Bronchiolar Regions Research Articles

Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1β contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1β-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1β-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

Paracelsian ‘Bergsucht’ – lung cancer or radiation-induced fibrosis?

Purpose Assessment of absorbed doses on organs and tissues of miners during radon exposure in the Schneeberg mines in the sixteenth century and calculation of the probability of occurrence of radiation-induced lung cancer and lung fibrosis, considering the life expectancy characteristic and the absence of smoking. Materials and methods The expected radon concentration at the Schneeberg mines has been estimated using published data. Modeling of the accumulation of radon in the working tunnels of mine workings was carried out using the RESRAD-Build 4.0, based on the radium concentration in soil and geometric parameters of the mining tunnel from the engravings in Agricola’s book. The dynamics of radionuclides in the human body were performed using the WinAct software in accordance with data from ICRP Publications 130 and 137. The values of absorbed doses on the tissues of the respiratory tract were obtained using the IDAC 2.1 program. Several models based on the epidemiology of uranium miners have been used to calculate radiation risks from radon exposure. The probability of male survival at birth and the age-specific frequency of spontaneous lung cancer not associated with radiation for miners of the sixteenth century (nonsmoking men aged 20–40 years) were estimated to properly calculate the radiation risks. Results The expected radon concentration in the Schneeberg mines was assessed in the range of 75–100 kBq m−3. The average value of the equilibrium factor was estimated as 0.49 ± 0.03. The annual exposure of miners to radon decay products was assessed as 125–165 WLM year−1. The annual values of absorbed doses to different sections of the respiratory tract were calculated, the maximum absorbed doses of α-radiation are formed on the bronchial and bronchiolar regions of the lungs (2.23 Gy year−1). The deterministic effects as radiation fibrosis of the lungs with 10 years of experience in the mines of Schneeberg have a probability of occurrence from 60 to 100%. All the models used for radiation risk assessments showed that the lifetime risk of developing lung cancer for nonsmoking Schneeberg miners is many times lower than the risk of developing deterministic radiation effects. In contrast, for the smoking cohort of miners in the nineteenth century lung cancer become the dominant cause of death. Conclusions The deterministic radiation effects of Schneeberg miners in sixteenth century, exposed to extremely high levels of radon, such as radiation pneumosclerosis or pulmonary fibrosis, are more likely than the development of radiation-induced lung cancer.

Development of Respiratory Tract Organs for ICRP Pediatric Mesh-type Reference Computational Phantoms.

As part of the activities of the International Commission on Radiological Protection (ICRP) Task Group 103, the present study developed a new set of respiratory tract organs consisting of the extrathoracic, bronchial, bronchiolar, and alveolar-interstitial regions for newborn, 1-, 5-, 10-, and 15-y-old males and females for use in pediatric mesh-type reference computational phantoms. The developed respiratory tract organs, while preserving the original topologies of those of the pediatric voxel-type reference computational phantoms of ICRP Publication 143, have improved anatomy and detailed structure and also include μm-thick target and source regions prescribed in ICRP Publication 66. The dosimetric impact of the developed respiratory tract organs was investigated by calculating the specific absorbed fraction for internal electron exposures, which were then compared with the ICRP Task Group 96 values. The results showed that except for the alveolar-interstitial region as a source region, the pediatric mesh phantoms showed larger specific absorbed fractions than the Task Group 96 values. The maximum difference was a factor of ~3.5 for the extrathoracic-2 basal cell and surface as target and source regions, respectively. These results reflect the differences in the target masses and geometry caused by the anatomical enhancement of the pediatric mesh phantoms. For the alveolar-interstitial region as a source region, the pediatric mesh phantoms showed larger values for low energy ranges and lower values with increasing energies, owing to the differences in the size and shape of the alveolar-interstitial region.

Progressive Lung Injury, Inflammation, and Fibrosis in Rats Following Inhalation of Sulfur Mustard.

