Quantification of PPAR-gamma protein in monocyte/macrophages from healthy smokers and non-smokers: a possible direct effect of nicotine

Previous observations demonstrated that Peroxisome Proliferator-Activated Receptor-gamma (PPAR-gamma), a key regulator of adipocyte differentiation, is expressed in a large variety of cells, including cells of the monocyte/macrophage lineage. This study was aimed to quantify both the constitutive and ligand-induced PPAR-gamma expression in monocytes and monocyte-derived macrophages (MDM) isolated from healthy smokers and non-smokers, and to evaluate the possible direct effect of nicotine. PPAR-gamma protein was detected by Western blot and quantification was performed by calculating the ratio between PPAR-gamma and beta-actin protein expression. Cytokine release was measured with enzyme-linked immunoassay kits. Constitutive PPAR-gamma protein was detected in human monocytes and its expression was up-regulated along with differentiation to MDM. The endogenous ligand 15-deoxy-delta(12,14)-prostaglandin J(2) and the synthetic agonist ciglitazone enhanced PPAR-gamma expression, the former being effective also at low micromolar concentrations. Both agonists significantly inhibited the basal secretion of pro-inflammatory cytokines (e.g., TNF-alpha, IL-6), ciglitazone being more potent. Monocytes and MDM from healthy smokers presented a significantly enhanced (4-fold and 2.5-fold, respectively) constitutive PPAR-gamma expression, as compared to those from healthy non-smokers. However, ligand-induced PPAR-gamma expression and inhibition of cytokine secretion were similar in healthy smokers and non-smokers. Nicotine dose-dependently enhanced PPAR-gamma expression with a maximum at 10 muM, and inhibited release of pro-inflammatory cytokines; these effects were reversed by alpha-bungarotoxin. Nicotine and PPAR-gamma agonists did not exert synergistic effects. In conclusion, monocytes and MDM from healthy smokers present a constitutively enhanced PPAR-gamma expression; this effect is reproduced, to some extent, by nicotine in vitro.     

Serum Clara cell protein (CC16) in healthy young smokers

The CC16 microprotein is the main secretory product of Clara cells, which are epithelial cells lining lung airways. In crossing through the bronchoalveolar/blood barrier, CC16 diffuses passively into plasma. Serum CC16 (sCC16) has recently been proposed as a biomarker for detecting Clara cell impairments. The aim of this study was to assess if sCC16 concentrations are reduced in a group of healthy young smokers. A group of 118 healthy young males volunteered to take part in the study. Each subject answered a questionnaire, and provided blood and urine samples. Serum CC16, urinary cotinine and creatinine were measured. Median serum CC16 concentrations were lower in smokers than in non-smokers (11.3 mug l^-1 vs 14.6 mug l^-1; p = 0.005; N = 89 and 29, respectively) but did not correlate with either the daily or the life-time cigarette consumption, or with urinary cotinine concentrations. sCC16 did not correlate with age or body mass index in the whole study population or in the groups of smokers and non-smokers. These results suggest the reduction in sCC16 concentrations in a group of healthy young smokers may be an early effect of cigarette smoking.     

Altered arterial stiffness and subendocardial viability ratio in young healthy light smokers after acute exercise

Background

Studies showed that long-standing smokers have stiffer arteries at rest. However, the effect of smoking on the ability of the vascular system to respond to increased demands (physical stress) has not been studied. The purpose of this study was to estimate the effect of smoking on arterial stiffness and subendocardial viability ratio, at rest and after acute exercise in young healthy individuals.

Methods/Results

Healthy light smokers (n = 24, pack-years = 2.9) and non-smokers (n = 53) underwent pulse wave analysis and carotid-femoral pulse wave velocity measurements at rest, and 2, 5, 10, and 15 minutes following an exercise test to exhaustion. Smokers were tested, 1) after 12h abstinence from smoking (chronic condition) and 2) immediately after smoking one cigarette (acute condition). At rest, chronic smokers had higher augmentation index and lower aortic pulse pressure than non-smokers, while subendocardial viability ratio was not significantly different. Acute smoking increased resting augmentation index and decreased subendocardial viability ratio compared with non-smokers, and decreased subendocardial viability ratio compared with the chronic condition. After exercise, subendocardial viability ratio was lower, and augmentation index and aortic pulse pressure were higher in non-smokers than smokers in the chronic and acute conditions. cfPWV rate of recovery of was greater in non-smokers than chronic smokers after exercise. Non-smokers were also able to achieve higher workloads than smokers in both conditions.

