A study of inflammatory biomarkers in crystalline silica exposed rock drillers.

BACKGROUND: Crystalline silica (CS) exposure can cause serious lung disease in humans, but mechanisms of pulmonary toxicity have not been completely elucidated. AIMS: To assess pro-inflammatory and anti-inflammatory biomarkers and biomarkers related to the development of chronic obstructive pulmonary disease and fibrosis in serum of rock drillers exposed to CS. METHODS: Rock drillers (N = 123) exposed to CS and non-specified particulate matter (PM) were compared to 48 referents without current or past exposure to PM in a cross-sectional study. RESULTS: The rock drillers had been exposed to CS for 10.7 years on average. Geometric mean (GM) current exposure was estimated to 36 µg/m3. Their GM concentration of matrix metalloproteinase 12 (MMP-12) was significantly higher (16 vs. 13 ng/L; p = 0.04), while interleukin (IL) 6 and IL-8 were significantly lower compared to the referents. Also pentraxin 3 was significantly lower (3558 vs. 4592 ng/L; p = 0.01) in the rock drillers. A dose-response relationship was observed between cumulative exposure to CS and MMP-12, the highest exposed subgroup having significantly higher MMP-12 concentrations than the referents. CONCLUSION: Exposure to CS may increase circulating MMP-12 concentrations in a dose-response related fashion. The results may also suggest a down-regulation of pro-inflammatory pathways.

Comparison of different reference values for lung function: implications of inconsistent use among centers.

BACKGROUND: For interpretation of pulmonary function tests (PFTs), reference values based on sex, age, height and ethnicity are needed. In Norway, the European Coal and Steel Community (ECSC) reference values remain widely used, in spite of recommendations to implement the more recent Global Lung Function Initiative (GLI) reference values. OBJECTIVE: To assess the effects of changing from ECSC to GLI reference values for spirometry, DLCO and static lung volumes, using a clinical cohort of adults with a broad range in age and lung function. METHODS: PFTs from 577 adults (18-85 years, 45% females) included in recent clinical studies were used to compare ECSC and GLI reference values for FVC, FEV1, DLCO, TLC and RV. Percent predicted and lower limit of normal (LLN) were calculated. Bland-Altman plots were used to assess agreement between GLI and ECSC % predicted values. RESULTS: In both sexes, GLI % predicted values were lower for FVC and FEV1, and higher for DLCO and RV, compared to ECSC. The disagreement was most pronounced in females, with mean (SD) difference 15 (5) percent points (pp) for DLCO and 17 (9) pp for RV (p < 0.001). With GLI, DLCO was below LLN in 23% of the females, with ECSC in 49% of the females. CONCLUSIONS: The observed differences between GLI and ECSC reference values are likely to entail significant consequences with respect to criteria for diagnostics and treatment, health care benefits and inclusion in clinical trials. To ensure equity of care, the same reference values should be consistently implemented across centers nationwide.

A study of pneumoproteins in crystalline silica exposed rock drillers.

Objective: The objective was to assess serum concentrations of club cell protein 16 (CC-16) and the surfactant proteins A (SPs-A) and D (SP-D) in male rock drillers (N = 123) exposed to crystalline silica and in 48 occupationally non-exposed. Methods: The arithmetic mean (AM) duration of exposure was 10.7 years. The geometric mean (GM) crystalline silica exposure was 36 µg/m3 at the time of the study. The GM cumulative exposure was 239 µg/m3. Results: The concentrations of SP-D (GM 12.7 vs. 8.8 µg/L, p < 0.001) and SP-A (AM 1847 vs. 1378 ng/L, p = 0.051) were higher among rock drillers than among occupationally non-exposed. A positive significant association was observed between cumulative crystalline silica exposure and the SP-D concentrations (ß = 0.07; p < 0.05). Rock drillers with small airway obstruction with maximal mid-expiratory flow % (MMEF%) <70% (N = 29) had higher SP-D concentrations than rock drillers with MMEF% ≥ 70% (N = 91) (GM 17.3 vs. 11.4 µg/L, p = 0.001). Rock drillers with MMEF% ≥70% (N = 91) had higher concentrations of SP-A (1957 vs. 1287 ng/L, p = 0.01) and SP-D (11.4 vs. 9.0 µg/L, p = 0.007) than non-exposed with MMEF% ≥70% (N = 39). Rock drillers with airway obstruction (FEV1/FVC < 0.70, N = 11) had significantly lower CC-16 concentrations than rock drillers with FEV1/FVC ≥0.70 (N = 109) after adjusting for relevant potential confounders (p = 0.02). Conclusion: The results indicate that pulmonary surfactant is a target for crystalline silica toxicity. The alterations appear to be driven by pulmonary alterations in the small airways and by exposure itself. Further studies on pneumoproteins and pulmonary function in other groups of workers exposed to crystalline silica are needed.

