Dosing intact birch pollen grains at the air-liquid interface (ALI) to the immortalized human bronchial epithelial cell line BEAS-2B.

In real life, humans are exposed to whole pollen grains at the air epithelial barrier. We developed a system for in vitro dosing of whole pollen grains at the Air-Liquid Interface (ALI) and studied their effect on the immortalized human bronchial epithelial cell line BEAS-2B. Pollen are sticky and large particles. Dosing pollen needs resuspension of single particles rather than clusters, and subsequent transportation to the cells with little loss to the walls of the instrumentation i.e. in a straight line. To avoid high speed impacting insults to cells we chose sedimentation by gravity as a delivery step. Pollen was resuspended into single particles by pressured air. A pollen dispersion unit including PTFE coating of the walls and reduced air pressure limited impaction loss to the walls. The loss of pollen to the system was still about 40%. A linear dose effect curve resulted in 327-2834 pollen/cm2 (± 6.1%), the latter concentration being calculated as the amount deposited on epithelial cells on high pollen days. After whole pollen exposure, the largest differential gene expression at the transcriptomic level was late, about 7 hours after exposure. Inflammatory and response to stimulus related genes were up-regulated. We developed a whole pollen exposure air-liquid interface system (Pollen-ALI), in which cells can be gently and reliably dosed.

Impaired Differentiation of Chronic Obstructive Pulmonary Disease Bronchial Epithelial Cells Grown on Bronchial Scaffolds.

Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation, small airway remodeling, and emphysema. Airway remodeling in patients with COPD involves both the airway epithelium and the subepithelial extracellular matrix (ECM). However, it is currently unknown how epithelial remodeling in COPD airways depends on the relative influence from inherent defects in the epithelial cells and alterations in the ECM. To address this, we analyzed global gene expression in COPD human bronchial epithelial cells (HBEC) and normal HBEC after repopulation on decellularized bronchial scaffolds derived from patients with COPD or donors without COPD. COPD HBEC grown on bronchial scaffolds showed an impaired ability to initiate ciliated-cell differentiation, which was evident on all scaffolds regardless of their origin. In addition, although normal HBEC were less affected by the disease state of the bronchial scaffolds, COPD HBEC showed a gene expression pattern indicating increased proliferation and a retained basal-cell phenotype when grown on COPD bronchial scaffolds compared with normal bronchial scaffolds. By using mass spectrometry, we identified 13 matrisome proteins as being differentially abundant between COPD bronchial scaffolds and normal bronchial scaffolds. These observations are consistent with COPD pathology and suggest that both epithelial cells and the ECM contribute to epithelial-cell remodeling in COPD airways.

Addressing the challenges of E-cigarette safety profiling by assessment of pulmonary toxicological response in bronchial and alveolar mucosa models.

Limited toxicity data on electronic cigarette (ECIG) impede evidence-based policy recommendations. We compared two popular mixed fruit flavored ECIG-liquids with and without nicotine aerosolized at 40 W (E-smoke) with respect to particle number concentrations, chemical composition, and response on physiologically relevant human bronchial and alveolar lung mucosa models cultured at air-liquid interface. E-smoke was characterized by significantly increased particle number concentrations with increased wattage (25, 40, and 55 W) and nicotine presence. The chemical composition of E-smoke differed across the two tested flavors in terms of cytotoxic compounds including p-benzoquinone, nicotyrine, and flavoring agents (for example vanillin, ethyl vanillin). Significant differences in the expression of markers for pro-inflammation, oxidative stress, tissue injury/repair, alarm anti-protease, anti-microbial defense, epithelial barrier function, and epigenetic modification were observed between the flavors, nicotine content, and/ or lung models (bronchial or alveolar). Our findings indicate that ECIG toxicity is influenced by combination of multiple factors including flavor, nicotine content, vaping regime, and the region of respiratory tree (bronchial or alveolar). Toxic chemicals and flavoring agents detected in high concentrations in the E-smoke of each flavor warrant independent evaluation for their specific role in imparting toxicity. Therefore, multi-disciplinary approaches are warranted for comprehensive safety profiling of ECIG.

Heterogeneous ozone effects on the DNA methylome of bronchial cells observed in a crossover study.

