Toxicological assessment of kretek cigarettes Part 5: mechanistic investigations, inhalation toxicity.

The biological effects of mainstream smoke (MS) from Indonesian-blended cigarettes with and without added cloves, cloves extracted with hot ethanol, and extracted cloves replenished with eugenol or clove oil were assessed in a 90-day inhalation study in rats. A separate 35-day inhalation study in rats was performed with MS from American-blended cigarettes with 0%, 2.5%, 5% or 10% added eugenol. Effects commonly seen in inhalation studies with MS were observed. These included histopathological changes indicative of irritation in the entire respiratory tract and inflammatory responses in the lung. Adding cloves to American- or Indonesian-blended cigarettes reduced the inflammatory response in the lung but with no difference between the two blend types. When the clove oil was extracted (∼ 75% reduction of eugenol achieved) from cloves, the inflammatory response in the lung was still reduced similarly to whole cloves but the severity of histopathological changes in the upper respiratory tract was less reduced. Add back of clove oil or pure eugenol reduced this response to a level similar to what was seen with whole cloves. When eugenol was added to American-blended cigarettes, similar findings of reduced lung inflammation and severity of histopathological changes in respiratory the tract was confirmed. These studies demonstrate a clear effect of cloves, and in particular eugenol, in explaining these findings.

Role of elastases in the pathogenesis of chronic obstructive pulmonary disease: implications for treatment.

Neutrophil elastase, metalloproteinases, and their inhibitors play an important role in the development of chronic obstructive pulmonary disease (COPD), resulting in extensive tissue damage and malfunctioning of the airways. Nearly fifty years after the protease-antiprotease imbalance hypothesis has been suggested for the cause of emphysema, it is still appealing, but it does not explain the considerable variation in the clinical expressions of emphysema. However, there are many recent research findings to support the imbalance hypothesis as will be shown in this review. Although limited, there might be openings for the treatment of the disease.

The ascent of the blessed: regulatory issues on health effects and health claims for probiotics in Europe and the rest of the world.

The outcome of the first series of health claim applications for probiotics in Europe as evaluated by the European Food Safety Authority (EFSA) has, up to 2013 almost completely yielded negative results. All recent applications also have been rejected, including the latest on prevention of mastitis in breastfeeding mothers. In other developed countries, such as Switzerland, Japan and Canada, the health effects of probiotics, for which scientific evidence has been provided, can be communicated to potential consumers. The number of clinical trials with probiotics over recent years shows a trend to level off or even decline. At the same time, clinical research into the role of (gut) microbiota in a wide variety of diseases and conditions is booming. Ultimately, this may offer new indications for gut microbiota management by probiotics, prebiotics or other food supplements.

The interrelationship between markers of inflammation and oxidative stress in chronic obstructive pulmonary disease: modulation by inhaled steroids and antioxidant.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is accompanied by both airway and systemic inflammation and by oxidative stress. This study aimed to characterise the relationship between oxidative stress and inflammatory components in induced sputum and blood. MATERIAL & METHODS: We studied blood and sputum samples from stable COPD patients (mean FEV1 60.5+/-7.5% predicted) at baseline (no treatment) and after 10 weeks treatment with either inhaled steroid, fluticasone propionate (FP) (1000 microg/d) or 10 weeks treatment with N-acetylcysteine (600mg/d) (NAC). We assessed the inflammatory markers (IL-8, ECP, sICAM-1, NE) in sputum and serum and we compared them with blood markers of oxidative stress (SOD, GPx, TEAC, albumin, vitamin E and A). RESULTS: At baseline blood sICAM-1 correlated with IL-8 levels (P<0.01, r = 0.62) and negatively with GPx (P<0.01, r = -0.63) and with TEAC (P<0.05, r = -0.53). TEAC correlated positively with GPx (P<0.01, r = 0.70). Correlation between sICAM and IL-8 disappeared after NAC treatment. The correlation between sICAM and GPx disappeared after FP treatment. The correlation between TEAC and GPx was maintained after both NAC and FP. CONCLUSIONS: The relationship between markers of inflammation, adhesion and antioxidant capacity is significantly modulated by treatment with N-acetylcysteine or inhaled corticosteroids.

Pterins inhibit nitric oxide synthase activity in rat alveolar macrophages.