DNA methylome-based validation of induced sputum as an effective protocol to study lung immunity: construction of a classifier of pulmonary cell types.

Flow cytometry is a classical approach used to define cell types in peripheral blood. While DNA methylation signatures have been extensively employed in recent years as an alternative to flow cytometry to define cell populations in peripheral blood, this approach has not been tested in lung-derived samples. Here, we compared bronchoalveolar lavage with a more cost-effective and less invasive technique based on sputum induction and developed a DNA methylome-based algorithm that can be used to deconvolute the cell types in such samples. We analysed the DNA methylome profiles of alveolar macrophages and lymphocytes cells isolated from the pulmonary compartment. The cells were isolated using two different methods, sputum induction and bronchoalveolar lavage. A strong positive correlation between the DNA methylome profiles of cells obtained with the two isolation methods was found. We observed the best correlation of the DNA methylomes when both isolation methods captured cells from the lower parts of the lungs. We also identified unique patterns of CpG methylation in DNA obtained from the two cell populations, which can be used as a signature to discriminate between the alveolar macrophages and lymphocytes by means of open-source algorithms. We validated our findings with external data and obtained results consistent with the previous findings. Our analysis opens up a new possibility to identify different cell populations from lung samples and promotes sputum induction as a tool to study immune cell populations from the lung.

Relation of sputum neutrophilia to the development of chronic lung allograft dysfunction after lung transplantation.

Chronic lung allograft dysfunction (CLAD) is a serious complication after lung transplantation (LuTx) and is associated with elevated proportions of neutrophils in bronchoalveolar lavage (BAL). Induced sputum is a less-invasive sampling method than BAL and assesses markers of inflammation on the surfaces of large central airways. We wanted to examine whether % neutrophil levels in induced sputum were elevated prior to CLAD diagnosis among LuTx recipients, and whether sputum markers of inflammation can be used as a tool for predicting the development of CLAD. Induced sputum samples were collected at 1, 3, 6, 12, and 24 months post-LuTx in 36 patients with a history of COPD or pulmonary fibrosis, and of these, 16 developed CLAD either during or after the sputum surveillance period. At 2 years, median (IQR) % neutrophils in induced sputum were significantly higher among patients with CLAD compared with those without CLAD [73 (52-80) % vs 59 (41-76) %, p = .01]. Interestingly, we found a significant increase in the rate of change in % neutrophils beginning at 90 days preceding the diagnosis of CLAD. This suggests using sputum neutrophil percentage as a surveillance modality for monitoring lung allograft function after LuTx.

Annual decline in forced expiratory volume and airway inflammatory cells and mediators in a general population-based sample.