Conclusion

Chronic and acute smoking appears to diminish the vascular response to physical stress. This can be seen as an impaired ‘vascular reserve’ or a blunted ability of the blood vessels to accommodate the changes required to achieve higher workloads. These changes were noted before changes in arterial stiffness or subendocardial viability ratio occurred at rest. Even light smoking in young healthy individuals appears to have harmful effects on vascular function, affecting the ability of the vascular bed to respond to increased demands.     

Serum Clara cell protein (CC16) in healthy young smokers

The CC16 microprotein is the main secretory product of Clara cells, which are epithelial cells lining lung airways. In crossing through the bronchoalveolar/blood barrier, CC16 diffuses passively into plasma. Serum CC16 (sCC16) has recently been proposed as a biomarker for detecting Clara cell impairments. The aim of this study was to assess if sCC16 concentrations are reduced in a group of healthy young smokers. A group of 118 healthy young males volunteered to take part in the study. Each subject answered a questionnaire, and provided blood and urine samples. Serum CC16, urinary cotinine and creatinine were measured. Median serum CC16 concentrations were lower in smokers than in non-smokers (11.3 mug l^-1 vs 14.6 mug l^-1; p = 0.005; N = 89 and 29, respectively) but did not correlate with either the daily or the life-time cigarette consumption, or with urinary cotinine concentrations. sCC16 did not correlate with age or body mass index in the whole study population or in the groups of smokers and non-smokers. These results suggest the reduction in sCC16 concentrations in a group of healthy young smokers may be an early effect of cigarette smoking.     

Smoking alters thromboxane metabolism in man

Chronic smoking is a major risk factor of atherosclerosis and coronary heart disease. The measurement of three major thromboxane A2 metabolites, 11-dehydrothromboxane B2, 2,3-dinorthromboxane B2 and thromboxane B2, in the urines of 13 apparently healthy smokers (average 39 years, range 27-56 years) showed significantly elevated excretion rates for all thromboxane A2 metabolites as compared to 10 apparently healthy age-matched non-smokers (average 37 years, range 26-56 years). Importantly, characteristic alterations in the thromboxane A2 metabolite pattern were found in the urines of smokers. The contribution of 2,3-dinorthromboxane B2 to total measured excretion of thromboxane A2 metabolites was 59.2% in smokers (404.0 +/- 53.0 pg/mg creatinine) versus 19.4% in non-smokers (85.2 +/- 8.3 pg/mg creatinine), that of 11-dehydrothromboxane B2 35.7% in smokers (673.2 +/- 88.9 pg/mg creatinine) as compared to 75.5% in non-smokers (332.6 +/- 30.9 pg/mg creatinine). The contribution of thromboxane B2 (57.5 +/- 7.7 pg/mg creatinine in smokers versus 21.9 +/- 1.5 pg/mg creatinine in non-smokers) was similar at 5.1%. The excretion of cotinine, the major urinary metabolite of nicotine that correlates well with the reported daily cigarette consumption (r = 0.97, P less than 0.0001), showed a good correlation to thromboxane A2 metabolite excretion (2,3-dinorthromboxane B2: r = 0.92, P less than 0.0001; 11-dehydrothromboxane B2; r = 0.87, P less than 0.0001).     