We used a randomized crossover experiment to estimate the effects of ozone (vs. clean air) exposure on genome-wide DNA methylation of target bronchial epithelial cells, using 17 volunteers, each randomly exposed on two separated occasions to clean air or 0.3-ppm ozone for two hours. Twenty-four hours after exposure, participants underwent bronchoscopy to collect epithelial cells whose DNA methylation was measured using the Illumina 450 K platform. We performed global and regional tests examining the ozone versus clean air effect on the DNA methylome and calculated Fisher-exact p-values for a series of univariate tests. We found little evidence of an overall effect of ozone on the DNA methylome but some suggestive changes in PLSCR1, HCAR1, and LINC00336 DNA methylation after ozone exposure relative to clean air. We observed some participant-to-participant heterogeneity in ozone responses.

Sonic hedgehog signalling as a potential endobronchial biomarker in COPD.

BACKGROUND: The hedgehog (HH) pathway has been associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies and recent studies suggest that HH signalling could be altered in COPD. We therefore used minimally invasive endobronchial procedures to assess activation of the HH pathway including the main transcription factor, Gli2, and the ligand, Sonic HH (Shh). METHODS: Thirty non-COPD patients and 28 COPD patients were included. Bronchial brushings, bronchoalveolar lavage fluid (BALF) and bronchial biopsies were obtained from fiberoptic bronchoscopy. Characterization of cell populations and subcellular localization were evaluated by immunostaining. ELISA and RNAseq analysis were performed to identify Shh proteins in BAL and transcripts on lung tissues from non-COPD and COPD patients with validation in an external and independent cohort. RESULTS: Compared to non-COPD patients, COPD patients exhibited a larger proportion of basal cells in bronchial brushings (26 ± 11% vs 13 ± 6%; p < 0.0001). Airway basal cells of COPD subjects presented less intense nuclear staining for Gli2 in bronchial brushings and biopsies (p < 0.05). Bronchial BALF from COPD patients contained lower Shh concentrations than non-COPD BALF (12.5 vs 40.9 pg/mL; p = 0.002); SHH transcripts were also reduced in COPD lungs in the validation cohort (p = 0.0001). CONCLUSION: This study demonstrates the feasibility of assessing HH pathway activation in respiratory samples collected by bronchoscopy and identifies impaired bronchial epithelial HH signalling in COPD.

Scent dog identification of samples from COVID-19 patients - a pilot study.

BACKGROUND: As the COVID-19 pandemic continues to spread, early, ideally real-time, identification of SARS-CoV-2 infected individuals is pivotal in interrupting infection chains. Volatile organic compounds produced during respiratory infections can cause specific scent imprints, which can be detected by trained dogs with a high rate of precision. METHODS: Eight detection dogs were trained for 1 week to detect saliva or tracheobronchial secretions of SARS-CoV-2 infected patients in a randomised, double-blinded and controlled study. RESULTS: The dogs were able to discriminate between samples of infected (positive) and non-infected (negative) individuals with average diagnostic sensitivity of 82.63% (95% confidence interval [CI]: 82.02-83.24%) and specificity of 96.35% (95% CI: 96.31-96.39%). During the presentation of 1012 randomised samples, the dogs achieved an overall average detection rate of 94% (±3.4%) with 157 correct indications of positive, 792 correct rejections of negative, 33 incorrect indications of negative or incorrect rejections of 30 positive sample presentations. CONCLUSIONS: These preliminary findings indicate that trained detection dogs can identify respiratory secretion samples from hospitalised and clinically diseased SARS-CoV-2 infected individuals by discriminating between samples from SARS-CoV-2 infected patients and negative controls. This data may form the basis for the reliable screening method of SARS-CoV-2 infected people.

Synthesis and Evaluation of Airway-Targeted PLGA-PEG Nanoparticles for Drug Delivery in Obstructive Lung Diseases.

Chronic airway inflammation is a hallmark of chronic obstructive airway diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and asthma. Airway inflammation and mucus obstruction present major challenges to drug or gene delivery and therapeutic efficacy of nano-based carriers in these chronic obstructive airway conditions. To achieve targeted drug delivery of NPs to the diseased cells, NPs need to bypass the obstructive airway and circumvent the airway's defense mechanisms. Although there has been increasing interest and significant progress in development of NPs for targeting cancer, relatively little progress has been made towards designing novel systems for targeted treatment of chronic inflammatory and obstructive airway conditions. Hence, we describe here methods for preparing drug loaded multifunctional nanoparticles for targeted delivery to specific airway cell types in obstructive lung diseases. The formulations and methods for selective drug delivery in the treatment of chronic airway conditions such as COPD, CF, and asthma have been evaluated using a variety of preclinical models by our laboratory and currently ongoing further clinical development for translation from bench to bedside.