Sulfur mustard (SM) inhalation causes debilitating pulmonary injury in humans which progresses to fibrosis. Herein, we developed a rat model of SM toxicity which parallels pathological changes in the respiratory tract observed in humans. SM vapor inhalation caused dose (0.2-0.6 mg/kg)-related damage to the respiratory tract within 3 days of exposure. At 0.4-0.6 mg/kg, ulceration of the proximal bronchioles, edema and inflammation were observed, along with a proteinaceous exudate containing inflammatory cells in alveolar regions. Time course studies revealed that the pathologic response was biphasic. Thus, changes observed at 3 days post-SM were reduced at 7-16 days; this was followed by more robust aberrations at 28 days, including epithelial necrosis and hyperplasia in the distal bronchioles, thickened alveolar walls, enlarged vacuolated macrophages, and interstitial fibrosis. Histopathologic changes were correlated with biphasic increases in bronchoalveolar lavage (BAL) cell and protein content and proliferating cell nuclear antigen expression. Proinflammatory proteins receptor for advanced glycation end product (RAGE), high-mobility group box protein (HMGB)-1, and matrix metalloproteinase (MMP)-9 also increased in a biphasic manner following SM inhalation, along with surfactant protein-D (SP-D). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS), inflammatory proteins implicated in mustard lung toxicity, and the proinflammatory/profibrotic protein, galectin (Gal)-3, were upregulated in alveolar macrophages and in bronchiolar regions at 3 and 28 days post-SM. Inflammatory changes in the lung were associated with oxidative stress, as reflected by increased expression of heme oxygenase (HO)-1. These data demonstrate a similar pathologic response to inhaled SM in rats and humans suggesting that this rodent model can be used for mechanistic studies and for the identification of efficacious therapeutics for mitigating toxicity.

Bacterial extracellular vesicles isolated from organic dust induce neutrophilic inflammation in the lung.

Inhalation of organic dust is an occupational hazard leading to the development of respiratory symptoms and respiratory diseases. Bioaerosols from concentrated animal feeding operations are rich in bacteria and could carry bacterial extracellular vesicles (EVs) that could induce lung inflammation. It is not known if organic dust contains bacterial EVs and whether they modulate lung inflammation. Herein, we show that poultry organic dust contains bacterial EVs (dust EVs) that induce lung inflammation. Treatment of airway epithelial cells, THP-1-monocytes and -macrophages with dust EVs rapidly induced IL-8, IL-6, ICAM-1, proIL-1β, and TNF-α levels. In airway epithelial cells, induction of inflammatory mediators was due to increased mRNA levels and NF-κB activation. Induction of inflammatory mediators by dust EVs was not inhibited by polymyxin B. Single and repeated treatments of mice with dust EVs increased lung KC, IL-6, and TNF-α levels without significantly altering IL-17A levels. Increases in cytokines were associated with enhanced neutrophil infiltration into the lung. Repeated treatments of mice with dust EVs increased lung mean linear intercept and increased collagen deposition around airways indicating lung remodeling. Peribronchial cell infiltrates and airway epithelial thickening were also observed in treated mice. Because bacterial EVs are nanometer-sized particles, they can reach and accumulate in the bronchiolar and alveolar regions causing lung injury leading to the development of respiratory diseases. Our studies have provided new evidence for the presence of bacterial EVs in organic dust and for their role as one of the causative agents of organic dust-induced lung inflammation and lung injury.