Humans Have Antibodies against a Plant Virus: Evidence from Tobacco Mosaic Virus

Tobacco mosaic virus (TMV), a widespread plant pathogen, is found in tobacco (including cigarettes and smokeless tobacco) as well as in many other plants. Plant viruses do not replicate or cause infection in humans or other mammals. This study was done to determine whether exposure to tobacco products induces an immune response to TMV in humans. Using a sandwich ELISA assay, we detected serum anti-TMV antibodies (IgG, IgG1, IgG3, IgG4, IgA, and IgM) in all subjects enrolled in the study (20 healthy smokers, 20 smokeless-tobacco users, and 20 non-smokers). Smokers had a higher level of serum anti-TMV IgG antibodies than non-smokers, while the serum level of anti-TMV IgA from smokeless tobacco users was lower than smokers and non-smokers. Using bioinformatics, we also found that the human protein TOMM40L (an outer mitochondrial membrane 40 homolog – like translocase) contains a strong homology of six contiguous amino acids to the TMV coat protein, and TOMM40L peptide exhibited cross-reactivity with anti-TMV antibodies. People who smoke cigarettes or other tobacco products experience a lower risk of developing Parkinson’s disease, but the mechanism by which this occurs is unclear. Our results showing molecular mimicry between TMV and human TOMM40L raise the question as to whether TMV has a potential role in smokers against Parkinson’s disease development. The potential mechanisms of molecular mimicry between plant viruses and human disease should be further explored.     

Adhesion molecules in subjects with COPD and healthy non-smokers: a cross sectional parallel group study

Background

The aim of the study was to investigate how the expression of adhesion molecules changes as neutrophils migrate from the circulation to the lung and if these changes differ between non-smoking subjects and smokers with and without COPD.

Methods

Non-smoking healthy subjects (n=22), smokers without (n=21) and with COPD (n=18) were included. Neutrophils from peripheral blood, sputum and bronchial biopsies were analysed for cell surface expression of adhesion molecules (CD11b, CD62L, CD162). Serum, sputum supernatant and BAL-fluid were analysed for soluble adhesion molecules (ICAM-1, -3, E-selectin, P-selectin, VCAM-1, PECAM-1).

Results

Expression of CD11b was increased on circulating neutrophils from smokers with COPD. It was also increased on sputum neutrophils in both smokers groups, but not in non-smokers, as compared to circulating neutrophils.Serum ICAM-1 was higher in the COPD group compared to the other two groups (p<0.05) and PECAM-1 was lower in smokers without COPD than in non-smoking controls and the COPD group (p<0.05). In BAL-fluid ICAM-1 was lower in the COPD group than in the other groups (p<0.05).

Conclusions

Thus, our data strongly support the involvement of a systemic component in COPD and demonstrate that in smokers neutrophils are activated to a greater extent at the point of transition from the circulation into the lungs than in non-smokers.     

Altered chemotactic peptide binding in tobacco smokers' neutrophils

The functions of polymorphonuclear leukocytes from tobacco smokers are altered compared to those from nonsmokers. Since neutrophil chemotaxis and oxidative metabolism are mediated by surface receptors, we studied the association of the chemotactic peptide formyl Met-Leu-Phe with neutrophils from smokers and non-smokers. An apparently single class of binding sites was observed in neutrophils from all the non-smokers, whereas upwardly curving Scatchard plots were obtained for binding to smokers' cells. Thus changes at the receptor level may be responsible for the previously observed alterations in smoker neutrophil function.     

Differential enhancement of sister-chromatid exchange frequencies by alpha-naphthoflavone in cultured lymphocytes from smokers and non-smokers

The sister-chromatid exchange (SCE) frequency was assessed in peripheral lymphocytes from 4 smokers and 8 non-smokers in the absence or presence of alpha-naphthoflavone (ANF) in the culture media. ANF produced a concentration-dependent increase in the frequency of SCEs in smoking individuals. At an ANF concentration of 11 micrograms/ml, average SCE levels were 54% and 13% above the baseline levels in smokers and non-smokers, respectively. The ANF-enhanced increase in the SCE frequency ranged from 3.12 to 5.72 among smokers, and from 0 to 1.96 among the non-smokers. No significant difference in the mean SCE baseline levels between smokers and non-smokers was detected. The mechanism responsible for the enhanced frequency of SCEs in smokers following in vitro exposure to ANF is not clear, but may reflect changes in metabolic activation/deactivation or increased sensitivity to genetic effects of ANF.     