Epigenome-wide effects of vitamin D on asthma bronchial epithelial cells.

Vitamin D is a nutrient and a hormone with multiple effects on immune regulation and respiratory viral infections, which can worsen asthma and lead to severe asthma exacerbations. We set up a complete experimental and analytical pipeline for ATAC-Seq and RNA-Seq to study genome-wide epigenetic changes in human bronchial epithelial cells of asthmatic subjects, following treatment of these cells with calcitriol (vitamin D3) and Poly (I:C)(a viral analogue). This approach led to the identification of biologically plausible candidate genes for viral infections and asthma, such as DUSP10 and SLC44A1.

Treatment of Cells and Tissues with Chromate Maximizes Mitochondrial 2Fe2S EPR Signals.

In a previous study on chromate toxicity, an increase in the 2Fe2S electron paramagnetic resonance (EPR) signal from mitochondria was found upon addition of chromate to human bronchial epithelial cells and bovine airway tissue ex vivo. This study was undertaken to show that a chromate-induced increase in the 2Fe2S EPR signal is a general phenomenon that can be used as a low-temperature EPR method to determine the maximum concentration of 2Fe2S centers in mitochondria. First, the low-temperature EPR method to determine the concentration of 2Fe2S clusters in cells and tissues is fully developed for other cells and tissues. The EPR signal for the 2Fe2S clusters N1b in Complex I and/or S1 in Complex II and the 2Fe2S cluster in xanthine oxidoreductase in rat liver tissue do not change in intensity because these clusters are already reduced; however, the EPR signals for N2, the terminal cluster in Complex I, and N4, the cluster preceding the terminal cluster, decrease upon adding chromate. More surprising to us, the EPR signals for N3, the cluster preceding the 2Fe2S cluster in Complex I, also decrease upon adding chromate. Moreover, this method is used to obtain the concentration of the 2Fe2S clusters in white blood cells where the 2Fe2S signal is mostly oxidized before treatment with chromate and becomes reduced and EPR detectable after treatment with chromate. The increase of the g = 1.94 2Fe2S EPR signal upon the addition of chromate can thus be used to obtain the relative steady-state concentration of the 2Fe2S clusters and steady-state concentration of Complex I and/or Complex II in mitochondria.

A case of spheroid-type localized lactoferrin amyloidosis in the bronchus.

We report a case of localized bronchial lactoferrin amyloidosis. A 47-year-old man presented with a complaint of persistent dry cough for two months. Chest computed-tomography revealed a calcification shadow of the right main bronchus; hence, a biopsy was performed, showing layered spheroid-type eosinophilic deposits in the bronchial wall. These deposits were positive for Congo red staining, exhibiting apple-green birefringence under polarized light. In addition, an electron microscopic examination demonstrated that this layered structure was formed by very thin cord-like amyloid deposits. By proteomics analysis using liquid chromatography-tandem mass spectrometry and immunohistochemistry, we confirmed that the deposited amyloid was composed of lactoferrin. While lactoferrin is known to be a precursor protein of localized corneal and seminal vesicle amyloidosis, localized lactoferrin amyloidosis of the bronchus has not been reported in the English literature. Our pathological findings suggested that localized lactoferrin amyloidosis may be caused by long-term tissue damage, and the characteristic spheroid-type appearance is thought to be associated with unique, thin cord-like amyloid deposits.

Differential bronchial epithelial response regulated by ΔNp63: a functional understanding of the epithelial shedding found in asthma.