Chronic model of sulfur mustard‐induced lung injury in Fischer 344 rats

Recent chemical attacks in Syria demonstrate the nondiscriminatory threat that chemical agents pose to both military personnel and civilians alike. Sulfur mustard (SM)is a highly reactive and toxic chemical agent that poses such a threat and is considered a potential weapon that could be employed in acts of terror. SM reacts with epithelial tissue of the respiratory tract, causing a progressive, debilitating injury that currently has no FDA‐approved medical therapeutic countermeasure. This study aimed to develop a chronic model of SM‐induced pulmonary morbidities in inbred Fischer 344 rats with the overall goal of evaluating mesenchymal stem cell therapy as a proof‐of‐concept study. Fifty‐one (n=15 per SM group, n=6vehicle group) male rats were anesthetized, underwent tracheal intubation and were exposed to one of three concentrations of sulfur mustard vapor (0.7, 1.0, and1.4 mg/kg) or vehicle (ethanol) via inhalation for 50 min. At euthanasia (21–28days PE), respiratory mechanics and lung pathology, on the left and accessory lobes, were assessed. Forced oscillation parameters of SM‐exposed animals showed similar significant reductions, despite the SM dose, in inspiratory capacity (IC), compliance of the entire respiratory system (Crs), and significant increases in the respiratory system elastance(Ers) and resistance (Rrs) in comparison to control (ethanol) animals. Pressure‐volume (PV) loop parameters showed that all three SM doses caused similar significant reductions in static compliance (Cst), forced vital capacity FVC, inspiratory capacity (A), K, forced expiratory volume at 200ms (FEV200) and peak inspiratory flow (PEF) in comparison to control animals. However, SM‐induced reductions in these parameters were non‐distinguishable between doses. Animals that received SM had pathology findings of proteinaceous edema fluid in the bronchial, bronchiolar, and alveolar regions of the lungs with mild to severe perivascular edema, hemorrhage and inflammation. Multifocal bronchiolar mucous cell hyperplasia was prominent after SM exposure with partial to extensive fibrosis of the bronchial wall and variable epithelial loss occurring. Although variable in severity and not widespread, alveolar fibrosis also occurred multifocally. In conclusion, a reduction in Crs and an increase in Ers of the forced oscillation measures, in animals that received SM, indicate an increased lung stiffness and elasticrecoil of the lung. PV‐loop analysis presented with a restrictive and obstructive pulmonary function pattern with an increase in Rrs and decreases in inspiratory capacity, FEV200 and PEF along with flattening (reduction) of the downward curvature of the deflating limb of the PV loop. Pathology results are consistent with the respiratory mechanics findings.Support or Funding InformationThe research described was supported by an interagency agreement (AOD18014‐001‐00000) between the NIH office of the Directory and the USAMRICD under the oversight of the Chemical Countermeasures Research Program within the Office of Biodefense Research at the National Institute of Allergy and Infectious Diseases. The views expressed are solely those of the authors and do not necessarily represent the official views of the CCRP, NIAID, NIH, HHS, USAMRICD or DOD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inclusion of thin target and source regions in alimentary and respiratory tract systems of mesh-type ICRP adult reference phantoms

It is not feasible to define very small or complex organs and tissues in the current voxel-type adult reference computational phantoms of the International Commission on Radiological Protection (ICRP), which limit dose coefficients for weakly penetrating radiations. To address the problem, the ICRP is converting the voxel-type reference phantoms into mesh-type phantoms. In the present study, as a part of the conversion project, the micrometer-thick target and source regions in the alimentary and respiratory tract systems as described in ICRP Publications 100 and 66 were included in the mesh-type ICRP reference adult male and female phantoms. In addition, realistic lung airway models were simulated to represent the bronchial (BB) and bronchiolar (bb) regions. The electron specific absorbed fraction (SAF) values for the alimentary and respiratory tract systems were then calculated and compared with the values calculated with the stylized models of ICRP Publications 100 and 66. The comparisons show generally good agreement for the oral cavity, oesophagus, and BB, whereas for the stomach, small intestine, large intestine, extrathoracic region, and bb, there are some differences (e.g. up to ~9 times in the large intestine). The difference is mainly due to anatomical difference in these organs between the realistic mesh-type phantoms and the simplified stylized models. The new alimentary and respiratory tract models in the mesh-type ICRP reference phantoms preserve the topology and dimensions of the voxel-type ICRP phantoms and provide more reliable SAF values than the simplified models adopted in previous ICRP Publications.

The Mayak Worker Dosimetry System (MWDS-2013): Plutonium Binding in the Lungs-An Analysis of Mayak Workers.

