Prediction of lung cancer risk using circulating pro-surfactant protein B and high-sensitivity C-reactive protein among Egyptian workers in the rubber industry.

OBJECTIVE: For several decades, there has been increasing evidence for excess incidence of lung cancer among workers in the rubber industry. The purpose of this study was to assess the risk of lung cancer occurrence among Egyptian workers involved in the rubber industry using two circulating protein biomarkers. METHODS: This study was performed in a rubber manufacturing factory in Shubra El-Kheima region in Greater Cairo, Egypt. Environmental assessment for the suspended particulate matter of size 10 µm (PM10) concentrations was done. Levels of plasma pro-surfactant protein B (pro-SFTPB) and serum high-sensitivity C-reactive protein (HsCRP) were measured among the studied population (n = 155) who were divided into two groups. The first group included 75 workers exposed to rubber manufacturing process while the control group involved 80 administrative subjects. RESULTS: The levels of PM10 neither exceeded the Egyptian nor the international permissible limits where the highest levels were observed in the mixing department. However, through medical history and clinical examination, it was observed that some general and respiratory manifestations were more prevalent among the exposed group when compared with their controls. Laboratory investigations revealed that the mean values of pro-SFTPB and HsCRP levels among exposed workers were significantly higher than those of the control group. These increased circulating proteins levels were strongly and positively correlated with each other and with the duration of employment of exposed workers. CONCLUSION: The study results support the conclusion that prolonged occupational exposure to rubber manufacturing process is associated with an elevated risk of lung cancer.

Gene polymorphisms of SFTPB rs7316, rs9752 and PAOX rs1046175 affect the diagnostic value of plasma Pro-SFTPB and DAS in Chinese Han non-small-cell lung cancer patients.

Plasma pro-surfactant protein B (pro-SFTPB) and N1,N12-diacetylspermine (DAS) can be used as markers for the diagnosis of non-small-cell lung carcinoma (NSCLC). Whether the genetic diversity affects the application value of Pro-SFTPB and DAS as a diagnostic marker for NSCLC is still unknown. This study aims to explore the relationship between SFTPB rs7316, rs9752 and PAOX rs1046175 gene polymorphisms and the diagnostic value of plasma Pro-SFTPB and DAS in patients with Chinese Han lung cancer. SFTPB rs7316, rs9752 and PAOX rs1046175 genotypes were analyzed by direct sequencing in 425 patients with NSCLC and 425 controls, and the levels of Pro-SFTPB and DAS in plasma were determined by enzyme-linked immunosorbent assay (ELISA). The area under the curve (AUC) of the SFTPB rs7316 locus TT genotype for the diagnosis of NSCLC was 0.758, and the AUC of the TC/CC genotype for the diagnosis of NSCLC was 0.872. The AUC of the SFTPB rs9752 locus GG genotype for the diagnosis of NSCLC was 0.935, and the AUC of the GC/CC genotype for the diagnosis of NSCLC was 0.648. The AUC of the PAOX rs1046175 locus GG for the diagnosis of NSCLC was 0.669, and the AUC of the GC/CC genotype for the diagnosis of NSCLC was 0.749. In conclusion, SFTPB rs7316, rs9752, and PAOX rs1046175 gene polymorphisms affect the diagnostic value of plasma Pro-SFTPB and DAS in patients with Chinese Han NSCLC.

Expression of Lung Surfactant Proteins SP-B and SP-C and Their Regulatory Factors in Fetal Lung of GDM Rats.