Bronchial epithelial cells serve as a physical barrier at the forefront of the immune system. Barrier disruption and an excessive immune response of the bronchial epithelium contribute to the pathophysiology of asthma, a chronic bronchial inflammatory disease. The purpose of this study was to investigate the functional significance of ΔNp63, a p53-like transcription factor expressed by the basal bronchial epithelium. The immunohistochemical expression profile of ΔNp63 was evaluated in human bronchial tissue derived from asthma patients. The role of ΔNp63 in apoptosis inhibition and production of soluble mediators was investigated in vitro with cultured BEAS-2B bronchial epithelial cells using molecular biological analysis. In healthy bronchial tissue, ΔNp63-positive basal epithelial cells were covered with differentiated ΔNp63-negative cells but in the asthmatic airway, ΔNp63-positive cells were directly exposed to the bronchial lumen due to severe epithelial shedding. ΔNp63 regulated bronchial apoptosis in response to Toll-like receptor 3 stimulation. On the other hand, expression of ΔNp63 was modulated by stimulation with trypsin and SLIGKV, protease-activated receptor 2 ligands. Further phenotypic analysis revealed that ΔNp63 controlled the transcriptional expression and protein release of some epithelium-derived proinflammatory cytokines and endogenous protease inhibitors. We conclude that ΔNp63 modulates the bronchial epithelial response to viral infection. At the same time, ΔNp63 expression is influenced by proteases, which are abundant in house dust mites. Therefore, the ΔNp63 axis would be intimately involved in these two major triggers of asthma exacerbations, viral infection and protease overload.

The effects of bisphenol A on some plasma cytokine levels and distribution of CD8+ and CD4+ T lymphocytes in spleen, ileal Peyer's patch and bronchus associated lymphoid tissue in rats.

The effects of bisphenol A on the some plasma cytokine levels and distribution of CD8+ and CD4+ T lymphocytes in spleen, ilealPeyer's patch and bronchus-associated lymphoid tissue in rats were investigated. A total of fourty male Wistar Albino rats were divided into five groups including 8 rats in each one: control, vehicle, BPA 5, BPA 50 and BPA 500 groups. Doses of 5, 50 and 500 µg/kg BPA were dissolved in ethanol, then mixed with corn oil. The control group received no treatment. The vehicle group was given the ethanol-corn oil mixture. BPA 5, BPA 50 and BPA 500 groups were given, respectively, 5, 50, and 500 µg/kg/day orally. In blood samples, IL-4, IL-6, IL-10 and TNF-α plasma levels were determined with ELISA. Tissue samples (spleen, ileal Peyer's patches and lung) were processed by means of routine histological techniques. CD4 and CD8 were stained immunohistochemically. Data obtained from this study showed that, BPA causes the alteration on immune parameters including cytokine profile, distribution of CD8+ and CD4+ T lymhpocytes in spleen and ileal Peyer's patches. Present study indicated that BPA may affect immune systems even at lower doses.Disruption of immun system cells and cytokine levels can result in harmful outcomes triggering autoimmune diseases and immunodeficiencies.

Club cell protein (CC16) in plasma, bronchial brushes, BAL and urine following an inhaled allergen challenge in allergic asthmatics.

BACKGROUND: Club cell protein (CC16) is a pneumoprotein secreted by epithelial club cells. CC16 possesses anti-inflammatory properties and is a potential biomarker for airway epithelial damage. We studied the effect of inhaled allergen on pulmonary and systemic CC16 levels. METHODS: Thirty-four subjects with allergic asthma underwent an inhaled allergen challenge. Bronchoscopy with bronchoalveolar lavage (BAL) and brushings was performed before and 24 h after the challenge. CC16 was quantified in BAL and CC16 positive cells and CC16 mRNA in bronchial brushings. CC16 was measured in plasma and urine before and repeatedly after the challenge. Thirty subjects performed a mannitol inhalation challenge prior to the allergen challenge. RESULTS: Compared to baseline, CC16 in plasma was significantly increased in all subjects 0-1 h after the allergen challenge, while CC16 in BAL was only increased in subjects without a late allergic response. Levels of CC16 in plasma and in the alveolar fraction of BAL correlated significantly after the challenge. There was no increase in urinary levels of CC16 post-challenge. Mannitol responsiveness was greater in subjects with lower baseline levels of CC16 in plasma. CONCLUSIONS: The increase in plasma CC16 following inhaled allergen supports the notion of CC16 as a biomarker of epithelial dysfunction.

Thermo-physical properties of synthetic mucus for the study of airway clearance.