1. The synthesis of nitrite and citrulline from L-arginine by immune-stimulated rat alveolar macrophages and the modulation of this synthesis were studied. 2,4-Diamino-6-hydroxypyrimidine (DAHP), 6R-5,6,7,8-tetrahydro-L-biopterin (BH4) and L-sepiapterin were potent inhibitors of the recombinant interferon-gamma induced production of nitrogen oxides in intact cultured cells with I50 values for BH4 and L-sepiapterin of approximately 10 microM. They were equally effective in inhibiting the induced production of citrulline. This inhibitory effect was concentration-dependent for all three modulators investigated. 2. The inhibitory effects were not dependent on incubation times of either 24 or 48 h, on the immune-stimulus used (lipopolysaccharide, interferon-gamma), or whether these stimuli were added during or after the induction period. 3. Pterin-6-carboxylic acid (PCA), which cannot be converted into BH4, and methotrexate (MTX), which inhibits dihydrofolatereductase but not de novo biosynthesis of BH4, did not change the production of nitrite. 4. The data indicate that DAHP, an inhibitor of the de novo biosynthesis of the co-factor BH4, blocks the nitric oxide synthase activity in intact cells. Since the pterins BH4 and L-sepiapterin blocked the L-arginine dependent production of nitrite and citrulline, the activity of nitric oxide synthase in phagocytic cells may be regulated by metabolic endproducts of the de novo biosynthesis of BH4.

Soybean trypsin inhibitor and beta-amylase induce alveolar macrophages to release nitrogen oxides.

Rat alveolar macrophages incubated with soybean trypsin inhibitor and beta-amylase produced nitrite in a dose- and time-dependent manner. This production depends on the presence of L-arginine (L-arg) in the culture medium. The precursor of this nitrite was demonstrated as being nitric oxide by bleaching ferredoxin at 410 nm when added to the culture medium. NG-Monomethyl-L-arginine and the tetrahydrobiopterin biosynthesis inhibitor 2,4-diamino-6-hydroxypyrimidine inhibited the release of nitrite in a dose-dependent manner. Dexamethasone was able to modulate this release. These data indicate that alveolar macrophages are capable of secreting L-arg-derived nitrogen oxides when stimulated with certain alimentary proteins.

N-acetylcysteine pretreatment of cardiac surgery patients influences plasma neutrophil elastase and neutrophil influx in bronchoalveolar lavage fluid.

OBJECTIVE: Study of leukocyte activation and release of toxic mediators during extracorporeal circulation (ECC). ECC can be used to study the potential protective effect of a pharmacon against neutrophil-mediated lung injury. Clinical studies have indicated that N-acetylcysteine (NAC) may improve systemic oxygenation and reduce the need for ventilatory support when given to patients with acute lung injury. DESIGN: Cardiac surgery patients were pretreated with high-dose NAC in order to assess the potential role of NAC to interfere with neutrophil-mediated inflammation and lung injury. PATIENTS: 18 patients who underwent ECC: group 1 (n = 8) no premedication (only placebo); group 2 (n = 10) NAC (72 mg/kg i.v. as a bolus, later 72 mg/kg over 12 h). MEASUREMENTS AND RESULTS: In group 2, the partial pressure of oxygen in arterial blood/fractional inspired oxygen 4 h after surgery was significantly higher than in group 1 (213 +/- 31 vs 123 +/- 22; p = 0.044). NAC pretreatment prevented an increase in plasma neutrophil elastase activity (18.9 +/- 6.9 vs 49.9 +/- 5.6 ng/ml in group 1 at the end of ECC; p = 0.027). Release of myeloperoxidase (MPO) was not affected (group 1:1105 +/- 225 ng/ml vs group 2:1127 +/- 81 at the end of ECC; p = 0.63). At the end of ECC, total antigenic human neutrophil elastase (group 1:671 +/- 72 ng/ml vs group 2:579 +/- 134; p = 0.37) and complex formation between elastase and alpha 1-proteinase inhibitor were no different in the two groups. There were no significant difference in cellular composition and mediators in the lavage fluid, although values for total number of neutrophils, elastase, MPO and interleukin-8 were lower in group 2. CONCLUSION: Pretreatment with NAC may prevent lung injury by diminishing elastase activity. Since the release of mediators, especially MPO, is not affected, this diminished activity of elastase may be achieved by enhanced inactivation by antiproteases after initial treatment.

Angiotensin-converting enzyme activity in serum and bronchoalveolar lavage fluid after damage to the alveolo-capillary barrier in the human lung.