BACKGROUND: Few studies have examined the relationships between sputum inflammatory markers and subsequent annual decline in forced expiratory volume in 1 s (dFEV1). This study investigated whether indices of airway inflammation are predictors of dFEV1 in a general population-based sample. METHODS: The study, conducted from 2003 to 2005, included 120 healthy Norwegian subjects aged 40 to 70 years old. At baseline, the participants completed a self-administered respiratory questionnaire and underwent a clinical examination that included spirometry, venous blood sampling, and induced sputum examination. From 2015 to 2016, 62 (52%) participants agreed to a follow-up examination that did not include induced sputum examination. Those with a FEV1/forced vital capacity (FVC) ratio <  0.70 underwent a bronchial reversibility test. The levels of cytokines, pro-inflammatory M1 macrophage phenotypes were measured in induced sputum using bead-based multiplex analysis. The associations between cytokine levels and dFEV1 were then analysed. RESULTS: The mean dFEV1 was 32.9 ml/year (standard deviation 26.3). We found no associations between dFEV1 and the baseline indices of sputum inflammation. Seven participants had irreversible airflow limitation at follow-up. They had lower FEV1 and gas diffusion at baseline compared with the remaining subjects. Moreover, two of these individuals had a positive reversibility test and sputum eosinophilia at baseline. CONCLUSIONS: In this cohort of presumably healthy subjects, we found no associations between sputum inflammatory cells or mediators and dFEV1 during 10 years of follow-up.

Isolating and culturing of sputum macrophages: A potential ex vivo/in vitro model.

PURPOSE: This paper aimed to test whether induced sputum samples acquired from human volunteers could be used to isolate and culture airway macrophages for in vitro exposures. This was assessed in terms of the culturing success rate, culture purity, viability and responsiveness of cultured cells. MATERIALS AND METHODS: The isolation and culturing procedure was performed over three days. On Day 1, induced sputum samples were obtained, processed and seeded in culture wells. Differential cell counts and viability tests were performed to allow for calculation of viable macrophage numbers and appropriate sample dilution. After a 1 h rest, seeded wells were washed to remove non-adherent cells, resulting in macrophage isolation. Then, cells rested overnight (Day 1-Day 2), before in vitro exposure for 2-24 h (Day 2-Day 3). The criteria for progressing into the culturing procedure was cell viability >40% and total cell number >106. Successful culturing was evaluated based on cell attachment (N = 40). Culture purity by differential cell analysis and viability was monitored during culturing (N = 4-8). Macrophage responsivity was assessed by measurement of inflammatory cytokine gene expression (N = 4) and cytokine levels (N = 6) following in vitro exposure to lipopolysaccharide (LPS) (2-24 h) and live bacteria (S. aureus) (4h). RESULTS: Overall, 88% (35/40) of the samples acquired were suitable for isolation, and 80% (32/40) were successfully progressed through the 2-3 day culturing protocol. Macrophage purity (88%) and viability (85%) were adequate. Moreover, cultured macrophages were responsive to in vitro stimulation with LPS and viable S. aureus showing positive mRNA responses for TNFα, IL-1ß and IL-8 and release of IL-1ß, respectively. CONCLUSION: Sputum macrophage isolation by plate adherence and subsequent culturing of sputum macrophages was successfully performed and represents a promising in vitro model for examination of airway macrophage behavior.

The Role of Airway Inflammation and Bronchial Hyperresponsiveness in Athlete's Asthma.

PURPOSE: Asthma is frequently reported in endurance athletes. The aim of the present study was to assess the long-term airway inflammatory response to endurance exercise in high-level athletes with and without asthma. METHODS: In a cross-sectional design, 20 asthmatic athletes (10 swimmers and 10 cross-country skiers), 19 athletes without asthma (10 swimmers and 9 cross-country skiers), and 24 healthy nonathletes completed methacholine bronchial challenge, lung function tests, and sputum induction on two separate days. All athletes competed on a national or international level and exercised ≥10 h·wk. The nonathletes exercised ≤5 h·wk and reported no previous lung disease. Bronchial hyperresponsiveness (BHR) was defined as a methacholine provocation dose causing 20% decrease in the forced expiratory volume in 1 s of ≤8 µmol. RESULTS: BHR was present in 13 asthmatic athletes (62%), 11 healthy athletes (58%), and 8 healthy nonathletes (32%), and the prevalence differed among groups (P = 0.005). Sputum inflammatory and epithelial cell counts did not differ between groups and were within the normal range. Median (25th to 75th percentiles) sputum interleukin-8 was elevated in both asthmatic (378.4 [167.0-1123.4]) and healthy (340.2 [175.5-892.4]) athletes as compared with healthy nonathletes (216.6 [129.5-314.0], P = 0.02). No correlations were found between provocation dose causing 20% decrease and sputum cell counts. CONCLUSION: Independent of asthma diagnosis, a high occurrence of BHR and an increased sputum interleukin-8 were found in athletes as compared with nonathletes. Airway inflammation or epithelial damage was not related to BHR.