In this article, dynamic viscosity, surface tension, density, heat capacity and thermal conductivity, of a bronchial mucus simulant proposed by Zahm et al., Eur Respir J 1991; 4: 311-315 were experiementally determined. This simulant is mainly composed of a galactomannan gum and a scleroglucan. It was shown that thermophysical properties of synthetic mucus are dependant of scleroglucan concentrations. More importantly and for some scleroglucan concentrations, the syntetic mucus, exhibits, somehow, comparable thermophysical properties to real bronchial mucus. An insight on the microstructure of this simulant is proposed and the different properties enounced previously have been measured for various scleroglucan concentrations and over a certain range of operating temperatures. This synthetic mucus is found to mimic well the rheological behavior and the surface tension of real mucus for different pathologies. Density and thermal properties have been measured for the first time. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3025-3033, 2017.

Carbon Nanotubes in the Human Respiratory Tract-Clearance Modeling.

Clearance of single-wall carbon nanotubes (SWCNT, diameter: 5 nm) and multi-wall carbon nanotubes (MWCNT, diameter: 50 nm) in the respiratory tract was predicted for various age groups (infants, children, adolescents, and adults). The model was founded on the assumption that lung clearance takes place in three distinct phases: (i) fast mucociliary clearance, (ii) slow bronchial clearance, and (iii) alveolar clearance. To each of these phases a specific fraction of deposited particles was attributed, the amount of which depended on particles' geometry and particles' deposition sites in the respiratory system. Clearance velocities were expressed by respective clearance half-times ranging from several hours in the case of fast clearance to tens of days in the case of slow clearance. Results of the simulations clearly demonstrate that for the specific deposition scenario (sitting, nasal breathing) considered here fast clearance fraction exhibits a slight decrease with increasing age, but total clearance times (i.e. time spans, within which 100% of the deposited particulate mass are removed) are rather constant among the age groups. Nanotubes deposited in the respiratory bronchioles and alveoli are usually subject to a long-term storage in these structures and, thus, may trigger malignant transformations in adjacent cells and tissues.

Effect of Polypeptides on Cell Proliferation and Apoptosis during Aging.

Polypeptide complexes derived from the bronchi, blood vessels, muscles, kidneys, ovaries, testes, and retina stimulated the processes of cell renewal in organotypic cultures of the corresponding organs of young and old animals. A correlation between the intensity of regeneration and animal' age was revealed. The polypeptide complexes reduced the expression of apoptotic factors p53 and caspase 3 and increased the expression of proliferation protein Ki-67. These results provide the basis for further study of the polypeptide complexes as stimulators of regenerative processes in different tissues during ageing.

Extracellular cadmium in the bronchoalveolar space of long-term tobacco smokers with and without COPD and its association with inflammation.

Tobacco contains cadmium, and this metal has been attributed a causative role in pulmonary emphysema among smokers, although extracellular cadmium has not to date been quantified in the bronchoalveolar space of tobacco smokers with or without COPD. We determined whether cadmium is enhanced in the bronchoalveolar space of long-term tobacco smokers with or without COPD in vivo, its association with inflammation, and its effect on chemokine release in macrophage-like cells in vitro. Bronchoalveolar lavage (BAL), sputum, and blood samples were collected from current, long-term smokers with and without COPD and from healthy nonsmokers. Cadmium concentrations were determined in cell-free BAL fluid using inductively coupled plasma mass spectrometry. Blood monocyte-derived macrophages were exposed to cadmium chloride in vitro. Depending upon the type of sample, molecular markers of inflammation were quantified either as protein (enzyme-linked immunosorbent assay) or as mRNA (real-time polymerase chain reaction). Cadmium concentrations were markedly increased in cell-free BAL fluid of smokers compared to that of nonsmokers (n=19-29; P<0.001), irrespective of COPD. In these smokers, the measured cadmium displayed positive correlations with macrophage TNF-α mRNA in BAL, neutrophil and CD8(+) cell concentrations in blood, and finally with IL-6, IL-8, and MMP-9 protein in sputum (n=10-20; P<0.05). The cadmium chloride exposure caused a concentration-dependent increase in extracellular IL-8 protein in monocyte-derived macrophages in vitro. In conclusion, extracellular cadmium is enhanced in the bronchoalveolar space of long-term smokers and displays pro-inflammatory features. Its pathogenic role in tobacco-induced disease deserves further evaluation.

Interleukin-31 expression and relation to disease severity in human asthma.