Estimates of plutonium lung doses from urine bioassay are highly dependent on the rate of absorption from the lungs to blood assumed for the inhaled aerosol. Absorption occurs by dissolution of particles in lung fluid followed by uptake to blood. The latter may occur either rapidly or dissolved ions may first become temporarily bound within airway tissue. The presence of long-term binding can greatly increase lung doses, particularly if it occurs in the bronchial and bronchiolar regions. Analyses of autopsy data from Beagle dogs and USTUR Case 0269, obtained following exposure to plutonium nitrate, suggest that a small fraction of 0.2-1.1 and 0.4-0.7%, respectively, of plutonium becomes permanently bound within the lungs. The present work performs a further analysis using autopsy data of former plutonium workers of the Mayak Production Association to determine values of the bound fraction that are supported by these data. The results suggest a bound fraction value of 0-0.3%. The results also indicate that the Mayak worker population median values of the particle transport clearance parameters from the alveolar-interstitial region are largely consistent with expected values, but suggest the rate from the alveolar region to the interstitium may be lower than initially thought.

The Mayak Worker Dosimetry System (MWDS-2013): A Bayesian Analysis to Quantify Pulmonary Binding of Plutonium in Lungs Using Historic Beagle Dog Data

The revised human respiratory tract model, published in Part 1 of the International Commission on Radiological Protection's (ICRP) report on Occupational Intakes of Radionuclides (OIR), includes a bound fraction, fb, to represent radionuclides that have become chemically bound in the lungs following dissolution of particulates in lung fluid. Bound radionuclides are not subject to particle transport clearance but can be absorbed to blood at a rate, sb. The occurrence of long-term binding of plutonium can greatly increase lung doses, particularly if it occurs in the bronchial and bronchiolar regions. However, there has been little evidence that currently supports the existence of a long-term bound state for plutonium. The present work describes the analysis of measurements of lung data obtained from a life span study of Beagle dogs that were exposed by inhalation to different concentrations of plutonium-239 (239Pu) nitrate aerosol at Pacific Northwest Laboratories, USA. The data have been analysed to assess whether a bound state was required to explain the data. A Bayesian approach was adopted for the analysis that accounts for uncertainties in model parameter values, including uncertainties in the rates of particle transport clearance. Furthermore, it performs the analysis using two different modelling hypotheses: a model based on the current ICRP human respiratory tract model and its treatment of alveolar particle transport clearance; and a model of particle transport clearance that is based on the updated model developed by ICRP to calculate dose coefficients for the OIR. The current model better represents clearance in dogs at early times (up to 1 year following intake) and the latter better represents retention at greater times (>5 years following intake). The results indicate that a long-term bound fraction of between 0.16 and 1.1%, with a mean value of between 0.24 and 0.8% (depending on the model) is required to explain the data.

THE MAYAK WORKER DOSIMETRY SYSTEM (MWDS 2013): A RE-ANALYSIS OF USTUR CASE 0269 TO DETERMINE WHETHER PLUTONIUM BINDS TO THE LUNGS.

Radionuclides in ionic form can become chemically bound in the airways of the lungs following dissolution of inhaled particulates in lung fluid. The presence of long-term binding can greatly increase lung doses from inhaled plutonium, particularly if it occurs in the bronchial and bronchiolar regions. However, the only published evidence that plutonium binding occurs in humans comes from an analysis of the autopsy and bioassay data of United States Transuranium and Uranium Registries Case 0269, a plutonium worker who experienced a very high (58 kBq) acute inhalation of plutonium nitrate. This analysis suggested a bound fraction of around 8 %, inferred from an unexpectedly low ratio of estimated total thoracic lymph node activity:total lung activity, at the time of death. However, there are some limitations with this study, the most significant being that measurements of the regional distribution of plutonium activity in the lungs, which provide more direct evidence of binding, were not available when the analysis was performed. The present work describes the analysis of new data, which includes measurements of plutonium activity in the alveolar-interstitial (AI) region, bronchial (BB) and bronchiolar (bb) regions, and extra-thoracic (ET) regions, at the time of death. A Bayesian approach is used that accounts for uncertainties in model parameter values, including particle transport clearance, which were not considered in the original analysis. The results indicate that a long-term bound fraction between 0.4 and 0.7 % is required to explain this data, largely because plutonium activity is present in the extra-thoracic (ET2), bronchial and bronchiolar airways at the time of death.