OBJECTIVE: Angiotensin-converting enzyme (ACE) is considered as a possible marker for endothelial cell damage in serum or bronchoalveolar lavage fluid. This hypothesis was tested during cardiac surgery and during the adult respiratory distress syndrome. DESIGN: We used patients with an expected different degree of endothelial cell damage. ACE levels in serum and bronchoalveolar lavage fluid were compared with indirect markers of alveolo-capillary barrier integrity. SETTING: Interdisciplinary team in a university hospital. METHODS: 13 Cardiac surgery patients received no glucocorticoids and 13 others received 2 g methylprednisolone before extracorporeal circulation. Thirteen patients were used as controls and 15 patients had nonseptic adult respiratory distress syndrome. All underwent bronchoalveolar lavage for ACE determination. RESULTS: At different times during surgery serum angiotensin-converting enzyme levels were not significantly different between the two groups. In post-operative bronchoalveolar lavage fluid, angiotensin-converting enzyme levels were significantly higher in patients who received corticoids (27.8 +/- 1.7 U/l, mean +/- SEM), compared to patients without corticoids (19.8 +/- 1.4 U/l), control patients (18.2 +/- 1.3 U/l) or patients with full blown non-septic adult respiratory distress syndrome (18.8 +/- 1.1 U/l). There were no correlations between lavage angiotensin-converting enzyme and other parameters for alveolo-capillary membrane integrity in the lavage fluid such as the number of neutrophil cells, albumin or protein concentration, and between lavage angiotensin-converting enzyme and PaO2/FIO2 ratio during lavage. CONCLUSION: Angiotensin-converting enzyme activity in serum or bronchoalveolar lavage fluid does not reflect damage of endothelial cells or damage of alveolocapillary integrity in acute pulmonary disease.

L-arginine-dependent production of nitrogen oxides by rat pulmonary macrophages.

Rat alveolar and pleural macrophages incubated with lipopolysaccharide, opsonized zymosan or recombinant interferon-gamma, but not with recombinant tumor necrosis factor-alpha, produced nitrite dose and time dependently. This production depends on the presence and amount of L-arginine in the culture medium. The precursor of the nitrite was demonstrated as being nitric oxide, by bleaching of ferredoxin at 410 nm when added to the culture medium. Addition of NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, and cycloheximide, a protein synthesis inhibitor, to the medium resulted in a decrease of nitrite production. Glucocorticoids were able to block the induction of nitrite production in alveolar macrophages. These data indicate that pulmonary macrophages are capable of secreting L-arginine-derived nitrogen oxides.

Synergism between interleukin-1 beta and interferon-gamma, an inducer of nitric oxide synthase, in rat lung fibroblasts.

An L-arginine-dependent pathway, by which L-arginine is metabolised to citrulline and nitrogen oxides, has been recently identified in some cell types. In cultured rat lung fibroblasts the presence of L-arginine was necessary for the production of nitrite to be induced by rat recombinant interferon-gamma and synergistically enhanced by lipopolysaccharide and interleukin-1 beta. Lipopolysaccharide and interleukin-1 beta did not induce nitrite biosynthesis by themselves. Biosynthesis was apparently dependent on tetrahydrobiopterin, since it could be blocked by diaminohydroxypyrimidine, an inhibitor of tetrahydrobiopterin synthesis. Dexamethasone blocked nitrite production by a receptor-mediated mechanism. These data indicate that rat lung fibroblasts express an L-arginine-dependent nitric oxide synthase which can be induced by some mediators of inflammation.

Plasma protein leakage and local secretion of proteins assessed in sputum in asthma and COPD. The effect of inhaled corticosteroids.

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation with cell infiltration, increased plasma exudation and abnormal local secretion of proteins. We have analysed whether sputum differs in this respect between asthma (n = 9) and COPD (n = 9), and whether inflammatory markers in sputum are affected by treatment. In non-smoking asthma patients there was more plasma protein leakage, based on the relative coefficient of excretion Q alpha 2macroglobulin/QIgG (P = 0.03). There was less local secretion of sIgA and lactoferrin than in COPD (P < 0.05). Tryptase was slightly higher in sputum from asthma than from COPD (P < 0.05), whereas eosinophil cationic protein and myeloperoxidase were similar. After treatment with glucocorticosteroids, there was a reduction in the Q alpha 2macroglobulin/Qalbumin (P < 0.015), but no effect was seen on the levels of products from local cells. We conclude that sputum analysis is useful to study the local inflammatory process in asthma and COPD.

The isolation of inflammatory cells from normal human lung tissue.