Comparison of the protective effects on the erythrocytes of ceruloplasmin from the blood of healthy donors and patients with hepatocerebral dystrophy

The binding of the ceruloplasmin (CP) from the healthy donor's blood and of ceruloplasmin--like protein (p-CP) isolated from the Wilson disease patient's blood with erythrocytes (RBC) of healthy donors and with RBC of Wilson's patients (p-RBC) was investigated. It was shown, that the CP number of binding sites both on the RBC and p-RBC was significantly lower than that for p-CP, but Kd value for p-CP binding of the both types of erythrocytes was approximately 10 times higher than Kd value for CP. The protective action of CP on copper stimulated hemolysis is almost 3 times higher than that of p-CP. The protective action of CP on ferrous ion stimulated hemolysis doesn't correlate with its ferroxidase activity. On the contrary the protective effect of p-CP which has no ferroxidase activity is more powerful than that of CP.     

Thiocyanate concentration in saliva of cystic fibrosis patients

Thiocyanates (SCN-) are ubiquitous in nature. There are indispensable part of host defense system that act as a substrate for lactoperoxidase (LPO). In our study we present initial data on SCN- concentration in saliva of CF patients in comparison to healthy non-smokers and healthy smokers. 5 ml of saliva was collected from each subject to a sterile tube and thiocyanate concentration was measured in each sample. The results of the measurements are presented on Fig. 1. Mean concentration of SCN- in saliva of CF patients was 0.031 +/- 0.0052 g/l, in healthy non-smokers 0.039 +/- 0.0048 g/l and in healthy smokers 0.048 +/- 0.0161 g/l. The differences between each group were statistically significant. Studies on larger group of patients and probably on different material (BALF or induced sputum) should present interesting data complementing the in vitro studies.     

Down-regulation of circulating microRNA let-7a in Egyptian smokers

Altered miRNAs were associated with cigarette smoking. The study aimed to examine the gene expression level of plasma let-7a among healthy smokers and compared it with the non-smokers. Forty subjects were recruited for the present study and classified into 21 smokers and 19 non-smokers, age, and sex were matched. The software that used to design functional primers was MIRprimer. Quantitative real-time PCR was employed to compare the relative expression of plasma let-7a. Results showed that the level of let-7a was down-regulated in smokers to 0.34fold (p?=?0.006) that of the non-smokers. Plasma let-7a showed an area under curve (AUC) of 0.749 with sensitivity 43% and specificity 100%. In conclusion, plasma let-7a was significantly down-regulated in the smokers, and it might be considered a candidate biomarker to discriminate between smokers and non-smokers.     

Study of the amniotic fluid from smokers and non-smokers in the Ames test

Amniotic fluid from smokers and non-smokers was tested by the Salmonella/mammalian microsome test. Concentrated amniotic fluid from heavy smokers at term showed an increase in the number of revertants with increasing exposure to tar. However, some of the non-smokers had a higher number of revertants than the smokers. No significant differences were found between second-trimester samples from smokers and non-smokers, but the limited volumes available at this stage of pregnancy may be a source of error.     

Effect of acidosis on bilirubin-induced toxicity to human erythrocytes

Unconjugated bilirubin binds to erythrocytes, eliciting crenation, lipid elution and hemolysis. The present work attempts to establish the role of acidosis on bilirubin-induced toxicity to human erythrocytes. To this end, pH values ranging from 7.0–8.0 were used to induce a different representation of acid and anionic bilirubin species, respectively. Erythrocytes from healthy donors were incubated with bilirubin and albumin (3:1, molar ratio), during 4 h. Erythrocyte-bound bilirubin was evaluated by albumin or chloroform extraction in an attempt to assess either mono- and dianion bilirubin adsorbed on the cell surface or colloidal aggregates, respectively. Cytotoxicity indicators, such as the morphological index, and the extent of phospholipids and hemoglobin release were also determined. The results showed that as pH drops from 8.0–7.0, less bilirubin is removed by albumin and more become recovered by chloroform. The data corroborate the predominance of anionic and non-aggregated bilirubin species at pH 8.0 with dimers and precipitates occurring at 7.0. In accordance, crenation and cell lysis were four times increased at acidic pH. In contrast, elution of phospholipids was 1.5 times less evident at the same pH, thus suggesting that formation of bilirubin complexes with membrane phospholipids may have contributed to prevent their release. In conclusion, both anionic and acid bilirubin species interact with human erythrocytes leading to cytotoxic alterations that may determine definitive lesions. Nevertheless, increased vulnerability to crenation and hemolysis are more likely to occur in acidic conditions pointing to the bilirubin precipitates as the main candidates of bilirubin-induced toxicity to erythrocytes.     