This study investigated the expression of lung surfactant proteins (SP-B and SP-C), and regulatory factors [forkhead box A2 (FOXA2) and nitrolyogenic FOXA2 (N-FOXA2)] in the fetal lung of rats with gestational diabetes mellitus (GDM) in order to study the mechanism of pulmonary dysplasia. The rat GDM model was established by using streptozotocin intraperitoneally in the first stage of pregnancy. There were 10 rats in the GDM group, and 10 healthy rats in normal control group without any treatment. Fetal lungs of two groups were taken at day 21 of pregnancy. Blood glucose levels of maternal rats and fetal rats were measured by Roche blood glucose meter. The histological changes in the fetal lung were observed under the light microscope in both groups. The SP-B, SP-C and FOXA2 were determined in the fetal lung of two groups immunohistochemically. The expression levels of SP-B, SP-C, total FOXA2, FOXA2 in nucleus (n-FOXA2), N-FOXA2 proteins were detected by Western blotting, and the relative expression levels of SP-B, SP-C, FOXA2 mRNA in the fetal lung of two groups were detected by RTPCR. The results showed that blood glucose levels of maternal rats and fetal rats in GDM group were higher than those in control group. The light microscope revealed fetal lung development retardation in GDM group. The expression of SP-B and SP-C in GDM group was significantly reduced as compared with control group (P<0.05). As compared with control group, the n-FOXA2 expression was significantly decreased in the fetal lung tissue, and N-FOXA2 was significantly increased in control group (P<0.05), but there was no significant changes in the total FOXA2 (P>0.05). It was concluded that GDM can cause fetal lung development and maturation disorders, and FOXA2 in fetal lung tissue decreases while nitrocellulose FOXA2 increases.

HIV Infection and Circulating Levels of Prosurfactant Protein B and Surfactant Protein D.

Prosurfactant protein B (pro-SFTPB) and surfactant protein D (SFTPD) are markers of lung inflammation and damage. We estimated geometric mean pro-SFTPB and SFTPD levels in 500 human immunodeficiency virus (HIV)-infected and 300 HIV-uninfected injection drug users, adjusting for smoking and other covariates. Pro-SFTPB levels were significantly higher among people with HIV (PWH) (adjusted geometric mean, 21.4 vs 18.1 ng/mL; P = .03), and were higher with lower CD4 counts (P trend = .001), higher HIV RNA (P trend = .05), and without highly active antiretroviral therapy (P = .03). These associations were not observed for SFTPD. Serum levels of pro-SFTPB are elevated among PWH and are associated with immunosuppression and uncontrolled viremia.

Pulmonary surfactant-associated proteins and inflammatory factors in obstructive sleep apnea.

PURPOSE: Pulmonary surfactant (PS) plays roles in promoting the removal of the liquid, host defense, and immune regulation in the tracheal, bronchial, and alveoli epithelium. PS protein expression level can be regulated by oxygen levels and related free radicals. Obstructive sleep apnea (OSA) is featured with oxygen free radical production for damaging epithelial tissues and thus may affect PS production. The study was to explore the relationship between PS protein and OSA severity. METHODS: We collected serum and bronchoalveolar lavage fluid (BALF) samples from 35 OSA patients and 22 healthy subjects. PS-associated proteins and inflammatory factors, including surfactant proteins, HIF-1α, NF-κB, and IL-6, were analyzed. Regression analysis was performed to reveal the relationship between biochemical factors and clinical indexes recorded during PSG monitor. RESULTS: Lower BALF and surfactant protein (except surfactant protein C or SPC) levels occurred in OSA patients (all p < 0.05 compared to control group). A strongly negative correlation was found between surfactant protein with apnea-hypopnea index (AHI) and other sleeping indexes including ODI3 and ODI4. Similar patterns were found in serum samples, which were strongly correlated with BALF counterparts. Surfactant proteins were further found to have negative regression with inflammatory factors such as HIF-1α, NF-κB, and IL-6. CONCLUSIONS: This study established the relationship between PS-related protein with severity of OSA, plus their relationship with inflammatory factors. Our results provided possibly novel markers in general circulation for disease evaluation of OSA.

Experimental study of peripheral-blood pro-surfactant protein B for screening non-small cell lung cancer.

PURPOSE:: To evaluate the possibility of using peripheral-blood presurfactant protein B (Pro-SFTPB) for screening non-small cell lung cancer (NSCLC). METHODS:: A total of 873 healthy volunteers and 165 lung cancer patients hospitalized in the Fifth People's Hospital of Dalian were tested Pro-SFTPB once every half year from January 2014 to September 2015. The healthy volunteers were also conducted spiral computed tomography (CT) examination once every year. The data were then com-pared and statistically analyzed. RESULTS:: The positive expression rate of Pro-SFTPB in NSCLC was significantly higher than that in healthy volunteers, and significantly higher in lung adenocarcinoma than in squamous cell carcinoma; additionally, the expression rate was increased with the in-crease of smoking index, and the intergroup differences showed statistical signifi-cance (p≤0.05). The positive rate of newly diagnosed lung cancer was 29.55%, higher than healthy volunteers (22.34%), but there was no significant difference (p>0.05). CONCLUSION:: Pro-SFTPB is over expressed in non-small cell lung cancer, especially in lung adeno-carcinoma, but it can't be used as a clinical screening tool for lung cancer.