Interleukin 31 (IL-31) is a novel T helper type 2 effector cytokine that plays an important role in the pathogenesis of allergic diseases. However, its role in human asthma remains unclear. The aim of this study was to measure IL-31 levels in the serum, bronchoalveolar lavage fluid (BALF) and bronchial tissue of asthmatics and healthy subjects, and identify its possible correlation to disease severity. We quantified IL-31 levels in the serum of patients with asthma (n = 44), as well as in controls (n = 22). Of these subjects, 9 asthmatics and five controls underwent bronchoscopy with endobronchial biopsy and BALF collection. Our data showed that serum and BALF IL-31 levels were significantly elevated in patients with asthma compared with controls. Expressions of IL-31 and IL-31 receptor (IL-31RA and OSMR) were more prominent in the bronchial tissue in severe compared to mild asthma and controls. Serum IL-31 levels correlated positively with Th2 related cytokines (IL-5, IL-13, and TSLP), asthma severity or total serum immunoglobulin E (IgE), and inversely with asthma control and the forced expiratory volume in 1 second (FEV1). The current data may provide insight into the underlying pathogenesis of asthma, in which IL-31 has an important pathogenic role.

Bronchial involvement in advanced stage lymphangioleiomyomatosis: histopathologic and molecular analyses.

Lymphangioleiomyomatosis (LAM), a rare progressive disease that almost exclusively affects women, is characterized by pulmonary cysts and neoplastic proliferation of smooth muscle-like cells (LAM cells). Airflow obstruction is a physiologic consequence that is commonly observed in LAM and has been attributed to narrowing of peripheral airways. However, histopathologic examinations of the entire airway have been precluded by the limited availability of such specimens. Here, we used explanted lung tissues from 30 LAM patients for a thorough histologic analysis with a special emphasis on the bronchi. We found bronchial involvement by LAM cells and lymphatics in all patients examined. Furthermore, a moderate to severe degree of chronic inflammation (73%), goblet cell hyperplasia (97%), squamous cell metaplasia (83%) of the epithelium, and thickening of basal lamina (93%) were identified in the bronchi. Because LAM cells are transformed by the functional loss of the TSC genes leading to a hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, we confirmed the expression of phospho-p70S6K, phospho-S6, phospho-4E-BP1, and vascular endothelial growth factor (VEGF)-D in LAM cells from all of the patients examined. In contrast, no protein expression of hypoxia-inducible factor 1α, a downstream molecule indicative of mTORC1 activation and leading to VEGF production, was detected in any patient. Our study indicates that late-stage LAM patients commonly have bronchi involved by the proliferation of both LAM cells and lymphatics and that chronic inflammation complicated their disease. Furthermore, the up-regulation of hypoxia-inducible factor 1α, a common event in mTORC1-driven tumor cells, does not occur in LAM cells and plays no role in VEGF-D expression in LAM cells.

Severe dysplasia can be distinguished from moderate and mild dysplasia of bronchial mucosa by changes in Ki-67 index.

Preneoplastic lesions on small bronchial biopsy specimens may cause a diagnostic dilemma. The aim of this study was to estimate karyometric variables and the Ki-67 index of preneoplastic bronchial lesions and squamous cell carcinoma of the lung. The study was performed on endoscopic samples of squamous cell carcinoma (n = 22), normal appearing mucosa surrounding carcinoma (n = 10), bronchial dysplasia of mild (n = 7), moderate (n = 6), and severe grade (n = 6), carcinoma in situ (n = 17), and normal mucosa from patients with chronic bronchitis (n = 26). Karyometric analysis was done using the image analyzer ImageJ 1.47q. Ki-67 activity was also quantified by ImageJ 1.47q with the plugin Cell Counter. The highest values of nuclear area were found in squamous cell carcinoma, and differences were statistically significant compared to normal mucosa, all grades of dysplasia and normal appearing mucosa surrounding carcinoma (p < 0.01). The Ki-67 index was significantly higher in squamous cell lung carcinoma compared to normal mucosa, mild and moderate dysplasia and normal appearing mucosa surrounding carcinoma (p < 0.01). The Ki-67 index was significantly higher in severe dysplasia than in mild and moderate dysplasia (p < 0.01). In conclusion, the Ki-67 index is a useful parameter for more objective grading and can be of prognostic value to determine the biological potential of preneoplastic bronchial lesions.