Increased frequency of micronuclei in B and T8 lymphocytes from smokers

The frequency of micronuclei was measured in peripheral B lymphocytes and some T lymphocyte subpopulations from 5 medium-tar cigarette smokers, and 5 non-smokers with no regular exposure to tobacco smoke. The peripheral lymphocytes were stimulated in vitro with phytohemagglutinin and B lymphocytes and the various T lymphocyte subsets were classified by a recently developed MAC (Morphology, Antibody, Chromosomes) method which allows the immunologic identification of different cell lineages. An increased frequency of micronuclei was observed in B and especially in the suppressor/cytotoxic T8 lymphocytes from smokers, as compared with non-smoker values. In non-smoker cultures, no differences in the frequency of micronuclei were observed among the different T lymphocyte subsets. In these cultures, B cells tended to have a higher frequency of micronuclei than did T cells. The proportions of B cells and the various T subpopulations among mitotic and interphasic lymphocytes from smokers and non-smokers were also determined. The proportions of B cells and T cell subsets among all mitotic lymphocytes were similar in smokers and non-smokers. Contrarily, a significant decrease in the proportion of T8 lymphocytes among all interphasic lymphocytes was observed in cultures derived from smokers.     

Comparison of insulin receptor binding to monocytes and erythrocytes in newly diagnosed type II diabetes mellitus

Insulin binding to circulating monocytes and erythrocytes was studied in 20 healthy volunteers and in 25 obese hyperinsulinemic newly diagnosed type-II diabetics. In type-II diabetics insulin binding to monocytes as well as to erythrocytes was significantly decreased in comparison with healthy individuals. The lowered insulin binding of the diabetics was mainly caused by a loss of receptor number. Individual analysis of the binding data, however, shows a marked discrepancy between receptor binding to circulating monocytes compared with erythrocytes. Since insulin binding to erythrocytes shows a great variation and seems to be influenced by other factors beside insulin concentrations it is suggested that insulin receptors on monocytes should be preferred for evaluation of peripheral insulin sensitivity.     

Association of Leisure-Time Physical Activity to Cardiovascular Disease Prevalence in Relation to Smoking among Adult Nevadans

It is well known that cigarette smoking and physical activity have significant impacts on cardiovascular disease (CVD) mortality and morbidity. Meanwhile, it is of interest to understand whether physical activity protects against CVD for smokers in a similar manner as it does for non-smokers. The present study examined how leisure-time physical activity (LTPA) is associated with the prevalence of CVD in relation to smoking status among adult Nevadans, using data from the 2010 Nevada Behavioral Risk Factor Surveillance System. Of the 3,913 survey respondents, 8.5% self-reported that they had ever been diagnosed with CVD. People with a history of CVD were significantly less likely to engage in LTPA than those with no history of CVD (p < 0.05). After adjusting for common sociodemographic variables, it was revealed that people with CVD were twice more likely to not engage in LTPA than their counterparts independent of smoking status. Without taking LTPA into account, the odds of having CVD for current and former smokers was 1.87–2.25 times higher than the odds for non-smokers. Interestingly, however, if LTPA was accounted for, there was no significant difference in the odds of having CVD between current and non-smokers. These results indicate that LTPA is inversely associated with the prevalence of CVD independent of smoking status, and that regular physical activity may protect against CVD for smokers as well as for non-smokers. Physical activity, along with smoking cessation, should be promoted to better prevent and control CVD among smokers.     