Seasonal size distribution of airborne culturable bacteria and fungi and preliminary estimation of their deposition in human lungs during non-haze and haze days

In recent years, haze events in Beijing have significantly increased in frequency. On haze days, airborne microorganisms are considered to be a potential risk factor for various health concerns. However, limited information on bioaerosols has prevented our proper understanding of the possible threat to human health due to these bioaerosols. In this study, we used a six-stage impactor for sampling culturable bioaerosols and the LUDEP 2.07 computer-based model for calculating their deposition on human lungs to investigate seasonal concentration, size distribution, and corresponding deposition efficiency and flux in the human respiratory tract during different haze-level events. The current results of the analysis of 398 samples over four seasons indicate that the concentration of culturable airborne bacteria decreased with increasing haze severity. The bioaerosol concentration ratio was skewed towards larger particle sizes on heavy haze days leading to larger bioaerosol aerodynamic diameters than on non-haze days. During nasal breathing by an adult male engaged in light exercise in an outdoor environment, the total deposition efficiency of culturable bioaerosols is 80–90% including approximately 70% in the upper respiratory tract, 5–7% in the alveoli, and about 3% in the bronchial couple with bronchiolar regions. Although the difference in culturable bioaerosol aerodynamic diameters at different haze levels was not large enough to cause obvious differences in lung deposition efficiency, the deposition fluxes clearly varied with the degree of haze owing to the varied concentration of culturable airborne bacteria and fungi. The results here could improve our understanding of the seasonal health threat due to culturable bioaerosols during non-haze and haze days.

Stochastic rat lung dosimetry for inhaled radon progeny: a surrogate for the human lung for lung cancer risk assessment.

Laboratory rats are frequently used in inhalation studies as a surrogate for human exposures. The objective of the present study was therefore to develop a stochastic dosimetry model for inhaled radon progeny in the rat lung, to predict bronchial dose distributions and to compare them with corresponding dose distributions in the human lung. The most significant difference between human and rat lungs is the branching structure of the bronchial tree, which is relatively symmetric in the human lung, but monopodial in the rat lung. Radon progeny aerosol characteristics used in the present study encompass conditions typical for PNNL and COGEMA rat inhalation studies, as well as uranium miners and human indoor exposure conditions. It is shown here that depending on exposure conditions and modeling assumptions, average bronchial doses in the rat lung ranged from 5.4 to 7.3 mGy WLM(-1). If plotted as a function of airway generation, bronchial dose distributions exhibit a significant maximum in large bronchial airways. If, however, plotted as a function of airway diameter, then bronchial doses are much more uniformly distributed throughout the bronchial tree. Comparisons between human and rat exposures indicate that rat bronchial doses are slightly higher than human bronchial doses by about a factor of 1.3, while lung doses, averaged over the bronchial (BB), bronchiolar (bb) and alveolar-interstitial (AI) regions, are higher by about a factor of about 1.6. This supports the current view that the rat lung is indeed an appropriate surrogate for the human lung in case of radon-induced lung cancers. Furthermore, airway diameter seems to be a more appropriate morphometric parameter than airway generations to relate bronchial doses to bronchial carcinomas.

Fatal acute interstitial pneumonia in a worker making chips from wooden debris generated by the Great East Japan earthquake and tsunami

A man was admitted to our hospital with shortness of breath. He was involved in making wood chips from contaminated debris created by the tsunami that occurred after the Great East Japan Earthquake. Fungi detected at his home and workplace were possible inducers of hypersensitivity pneumonitis, but the absence of precipitating antibodies countered this diagnosis. His rapid and progressive clinical course and surgical lung biopsy and bronchoalveolar lavage findings suggested acute interstitial pneumonia. Electron probe X-ray microanalysis revealed the deposition of excessive exogenous substances in bronchiolar regions. Inhalation of harmful materials was suspected to be the cause of acute lung injury.