Military small arms fire in association with acute decrements in lung function.

OBJECTIVE: After introduction of unleaded ammunition, Norwegian Armed Forces received reports of acute respiratory symptoms in soldiers after exposure to fumes from firing the standard weapon, HK416. The aim of the present study was to examine lung function before and after exposure to fumes from HK416 in a double-blinded standardised study design using three different types of ammunition. METHODS: Fifty-four healthy, non-smoking male volunteers (19-62 years) fired the weapons for 60 min with either leaded, unleaded or 'modified' unleaded ammunition. Gaseous and particulate emissions were monitored. Spirometry and exhaled nitric oxide (eNO) were performed within 14 days before (T0), shortly after (T1) and 24 hours after (T2) shooting. Methacholine provocation and diffusing capacity of carbon monoxide (DLCO) were carried out at T0 and T2. RESULTS: The mean forced expiratory volume in 1 s on a group level was significantly reduced both at T1 and T2 compared with T0, with means and 95% CI of 226 mL (158 to 294 mL) and 285 mL (218 to 351 mL), respectively. The same significant pattern was seen for DLCO, forced vital capacity and eNO. The methacholine test indicated a slight increase in bronchial hyper-reactivity. However, there were no significant differences between types of ammunition used. CONCLUSION: Exposure to fumes from military weapons might be a respiratory hazard for soldiers who do live-fire training regularly or are in a closed combat environment.

Sputum neutrophils are elevated in smelter workers, and systemic neutrophils are associated with rapid decline in FEV1.

OBJECTIVES: In a previous study on smelter workers we, found significant relationship between exposure to dust and accelerated annual decline in forced expiratory volume in 1 s (FEV1). In this cross-sectional study at the end of a follow-up, we aimed to investigate the possible association between annual decline in FEV1 and markers of airways, and systemic inflammation in smelter workers. METHODS: Employees (n=76 (27 current smokers)) who had been part of a longitudinal study (9-13 years) that included spirometry (>6 measurements) and respiratory questionnaires, performed induced sputum, exhaled NO and had blood drawn. Participants with annual decline in FEV1≥45 mL were compared with participants with annual decline <45 mL; also 26 non-exposed controls were included. RESULTS: Compared with non-exposed controls, smelter workers demonstrated a significantly increased percentage of neutrophils (mean (SD)) (57% (17) vs 31% (15)) and matrix metalloproteinases 8 (MMP-8) levels in sputum, and MMP-9, surfactant protein D (SpD) and transforming growth factor ß (TGFb) levels in blood. A significant association in FEV1≥45 mL was found for blood neutrophils when controlling for smoking habits (OR=1.7 (95% CI 1.0 to 2.8), p=0.045). Airway and blood protein markers were not associated with annual decline in FEV1. CONCLUSIONS: All workers displayed airway and systemic inflammation characterised by increased levels of neutrophils and MMP-8 in sputum, and MMP-9, SpD and TGFß in blood compared with non-exposed controls. Blood neutrophils in particular were significantly elevated in those workers with the most rapid decline in lung function. A similar observation was not seen with airway neutrophils. In the present study, we were able to identify systemic but not airway inflammatory markers that can predict increased decline in FEV1 in smelter workers.

Health effects after firing small arms comparing leaded and unleaded ammunition.

A number of Norwegian soldiers have reported health problems after live-fire training using the HK416 rifle. The objective of this study was to characterize gaseous and particulate emissions from three different types of ammunition, and record the health effects after exposure to emissions from live-firing. Fifty-five healthy, non-smoking men (mean age 40 years) were recruited and divided randomly into three groups, one for each type of ammunition. All subjects fired the HK416 rifle in a semi-airtight tent for 60 min using leaded ammunition, unleaded ammunition and modified unleaded ammunition. Gaseous and particulate emissions were monitored within the tent. The symptoms experienced by the subjects were recorded immediately after and the day after firing using a standardized questionnaire. The concentrations of particulate matter and copper exceeded their respective occupational exposure limits (eight hours per day, five days a week) by a factor of 3 and 27, respectively. Of the 55 subjects, 54 reported general and respiratory symptoms. The total number of symptoms reported was significantly higher among shooters using unleaded ammunition as compared with the use of leaded and modified unleaded ammunition. Copper was the substance that had the highest concentration relative to its toxicity. Although the general symptoms were found to be consistent with the development of metal fume fever, the respiratory symptoms indicated an irritant effect of the airways different from that seen in metal fume fever. More symptoms were reported when unleaded ammunition was used compared with leaded and modified unleaded ammunition.

Recovery from workplace-induced airway inflammation 1 year after cessation of exposure.

BACKGROUND: Bacterial single-cell protein (BSCP) constitutes of dried bacterial mass which is used as protein enrichment in animal and fish feed. In the production of BSCP, workers are exposed to organic dust containing high levels of endotoxins (arithmetic mean 390 EU/m3 in the moderate exposure and 5800-11,000 EU/m3 in the high exposure groups) and have elevated levels of sputum neutrophils and cytokines associated with exposure. OBJECTIVE: The aim of the present study was to investigate if airway inflammation among the workers had declined 1 year after cessation of exposure. METHODS: Twenty-four non-smoking production workers (age 28-52) with a work history of 2-7 years were included in the study. MEASUREMENTS: Markers of airways inflammation and innate immune function (using flow cytometry) were assessed in an exposure period and 1 year after cessation of exposure. RESULTS: Sputum neutrophil proportion and numbers were significantly decreased 1 year after cessation of exposure to BSCP (43% vs. 71%, 186 vs. 598 neutrophils/mg sputum; p<0.001) as were eNO (17 ppb vs. 21 ppb (p=0.01) and interleukin-1ß (p<0.05) and interleukin-8 (p<0.05). Neutrophils had enhanced expression of CD11b/CR3 (p<0.01) and CD16/FcγRIII (p<0.001) and macrophages had lower expression of CD86 (p<0.01) 1 year after cessation of exposure. CONCLUSIONS: One year after closure of the plant airway neutrophils and exhaled NO levels resolved to lower levels and cell surface phenotypes associated with innate immune function recovered to higher levels, indicating that these changes were partly reversible among workers who were no longer exposed to endotoxins in a BSCP plant.

Long-term occupational outcomes of endotoxin exposure and the effect of exposure cessation.

OBJECTIVE: To study the possible respiratory and haematological effects of endotoxin exposure to bacterial single-cell protein (BSCP) in workers during a follow-up period of 5 years including 4 years of exposure and 1 year without exposure. METHODS: The study included 28 workers examined in 2002-2005 and 1 year after exposure termination in 2007. The arithmetic mean endotoxin exposure was 5800-11,000 EU/m(3) among the high exposure group and 390 EU/m(3) in the low exposure group. Assessment of lung function included spirometry and gas diffusion in 2003, 2004 and 2007. Rhinometry was performed in 2004 and 2007. Blood analysis included leukocyte cell count and measurement of the acute phase proteins: C-reactive protein, interleukin-6, eosinophilic cationic protein, macrophage inflammatory protein-1α, monocyte chemoattractant protein-1, chemoattractant protein RANTES, platelet-derived growth factor BB, fibrinogen and D-dimer. RESULTS: In the low exposure group, but not in the high exposure group, there were significant improvements in both forced vital capacity (FVC) (290 ml) and forced expiratory volume in 1 s (FEV(1)) (180-210 ml) (p=0.004-0.03) 1 year after the end of exposure. The number of leukocytes and eosinophilic cationic protein and D-dimer levels increased significantly with increasing endotoxin exposures and decreased significantly 1 year after exposure termination. Changes in acute phase proteins suggested exposure-related tolerance. CONCLUSIONS: An inflammatory tendency during an exposure period of 4 years seems to reverse 1 year after cessation of exposure to endotoxins from a single species. Lung function improved significantly among workers exposed to low levels of endotoxin but not among the highly exposed workers.

Adrenomedullin is increased in alveolar macrophages and released from the lungs into the circulation in severe heart failure.

Adrenomedullin (AM) is a potent vasorelaxing peptide with natriuretic, diuretic, and growth inhibitory properties. Plasma concentrations and myocardial AM expression are increased in heart failure (HF). Since AM and AM binding sites are abundantly expressed in the lungs, we investigated to what extent pulmonary AM and AM receptor subtypes [CRLR/RAMP2 (AM1) and CRLR/RAMP3 (AM2)] are changed in HF and whether the lungs contribute to the increased plasma concentrations of AM reported in HF. Pulmonary AM mRNA and protein expression were increased by 2.8- and 2.6-fold, respectively, whereas mRNA expression of RAMP2 and CRLR was decreased in rats with HF 7 days after induction of MI compared to sham-operated rats (P < 0.05). Pulmonary AM receptor density was substantially decreased in HF rats compared to sham (3.7 +/-0.6 vs. 29.9 +/- 1.1 fmol/mg membrane protein; P < 0.05). Immunoreactivities against AM and the AM receptor components CRLR, RAMP2, and RAMP3 in the pulmonary tissue were seen in vascular smooth muscle cells, vascular endothelial cells, and in alveolar macrophages. AM mRNA expression in alveolar macrophages obtained from HF rats by bronchoalveolar lavage was 2.9-fold higher than in sham-operated rats (P < 0.05). An even more substantial increase of AM mRNA expression was found in alveolar macrophages from patients with HF (10-fold, P < 0.05), and this increase displayed a negative correlation to left ventricular systolic function (P < 0.05). Furthermore, a net release of AM from the lungs into the circulation was only found in HF patients with the most severe left ventricular systolic dysfunction. Thus, our data demonstrate increased expression and decreased receptor binding of AM in the lungs in severe HF. Furthermore, our data indicate that alveolar macrophages are an important source of pulmonary AM in both experimental and clinical HF. Finally, a net release of AM from the lungs into the circulation was only found in patients with severe systolic dysfunction.

DNA transfection of mononuclear cells in muscle tissue.

BACKGROUND: Genes encoding non-self proteins may be injected into skeletal muscles in vivo to obtain induction of cellular and humoral immune responses against the encoded antigens (DNA vaccination). Bone marrow derived professional antigen-presenting cells (APCs) play a key role in the induction of immunity by DNA vaccination. In the present work we have investigated whether the APCs are transfected by DNA injection into muscle. METHODS: DNA encoding green fluorescent protein (GFP) was injected into rat and mouse limb muscle and followed by electroporation. Whole mount muscle tissue with GFP-positive mononuclear cells (MNCs) were treated with immunocytochemical markers specific for leukocytes, and studied with fluorescent microscopy. To detect transfected cells migrating to peripheral lymphoid tissue RT-PCR was applied on RNA isolated from the draining popliteal lymph node and spleen. Lymphoid tissue was also analyzed with real-time PCR for distribution of the injected plasmid. RESULTS: MNCs were transfected after intramuscular DNA injection, and, following DNA injection with electroporation, the number of GFP-positive MNCs increased 6-fold in rats and 14-fold in mice. None of the GFP-positive MNCs were stained with leukocyte-specific antibodies. Even though GFP encoding DNA was detected in the popliteal lymph node, no RNA encoding GFP was found in the lymph node or spleen. However, MHC II-positive cells in the muscle tissue appeared preferentially around the transfected MNCs. CONCLUSIONS: Many MNCs in the muscle are transfected after intramuscular DNA injection. Electroporation significantly increases the number of transfected MNCs. None of the observed transfected MNCs however were leukocytes. MHC II-positive cells accumulated around transfected MNCs; this suggests that transfer of antigen from transfected MNCs to APCs may contribute to the immune response.