Investigations on the effect of cigarette smoking in the comet assay

The comet assay (single-cell gel electrophoresis, SCG) is widely accepted as an in vitro and in vivo genotoxicity test. Because of its demonstrated ability to detect various kinds of DNA damage and its ease of application, the technique is being increasingly used in human biomonitoring. However, the assessment of small genotoxic effects as typically obtained in biomonitoring may be limited by the different sources of assay variability and the lack of an optimal protocol with high sensitivity. To better characterize the suitability of the comet assay for biomonitoring, we are performing a comprehensive investigation on blood samples from smokers and non-smokers. Because tobacco smoke is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds, smokers should be a suitable study group with relevant mutagen exposure. Here, we report our results for the first sample of 20 healthy male smokers and 20 healthy male non-smokers. Baseline and benzo[a]pyrene diolepoxide (BPDE)-induced effects were analysed by two investigators using two image analysis systems. The study was repeated within 4 months. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline and BPDE-induced DNA damage was comparatively analysed. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. None of these approaches revealed significant differences between smokers and non-smokers. Although more data is needed for a final conclusion, this study indicates some limitations of the comet assay with regard to the detection of DNA damage induced by environmental mutagens in peripheral blood cells.     

Investigations on the effect of cigarette smoking in the comet assay

The comet assay (single-cell gel electrophoresis, SCG) is widely accepted as an in vitro and in vivo genotoxicity test. Because of its demonstrated ability to detect various kinds of DNA damage and its ease of application, the technique is being increasingly used in human biomonitoring. However, the assessment of small genotoxic effects as typically obtained in biomonitoring may be limited by the different sources of assay variability and the lack of an optimal protocol with high sensitivity. To better characterize the suitability of the comet assay for biomonitoring, we are performing a comprehensive investigation on blood samples from smokers and non-smokers. Because tobacco smoke is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds, smokers should be a suitable study group with relevant mutagen exposure. Here, we report our results for the first sample of 20 healthy male smokers and 20 healthy male non-smokers. Baseline and benzo[a]pyrene diolepoxide (BPDE)-induced effects were analysed by two investigators using two image analysis systems. The study was repeated within 4 months. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline and BPDE-induced DNA damage was comparatively analysed. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. None of these approaches revealed significant differences between smokers and non-smokers. Although more data is needed for a final conclusion, this study indicates some limitations of the comet assay with regard to the detection of DNA damage induced by environmental mutagens in peripheral blood cells.     

Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients

Backround

Cigarette smoke exposure including biologically active lipopolysaccharide (LPS) in the particulate phase of cigarette smoke induces activation of alveolar macrophages (AM) and alveolar epithelial cells leading to production of inflammatory mediators. This represents a crucial mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Respiratory pathogens are a major cause of exacerbations leading to recurrent cycles of injury and repair. The interaction between pathogen-associated molecular patterns and the host is mediated by pattern recognition receptors (PRR''s). In the present study we characterized the expression of Toll-like receptor (TLR)- 2, TLR4 and CD14 on human AM compared to autologous monocytes obtained from patients with COPD, healthy smokers and non-smokers.

Methods

The study population consisted of 14 COPD patients without evidence for acute exacerbation, 10 healthy smokers and 17 healthy non-smokers stratified according to age. The expression of TLR2, TLR4 and CD14 surface molecules on human AM compared to autologous monocytes was assessed ex vivo using FACS analysis. In situ hybridization was performed on bronchoalveolar lavage (BAL) cells by application of the new developed HOPE-fixative.

Results

The expression of TLR2, TLR4 and CD14 on AM from COPD patients, smokers and non-smokers was reduced as compared to autologous monocytes. Comparing AM we detected a reduced expression of TLR2 in COPD patients and smokers. In addition TLR2 mRNA and protein expression was increased after LPS stimulation on non-smokers AM in contrast to smokers and COPD patients.

Conclusion

Our data suggest a smoke related change in the phenotype of AM''s and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract.