.