Experimental study of peripheral-blood pro-surfactant protein B for screening non-small cell lung cancer

Abstract Purpose: To evaluate the possibility of using peripheral-blood presurfactant protein B (Pro-SFTPB) for screening non-small cell lung cancer (NSCLC). Methods: A total of 873 healthy volunteers and 165 lung cancer patients hospitalized in the Fifth People's Hospital of Dalian were tested Pro-SFTPB once every half year from January 2014 to September 2015. The healthy volunteers were also conducted spiral computed tomography (CT) examination once every year. The data were then com-pared and statistically analyzed. Results: The positive expression rate of Pro-SFTPB in NSCLC was significantly higher than that in healthy volunteers, and significantly higher in lung adenocarcinoma than in squamous cell carcinoma; additionally, the expression rate was increased with the in-crease of smoking index, and the intergroup differences showed statistical signifi-cance (p≤0.05). The positive rate of newly diagnosed lung cancer was 29.55%, higher than healthy volunteers (22.34%), but there was no significant difference (p>0.05). Conclusion: Pro-SFTPB is over expressed in non-small cell lung cancer, especially in lung adeno-carcinoma, but it can't be used as a clinical screening tool for lung cancer.

Effects of Bufei Yishen Granules Combined with Acupoint Sticking Therapy on Pulmonary Surfactant Proteins in Chronic Obstructive Pulmonary Disease Rats.

Our previous studies have demonstrated the beneficial effects of Bufei Yishen granules combined with acupoint sticking therapy (the integrated therapy) in chronic obstructive pulmonary disease (COPD), but the underlying mechanism remains unclear. Dysfunction of pulmonary surfactant proteins (SPs, including SP-A, SP-B, SP-C, and SP-D) may be included in pathophysiology of COPD. This study aimed to explore the mechanism of the integrated therapy on SPs. COPD rat models were established. The treatment groups received Bufei Yishen granules or acupoint sticking or their combination. Using aminophylline as a positive control drug. The levels of SPs in serum, BALF, and lung were measured. The results showed that the integrated therapy markedly reduced the levels of SPs in serum and increased these indicators in the lung. The integrated therapy was better than aminophylline in reducing the levels of SPs and was better than Bufei Yishen granules in reducing SP-A, SP-C, and SP-D in serum. The integrated therapy was better than aminophylline and Bufei Yishen granules in increasing SP-A, SP-B, and SP-D mRNA in the lung. SP-A and SP-D in BALF were positively correlated with PEF and EF50. The levels of SPs are associated with airway limitation. The beneficial effects of the integrated therapy may be involved in regulating pulmonary surfactant proteins.

Serum Levels of Surfactant Proteins in Patients with Combined Pulmonary Fibrosis and Emphysema (CPFE).

INTRODUCTION: Emphysema and idiopathic pulmonary fibrosis (IPF) present either per se or coexist in combined pulmonary fibrosis and emphysema (CPFE). Serum surfactant proteins (SPs) A, B, C and D levels may reflect lung damage. We evaluated serum SP levels in healthy controls, emphysema, IPF, and CPFE patients and their associations to disease severity and survival. METHODS: 122 consecutive patients (31 emphysema, 62 IPF, and 29 CPFE) and 25 healthy controls underwent PFTs, ABG-measurements, 6MWT and chest HRCT. Serum levels of SPs were measured. Patients were followed-up for 1-year. RESULTS: SP-A and SP-D levels differed between groups (p = 0.006 and p<0.001 respectively). In post-hoc analysis, SP-A levels differed only between controls and CPFE (p<0.05) and CPFE and emphysema (p<0.05). SP-D differed between controls and IPF or CPFE (p<0.001 for both comparisons). In IPF SP-B correlated to pulmonary function while SP-A, correlated to the Composite Physiological Index (CPI). Controls current smokers had higher SP-A and SP-D levels compared to non-smokers (p = 0.026 and p = 0.023 respectively). SP-D levels were higher in CPFE patients with extended emphysema (p = 0.042). In patients with IPF, SP-B levels at the upper quartile of its range (≥26 ng/mL) presented a weak association with reduced survival (p = 0.05). CONCLUSION: In conclusion, serum SP-A and SP-D levels were higher where fibrosis exists or coexists and related to disease severity, suggesting that serum SPs relate to alveolar damage in fibrotic lungs and may reflect either local overproduction or overleakage. The weak association between high levels of SP-B and survival needs further validation in clinical trials.