Siderite (FeCO3) and magnetite (Fe3O4) overload-dependent pulmonary toxicity is determined by the poorly soluble particle not the iron content

The two poorly soluble iron containing solid aerosols of siderite (FeCO3) and magnetite (Fe3O4) were compared in a 4-week inhalation study on rats at similar particle mass concentrations of approximately 30 or 100 mg/m3. The particle size distributions were essentially identical (MMAD ≈1.4 μm). The iron-based concentrations were 12 or 38 and 22 or 66 mg Fe/m3 for FeCO3 and Fe3O4, respectively. Modeled and empirically determined iron lung burdens were compared with endpoints suggestive of pulmonary inflammation by determinations in bronchoalveolar lavage (BAL) and oxidative stress in lung tissue during a postexposure period of 3 months. The objective of study was to identify the most germane exposure metrics, that are the concentration of elemental iron (mg Fe/m3), total particle mass (mg PM/m3) or particle volume (μl PM/m3) and their associations with the effects observed. From this analysis it was apparent that the intensity of pulmonary inflammation was clearly dependent on the concentration of particle-mass or -volume and not of iron. Despite its lower iron content, the exposure to FeCO3 caused a more pronounced and sustained inflammation as compared to Fe3O4. Similarly, borderline evidence of increased oxidative stress and inflammation occurred especially following exposure to FeCO3 at moderate lung overload levels. The in situ analysis of 8-oxoguanine in epithelial cells of alveolar and bronchiolar regions supports the conclusion that both FeCO3 and Fe3O4 particles are effectively endocytosed by macrophages as opposed to epithelial cells. Evidence of intracellular or nuclear sources of redox-active iron did not exist. In summary, this mechanistic study supports previous conclusions, namely that the repeated inhalation exposure of rats to highly respirable pigment-type iron oxides cause nonspecific pulmonary inflammation which shows a clear dependence on the particle volume-dependent lung overload rather than any increased dissolution and/or bioavailability of redox-active iron.

Jaagsiekte sheep retrovirus infects multiple cell types in the ovine lung.

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The details of early events in the pathogenesis of OPA are not fully understood. For example, the identity of the JSRV target cell in the lung has not yet been determined. Mature OPA tumors express surfactant protein-C (SP-C) or Clara cell-specific protein (CCSP), which are specific markers of type II pneumocytes or Clara cells, respectively. However, it is unclear whether these are the cell types initially infected and transformed by JSRV or whether the virus targets stem cells in the lung that subsequently acquire a differentiated phenotype during tumor growth. To examine this question, JSRV-infected lung tissue from experimentally infected lambs was studied at early time points after infection. Single JSRV-infected cells were detectable 10 days postinfection in bronchiolar and alveolar regions. These infected cells were labeled with anti-SP-C or anti-CCSP antibodies, indicating that differentiated epithelial cells are early targets for JSRV infection in the ovine lung. In addition, undifferentiated cells that expressed neither SP-C nor CCSP were also found to express the JSRV Env protein. These results enhance the understanding of OPA pathogenesis and may have comparative relevance to human lung cancer, for which samples representing early stages of tumor growth are difficult to obtain.

Pulmonary Gene Silencing in Transgenic EGFP Mice Using Aerosolised Chitosan/siRNA Nanoparticles

This work describes the production and application of an aerosolised formulation of chitosan nanoparticles for improved pulmonary siRNA delivery and gene silencing in mice. Aerosolised chitosan/siRNA nanoparticles were pneumatically formed using a nebulising catheter and sized by laser diffraction. In vitro silencing of aerosolised and non-aerosolised formulations was evaluated in an EGFP endogenous-expressing H1299 cell line by flow cytometry. Non-invasive intratracheal insertion of the catheter was used to study nanoparticle deposition by histological detection of Cy3-labeled siRNA and gene silencing in transgenic EGFP mouse lungs using a flow cytometric method. Flow cytometric analysis demonstrated minimal alteration in gene silencing efficiency before (68%) and after (62%) aerosolisation in EGFP-expressing H1299 cells. Intratracheal catheter administration in mice resulted in nanoparticle deposition throughout the entire lung in both alveoli and bronchiolar regions using low amounts of siRNA. Transgenic EGFP mice dosed with the aerosolised nanoparticle formulation showed significant EGFP gene silencing (68% reduction compared to mismatch group). This work provides a technology platform for effective pulmonary delivery and gene silencing of RNAi therapeutics with potential use in preclinical studies of respiratory disease treatment.

C5 contents and neutrophil chemotactic activities in bronchiolar and alveolar regions

To clarify the localization and mechanism of neutrophil infiltration in the lower respiratory tract, we measured neutrophil number, neutrophil chemotactic factor (NCF) activity and content of C5 in bronchial lavage (BL) fluid and bronchoalveolar lavage (BAL) fluid. Numbers of neutrophils, NCF activity and C5 content were higher in the BL fluid from normal volunteers (NV) and control patients (CP) than those in the BAL fluid from the same subjects. The NCF activity in the BL fluid was inhibited approximately 40% by anti-C5 antiserum, and correlated with C5 content in the BL fluid. In the BAL fluids of patients with chronic airway diseases (CAD) and patients with idiopathic interstitial pneumonia (IIP), neutrophil number, NCF activity and C5 content were increased compared to those in BAL fluid from NV or CP. These results indicated that neutrophils are predominant in the bronchial region compared to the alveolar region, and that C5-derived NCF play important roles in the accumulation of neutrophils in the bronchial region. Also C5-derived NCF are thought to be related to, at least, a part of the neutrophil infiltration in the respiratory tract of patients with CAD and IIP.

Evaluation of protease inhibitors and an antioxidant for treatment of sulfur mustard-induced toxic lung injury

Sulfur mustard (SM)-induced lung injury has been associated with protease activation, oxidative injury and inflammatory response culminating in tissue necrosis. The protease inhibitors aprotinin and ilomastat and the antioxidant trolox were evaluated for efficacy in ameliorating SM-induced lung injury. Anesthetized spontaneously breathing rats ( N = 6–8/group) were intratracheally intubated and exposed to 1.4 mg/kg SM (0.35 mg SM in 0.1 ml of ethanol) or ethanol alone by vapor inhalation for 50 min. At 1 min before the exposure rats were treated with one of the following: intravenous aprotinin, 4.4 mg/kg; intraperitoneal (ip) ilomastat, 25 mg/kg; or ip trolox, 500 μg/kg. Aprotinin-treated animals received supplemental 2.2 mg/kg doses at 1 min and 6 h post-exposure (PE). A whole body plethysmograph system was used to monitor pulmonary function (PF) parameters for 1 h before exposure (baseline), and from 5–6 and 23–24 h post-exposure. SM inhalation caused significant increases in several PF parameters, including tidal volume, peak inspiratory flow, peak expiratory flow, end expiratory pause and enhanced pause. Consistent with the reported development of SM-induced pathology, these changes were minimal at the 5–6-h time and significant at the 23–24-h timepoint. At the later time it is known from previous work that airways are becoming obstructed with loose cellular debris, damaged cells and exudate, which contributed to the changes in PF parameters. Treatment with aprotinin or ilomastat eliminated these PF changes, yielding results comparable with controls for each of these parameters. Lung lavage fluid analysis showed that SM caused a significant increase in total protein (TP) and in the cytokines IL-1α and IL-13. Aprotinin treatment prevented the increases in TP and IL-1α production, ilomastat prevented the increased production of IL-13, and trolox treatment did not significantly prevent the SM-related increases in TP, IL-1α or IL-13. Histopathologic examination of lung tissue 24 h post-exposure showed minimal alveolar effects caused by SM, while damage to bronchiolar regions was much more severe due to the highly reactive nature of SM. While aprotinin and ilomastat both alleviated the PF perturbations, surprisingly only aprotinin reduced the observed pathology, both grossly and histologically. These early results indicate that treatment with aprotinin and to a lesser extent ilomastat reduces some of the direct inflammatory response and damage associated with SM-induced lung injury. This research was supported by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.