An isolation procedure to obtain inflammatory cells from normal human lung tissue is described, consisting of four steps: an enzymatic digestion, centrifugal elutriation, gradient centrifugation and additional affinity chromatography (to obtain mast cells). The dispersed cell population consisted mainly of macrophages, pneumocytes type II, neutrophils and lymphocytes. Further separation by elutriation yielded pure macrophages and pure lymphocytes. The heterogeneous cell mixtures obtained at elutriation were separated by gradient centrifugation. This method yielded pure macrophages, lymphocytes and pneumocytes, and fractions containing eosinophils, mast cells or neutrophils were enriched considerably. Additional affinity chromatography with anti-human-IgE yielded a mast-cell fraction of 75%.

Differences in responses upon corticosteroid therapy between smoking and non-smoking patients with COPD.

Inhaled corticosteroids have a high level of topical anti-inflammatory activity. However, in patients with COPD these drugs have been reported to exert limited effects. A reduction in histone deacetylase (HDAC) activity is suggested to prevent the anti-inflammatory action of corticosteroids. Cigarette smoke is known to reduce HDAC expression. The aim of this study is to compare the outcome of corticosteroid therapy in both smoking and non-smoking COPD patients. Twenty-three smoking patients and 18 ex-smoking patients with COPD were treated with inhaled corticosteroids for a period of 2 months. Blood and induced sputum samples were collected before and after treatment. Values of FEV(1) %-predicted did not change upon the therapy, but there was a trend to improve in the ex-smokers (63.1 -> 64.8%-pred.), compared with a decrease in the smokers (63.3 -> 61.6%-pred.). The levels of the pro-inflammatory cytokine IL-8 increased in the group of smokers from 379 +/-78 to 526 +/-118 ng/ml. Although not significant, a slight decrease from 382 +/-70 to 342 +/-62 ng/ml was observed in the group of ex-smokers. The neutrophil related elastase activity showed similar effects after steroid treatment, it went up from 36.4 +/-12.0 to 113.5 +/-9.7 nmol/l in smokers, and decreased from 346.2 +/-72.1 to 131.1 +/-6.5 nmol/l in ex-smokers with COPD. These results support the evidence that inhaled corticosteroids have no anti-inflammatory effects in COPD patients, but only when these patients are still smoking. Smoking cessation seems the best therapy for COPD patients.

Influence of N-acetylcysteine on ICAM-1 expression and IL-8 release from endothelial and epithelial cells.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. The initial step in the inflammatory process is overexpression of adhesion molecules, which leads to excessive transmigration of neutrophils. One of these adhesion molecules is ICAM-1 which is elevated in COPD patients. In this study we evaluated the influence of N-acetylcysteine (NAC) (0.01 mM-30 mM) on the cytokine-induced (TNF-alpha/IL-1 beta) expression of the ICAM-1 adhesion molecule and on IL-8 release in endothelial (ECV-304) and bronchial epithelial (H292) cell lines. The methodology used consisted of immunochemistry for the assessment of surface ICAM-1 and ELISA method for that of soluble ICAM-1 and IL-8. NAC inhibited the TNF-alpha/IL-1 beta-stimulated ICAM-1 expression and IL-8 release from both cell lines in a concentration dependent manner. The most effective concentrations were 30 mM and 20 mM (99 and 90% inhibition respectively, P<0.01). We conclude that NAC is an effective inhibitor of TNF-alpha/IL-1 beta- stimulated ICAM-1 and IL-8 release in endothelial and epithelial cells. This fact highlights the anti-inflammatory potential of NAC in COPD.

New developments in the treatment of COPD: comparing the effects of inhaled corticosteroids and N-acetylcysteine.

Inhaled corticosteroids (ICS) are widely used for the treatment of COPD despite of controversial statements concerning their efficacy. The use of N-acetylcysteine (NAC), a mucolytic drug with antioxidant properties, is less clear, but it may counteract the oxidant-antioxidant imbalance in COPD. The aim of this study was to evaluate whether treatment of COPD patients with ICS or NAC is able to improve inflammatory indices and to enhance lung function. ICS treatment enhanced protective markers for oxidative stress such as glutathione peroxidase (GPx) (51.2 +/-5.8 vs. 62.2 +/-8.6 U/g Hb, P<0.02) and trolox-equivalent antioxidant capacity (TEAC) (1.44 +/-0.05 vs. 1.52 +/-0.06 mM, P<0.05). NAC decreased sputum eosinophil cationic protein (318 +/-73 vs. 163 +/-30 ng/ml, P<0.01) and sputum IL-8 (429 +/-80 vs. 347 +/-70 ng/ml, P<0.05). The increased antioxidant capacity prevented an up-regulation of adhesion molecules, since the levels of intracellular adhesion molecule 1 (ICAM-1) correlated negatively with GPx (P<0.0001) and TEAC (P<0.0001). On the other hand, expression of adhesion molecules was promoted by inflammation, reflected by a positive correlation between the levels of IL-8 and ICAM-1 (P<0.0001). The effects of treatment on lung function were only reflected in the FEV(1) values. The absolute value of FEV(1), both before and after salbutamol inhalation, increased from 1690 +/-98 to 1764 +/-110 ml, and 1818 +/-106 to 1906 +/-116 ml, respectively, after ICS (P<0.05) . Ten weeks after treatment, FEV(1) values dropped to 1716 +/-120 ml post-salbutamol (P<0.05). When followed by treatment with NAC, these values decreased even further to 1666 +/-84 ml. These results suggest that ICS improved lung function in COPD patients with moderate airflow obstruction, beside a minor improvement in the oxidant-antioxidant imbalance leading to a lesser expression of ICAM-1. Treatment with NAC decreased some inflammatory parameters and had indirectly an inhibitory effect on the expression of adhesion molecules.

Effects of homogenization of induced sputum by dithiothreitol on polymorphonuclear cells.

Induced sputum represents a useful and non-invasive tool to isolate different cells from the airways. Complete homogenization of sputum is important for dispersion of cells and is usually achieved by use of dithiothreitol (DTT). However, it is not known if DTT will influence the viability and functionality of cells obtained by induced sputum. In the present study, induced sputum was processed by DTT or by PBS treatment. The obtained neutrophils were compared with neutrophils obtained from peripheral blood and from bronchoalveolar lavage fluid (BAL). These isolated neutrophils were treated in a similar way as the sputum neutrophils with DTT or PBS. All isolated cells were used for chemiluminescence tests and for the measurement of elastase and myeloperoxidase release after stimulation with fMLP. The results showed that the maximum chemiluminescence response was always significantly lower after DTT treatment: blood, 16.68 +/-1.89 vs. 2.62 +/-0.43 mV, P<0.0001; sputum, 2.96 +/-0.30 vs. 1.09 +/-0.01 mV, P<0.01; BAL, 25.47 +/-0.88 vs. 8.22+/-0.20 mV, P<0.0001. Both spontaneous and fMLP-induced release of elastase and myeloperoxidase (MPO) was in most cases enhanced after DTT-treatment (P-values range from 0.24 to <0.01). We conclude that the use of DTT to homogenize sputum for dispersion of cells is harmful to cell functions and these cells are hampered for the evaluation of their normal functional characteristics.

Mediator release from human lung mast cell subtypes in chronic bronchitis and emphysema.

Human lung mast cells were obtained from pulmonary tissue of normal individuals and patients with chronic bronchitis or emphysema by enzymatic dispersion. Based on their density two mast cell subtypes, a formalin-sensitive (FS) and a formalin-insensitive (FI) cell type, could be separated. Although differences in anti-IgE-induced histamine release could be demonstrated for the mast cell subtypes of normal individuals, these experiments could not be performed for both mast cell subtypes from both patient groups. LTC4 and PGD2 release could be demonstrated for the FS- and FI-mast cell respectively. The release of PGD2 from FI-mast cells of patients with chronic bronchitis was enhanced as compared with normal subjects.

[Mast cells and basophilic granulocytes: their significance in allergic diseases]. / Mastzellen und basophile Granulozyten: Ihre Bedeutung bei allergischen Erkrankungen.

Mast cells, and to a lesser extent basophilic granulocytes, have long determined models in the field of pathogenetic mechanisms in allergic disease. Unfortunately this has not resulted in adequate therapy for these diseases, and other cell types such as eosinophils and lymphocytes are now thought to influence allergic mechanisms too. However, knowledge of mast cells has increased to such an extent over the last decade that there may be a "revival" of their importance for allergic disease. In many organs in the body heterogeneity of mast cell morphology has been demonstrated. These different types of mast cells may also release different types of mediators. Moreover, mast cells have recently been shown to be capable of releasing cytokines, which may have a very important bearing on the pathogenesis of allergic diseases. Because mast cells and basophils are able to release a wide variety of mediators which may be responsible for many different physiological responses, they remain important cell types for allergy.