Serum Glycans as Risk Markers for Non-Small Cell Lung Cancer.

Previous studies have suggested occurrence of altered serum glycan profiles in patients with lung cancer. Here, we aimed to determine the predictive value of serum glycans to distinguish non-small cell lung cancer (NSCLC) cases from controls in prediagnostic samples using a previously validated predictive protein marker pro-SFTPB, as anchor. Blinded prediagnostic serum samples were obtained from the Carotene and Retinol Efficacy Trial (CARET), and included a discovery set of 100 NSCLC cases and 199 healthy controls. A second test set consisted of 108 cases and 216 controls. Cases and controls were matched for age at baseline (5-year groups), sex, smoking status (current vs. former), study enrollment cohort, and date of blood draw. Serum glycan profiles were determined by mass spectrometry. Twelve glycan variables were identified to have significant discriminatory power between cases and controls in the discovery set (AUC > 0.6). Of these, four were confirmed in the independent validation set. A combination marker yielded AUCs of 0.74 and 0.64 in the discovery and test set, respectively. Four glycan variables exhibited significant incremental value when combined with pro-SFTPB compared with pro-SFTPB alone with AUCs of 0.73, 0.72, 0.72, and 0.72 in the test set, indicating that serum glycan signatures have relevance to risk assessment for NSCLC.

Diacetylspermine Is a Novel Prediagnostic Serum Biomarker for Non-Small-Cell Lung Cancer and Has Additive Performance With Pro-Surfactant Protein B.

PURPOSE: We have investigated the potential of metabolomics to discover blood-based biomarkers relevant to lung cancer screening and early detection. An untargeted metabolomics approach was applied to identify biomarker candidates using prediagnostic sera from the Beta-Carotene and Retinol Efficacy Trial (CARET) study. PATIENTS AND METHODS: A liquid chromatography/mass spectrometry hydrophilic interaction method designed to profile a wide range of metabolites was applied to prediagnostic serum samples from CARET participants (current or former heavy smokers), consisting of 100 patients who subsequently developed non-small-cell lung cancer (NSCLC) and 199 matched controls. A separate aliquot was used to quantify levels of pro-surfactant protein B (pro-SFTPB), a previously established protein biomarker for NSCLC. On the basis of the results from the discovery set, blinded validation of a metabolite, identified as N(1),N(12)-diacetylspermine (DAS), and pro-SFTPB was performed using an independent set of CARET prediagnostic sera from 108 patients with NSCLC and 216 matched controls. RESULTS: Serum DAS was elevated by 1.9-fold, demonstrating significant specificity and sensitivity in the discovery set for samples collected up to 6 months before diagnosis of NSCLC. In addition, DAS significantly complemented performance of pro-SFTPB in both the discovery and validations sets, with a combined area under the curve in the validation set of 0.808 (P < .001 v pro-SFTPB). CONCLUSION: DAS is a novel serum metabolite with significant performance in prediagnostic NSCLC and has additive performance with pro-SFTPB.

Plasma pro-surfactant protein B and lung function decline in smokers.

Plasma pro-surfactant protein B (pro-SFTPB) levels have recently been shown to predict the development of lung cancer in current and ex-smokers, but the ability of pro-SFTPB to predict measures of chronic obstructive pulmonary disease (COPD) severity is unknown. We evaluated the performance characteristics of pro-SFTPB as a biomarker of lung function decline in a population of current and ex-smokers. Plasma pro-SFTPB levels were measured in 2503 current and ex-smokers enrolled in the Pan-Canadian Early Detection of Lung Cancer Study. Linear regression was performed to determine the relationship of pro-SFTPB levels to changes in forced expiratory volume in 1 s (FEV1) over a 2-year period as well as to baseline FEV1 and the burden of emphysema observed in computed tomography (CT) scans. Plasma pro-SFTPB levels were inversely related to both FEV1 % predicted (p=0.024) and FEV1/forced vital capacity (FVC) (p<0.001), and were positively related to the burden of emphysema on CT scans (p<0.001). Higher plasma pro-SFTPB levels were also associated with a more rapid decline in FEV1 at 1 year (p=0.024) and over 2 years of follow-up (p=0.004). Higher plasma pro-SFTPB levels are associated with increased severity of airflow limitation and accelerated decline in lung function. Pro-SFTPB is a promising biomarker for COPD severity and progression.