Health assessment of phosgene: Approaches for derivation of reference concentration

This paper describes the derivation of the chronic reference concentration (RfC) for human inhalation of phosgene that was recently added to the Environmental Protection Agency’s (EPA) Integrated Risk Information System (IRIS) data base (U.S. EPA, 2005. Toxicological Review of Phosgene: In Support of Summary Information on the Integrated Risk Information System (IRIS). Available online at: < http://www.epa.gov/IRIS>). The RfC is an estimate of daily phosgene exposure to the human population that is likely to be without appreciable risk of deleterious effects during a lifetime. [For this and other definitions relevant to EPA risk assessments refer to the glossary of terms in the US EPA IRIS website ( http://www.epa.gov/IRIS).] Phosgene is a potential environmental pollutant that is primarily used as a catalyst in the polyurethane industry. It is a gas at room temperature, and in aqueous solution it rapidly hydrolyzes to CO 2 and HCl. In the absence of chronic human health effects information and lifetime animal cancer bioassays, the RfC is based on two 12-week inhalation studies in F344 rats which measured immune response and pulmonary effects, respectively. The immune response study showed impaired clearance of bacteria that was administered into the lungs of rats immediately after exposure to phosgene at concentrations of 0.1, 0.2 and 0.5 ppm. It also showed that the immune response in uninfected rats was stimulated by phosgene exposure at all concentrations. The pulmonary effects study showed a progressive concentration-related thickening and inflammation in the bronchiolar regions of the lung that was mild at 0.1 ppm and severe at 1.0 ppm. An increase in collagen content, as observed with histological collagen stains, was observed at 0.2 ppm and above. Though there is considerable uncertainty associated with the species and exposure duration employed, this endpoint is considered an indication of chronic lung injury of potential relevance to humans. Three different approaches for RfC derivation were taken in analyzing these studies: (1) the traditional NOAEL/LOAEL method; (2) the benchmark dose (BMD); and (3) the categorical regression for the analysis of severity-graded pulmonary damage data using the recently revised USEPA CatReg software. The BMD approach was selected as the method of choice to determine the RfC for phosgene because it has several advantages compared to the NOAEL/LOAEL: (1) it is not restricted to the set of doses used in the experiments; (2) the result is not dependent on sample size; (3) it incorporates information on statistical uncertainty. The CatReg approach allowed the incorporation of data on the severity of the pathological lesions, and therefore it complemented the other approaches. The BMD approach could not be applied to the immune response data because it was not possible to define an adverse effect level for bacterial resistance. However, NOAEL/LOAEL values for immune responses were consistent with benchmark dose levels derived from lung pathology data and used in the derivation of the RfC. The preferred RfC method and derivation involved dividing the benchmark dose from the collagen staining data (0.03 mg/m 3) by a composite uncertainty factor of 100: RfC = 0.03/100 = 3E − 4 mg/m 3.

Further study of the effect of particle size on slow particle clearance from the bronchial tree

The ICRP human respiratory tract model (HRTM) assumes that the 'slow-cleared fraction' fs in the bronchial (BB) and bronchiolar (bb) regions decreases with increasing particle geometric diameter dp. To test this assumption, five volunteers inhaled 8-microm aerodynamic diameter, dae, (111)In-polystyrene (PSL) and (198)Au particles simultaneously. The particles were administered at the end of each breath to maximise deposition in BB and bb. Because of the difference in densities (1.05 vs. 19.3 g cm(-3)), corresponding dp values are approximately 8 and 1.8 microm, and fs values are approximately 2 and 50%, respectively. However, lung retention was somewhat greater for PSL than for gold, contrary to the HRTM prediction. Other recent human experimental studies found no difference in retention for particles with the same dae but different dp values. It is concluded that the HRTM model of particle clearance from BB and bb needs revision.