Serum pneumoproteins in tunnel construction workers.

PURPOSE: The aim was to study inflammatory biomarkers in tunnel construction workers (TCW). METHODS: Surfactant protein D (SP-D), Clara cell protein 16 (CC-16) and C-reactive protein (CRP) were studied in serum of 90 TCW and 50 referents before and at the end of an 11-day work period. Personal air sampling was carried out on the two consecutive days before follow-up. RESULTS: The TCW's geometric mean exposure to particulate matter and α-quartz were 604 and 74 µg/m(3), respectively. The arithmetic mean concentration of elemental carbon was 51 µg/m(3). The arithmetic mean concentration of SP-D was reduced by 7.6 µg/L in the TCWs and 0.6 µg/L in the referents (p = 0.04) at the end as compared to before the work period. Subjects who had ever been TCW had lower arithmetic mean CC-16 concentrations at baseline (5.4 µg/L) than subjects who had never worked as TCW (6.4 µg/L). Years worked as TCW was significantly associated with an annual mean decline of the CC-16 concentration of 0.04 µg/L. The concentrations of the biomarker of systemic inflammation, CRP, were not affected by exposure in the TCWs. Current smoking and body mass index have a large impact on the measured biomarker concentrations. CONCLUSIONS: The results suggest that former and current TCWs have lower serum CC-16 concentrations than referents, while the concentrations of SP-D decreased during exposure. The serum biomarker of systemic inflammation, CRP, was not altered during exposure. Current smoking and BMI were related to the concentrations of all measured biomarkers.

Pro-surfactant protein B as a biomarker for lung cancer prediction.

PURPOSE: Preliminary studies have identified pro-surfactant protein B (pro-SFTPB) to be a promising blood biomarker for non-small-cell lung cancer. We conducted a study to determine the independent predictive potential of pro-SFTPB in identifying individuals who are subsequently diagnosed with lung cancer. PATIENTS AND METHODS: Pro-SFTPB levels were measured in 2,485 individuals, who enrolled onto the Pan-Canadian Early Detection of Lung Cancer Study by using plasma sample collected at the baseline visit. Multivariable logistic regression models were used to evaluate the predictive ability of pro-SFTPB in addition to known lung cancer risk factors. Calibration and discrimination were evaluated, the latter by an area under the receiver operating characteristic curve (AUC). External validation was performed with samples collected in the Carotene and Retinol Efficacy Trial (CARET) participants using a case-control study design. RESULTS: Adjusted for age, sex, body mass index, personal history of cancer, family history of lung cancer, forced expiratory volume in one second percent predicted, average number of cigarettes smoked per day, and smoking duration, pro-SFTPB (log transformed) had an odds ratio of 2.220 (95% CI, 1.727 to 2.853; P < .001). The AUCs of the full model with and without pro-SFTPB were 0.741 (95% CI, 0.696 to 0.783) and 0.669 (95% CI, 0.620 to 0.717; difference in AUC P < .001). In the CARET Study, the use of pro-SFPTB yielded an AUC of 0.683 (95% CI, 0.604 to 0.761). CONCLUSION: Pro-SFTPB in plasma is an independent predictor of lung cancer and may be a valuable addition to existing lung cancer risk prediction models.

[Relationship between total bile acid concentration and fetal pulmonary surfactant in intrahepatic cholestasis of pregnancy].

OBJECTIVE: To explore the relationship between total bile acid (TBA) concentration and fetal pulmonary surfactant in intrahepatic cholestasis of pregnancy (ICP). METHODS: Fifty five patients with ICP (ICP group) who received cesarean section from April 2008 to February 2010 in Second Xiangya Hospital, Central South University, were recruited. The general conditions of the neonates within 7 days after birth in ICP group were recorded. Those with fetal distress, neonatal asphyxia, or neonatal respiratory distress syndrome were referred as pathological neonates, others were referred as normal neonates. Over the same period, 23 healthy gravidas were recruited as control group. Enzymatic method was used to detect the TBA concentrations in maternal blood, cord blood and amniotic fluid. ELISA was employed to measure the urfactant protein A (SP-A) concentration in cord blood. High performance liquid chromatography system was used to detect the concentrations of phosphatidylcholine (PC), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), and sphingomyelin (SM) in amniotic fluid. RESULTS: (1) The concentrations of TBA in maternal blood, cord blood and amniotic fluid were (30.1 ± 7.9), (9.3 ± 3.3) and (4.4 ± 1.5) mmol/L in ICP group, (4.8 ± 2.2), (4.9 ± 0.9) and (1.4 ± 1.1) mmol/L in control group, respectively. The differences between the two groups were significant (P < 0.05). (2) The SP-A concentration in cord blood in ICP group was (29.5 ± 6.4) µg/L, significantly higher than that in control group, which was (22.6 ± 7.4) µg/L (P < 0.05). (3) There were 20 pathological neonates and 35 normal neonates in ICP group. In pathological neonates, the concentrations of TBA and SP-A in cord blood were (10.9 ± 2.2) mmol/L, (37.0 ± 5.9) µg/L, respectively; and were (8.0 ± 2.8) mmol/L, (26.7 ± 4.8) µg/L in normal neonates. The differences were significant (P < 0.05). (4) There was a positive correlation between TBA concentration in cord blood and in maternal blood (r(1) = 0.706, P < 0.05). The TBA concentration in cord blood was positively correlated with SP-A concentration as well (r(3) = 0.494, P < 0.05). (5) The PC and PI concentrations in amniotic fluid were (65.4 ± 7.2) mg/L and (3.8 ± 0.6) mg/L in ICP group, (69.7 ± 3.7) mg/L and (4.3 ± 0.7) mg/L in control group, respectively. The differences were significant (P < 0.05). The concentration of LPC in amniotic fluid in ICP group was (4.8 ± 0.9) mg/L, significantly higher than that in control group (P < 0.05), which was (4.2 ± 0.6) mg/L. The concentration of SM in amniotic fluid was (3.5 ± 0.8) mg/L in ICP group, (4.0 ± 0.5) mg/L in control group, with no significant difference (P > 0.05). (6)The ratio of PC/LPC in ICP group (14.2 ± 3.2) was significantly lower than that in control group (16.9 ± 2.5) (P < 0.05). (7)The TBA concentration in cord blood was negatively correlated with PC and PI concentrations (r(1) = -0.561, r(2) = -0.407, P < 0.05), and had no correlation with LPC concentration (r(3) = 0.260, P > 0.05). CONCLUSIONS: (1) The fetal TBA concentrations in both cord blood and amniotic fluid of patients with ICP was higher than those of healthy gravidas, they were also positively correlated with maternal TBA concentration. (2) ICP resulted in the change of fetal pulmonary surfactant and this change was associated with TBA concentrations in both cord blood and amniotic fluid.

Serum pneumoproteins in firefighters.

Serum Clara cell protein (CC16) and surfactant-associated protein A (SP-A) were measured in a cross-sectional study in 402 firefighters. For the population as a whole, no associations were detected between serum pneumoproteins and smoke exposure. SP-A levels were increased in symptomatic subjects exposed to fire smoke within 2 days before testing. SP-A levels were higher after an inhalation incident ever. CC16 was negatively associated with the number of fires fought in the last 12 months in current nonsmokers. These associations between pneumoprotein levels reiterate the importance of adequate use of self-contained breathing apparatus by firefighters.

Exposure to bioaerosols, respiratory health and lung-specific proteins: a prospective study in garbage and wastewater workers.

OBJECTIVES: To prospectively assess respiratory health in wastewater workers and garbage collectors over 5 years. METHODS: Exposure, respiratory symptoms and conditions, spirometry and lung-specific proteins were assessed yearly in a cohort of 304 controls, 247 wastewater workers and 52 garbage collectors. Results were analysed with random coefficient models and linear regression taking into account several potential confounders. RESULTS: Symptoms, spirometry and lung-specific proteins were not affected by occupational exposure. CONCLUSIONS: In this population no effects of occupational exposure to bioaerosols were found, probably because of good working conditions.