Modification of the proteinase/anti-proteinase balance in the respiratory tract of Sprague-Dawley rats after single intratracheal instillation of benzo[A]pyrene-coated onto Fe(2)O(3) particles.

Available data suggest that repeated concurrent exposure to haematite (Fe(2)O(3)) and benzo[A]pyrene (B[A]P) results in a decreased latency and an increased incidence of lung tumours in rodents compared to exposure to B[A]P alone. Moreover, the reactive oxygen species (ROS) formed by the lung cells themselves and/or by activated inflammatory cells may possibly contribute to the development of pulmonary disorders such as cancer formation. In order to investigate the precise role of iron in the injury induced by B[A]P-coated onto Fe(2)O(3) particles, we tend to address the hypothesis that Fe(2)O(3) and B[A]P, alone or in association, can induce oxidative stress conditions (malondialdehyde) and/or inflammatory reactions (interleukin-6) and thereby disrupt the proteinase/anti-proteinase balance (cathepsins B and L, polynuclear neutrophil (PNN) elastase, alpha-1 proteinase inhibitor (alpha(1)PI) and its inhibitory capacity) in the rat respiratory tract. Thus, Fe(2)O(3) or B[A]P-coated onto Fe(2)O(3) particles produce oxidative stress conditions through not only iron-catalysed oxidative reactions but also inflammatory processes. However, B[A]P initiates only inflammatory responses. These pollutants generate increased levels of proteases and decrease the concentrations of free alpha(1)PI. There is also a clear relationship between the partial inactivation of alpha(1)PI and the occurrence of ROS after exposure to Fe(2)O(3), alone or as a carrier of B[A]P. Hence, the proteinase/anti-proteinase balance might be more disrupted by Fe(2)O(3) or B[A]P-coated onto Fe(2)O(3) particles than by B[A]P alone. These results suggest a mechanism that can explain why B[A]P-coated onto Fe(2)O(3) particles are more injurious than B[A]P alone.

Effects of creatine monohydrate ingestion in sedentary and weight-trained older adults.

To investigate the effects of an oral creatine supplementation in older adults, 32 elderly subjects (67-80 years; 16 females, 16 males) were randomly assigned to four equivalent subgroups (control-creatine; control-placebo; trained-creatine; trained-placebo) based on whether or not they took part in an 8-week strength training programme and an 8-week oral creatine monohydrate creatine supplementation programme. The strength training programme consisted of three sets of eight repetitions at 80% of one-repetition maximum, for leg press, leg extension and chest press, 3 days a week. The 52-day supplementation programme consisted of 20 g of creatine monohydrate (or glucose) and 8 g of glucose per day for the initial 5 days followed by 3 g of creatine monohydrate (or glucose), and 2 g of glucose per day. Prior to and after the training and supplementation periods, body mass, body fat, lower limb muscular volume, 1-, 12-repetitions maxima and isometric intermittent endurance tests for leg press, leg extension and chest press were determined. In all groups, no significant changes in anthropometric parameters were observed. For all movements, the increases in 1- and 12-repetitions maxima were greater (P < 0.02) in trained than control subjects. No significant interactions (supplementation/training/time) were observed for the 1-, 12-repetitions maxima, and the isometric intermittent endurance, whatever the movement considered. We conclude that oral creatine supplementation does not provide additional benefits for body composition, maximal dynamical strength, and dynamical and isometric endurances of healthy elderly subjects, whether or not it is associated with an effective strength training.

Oncostatin M is a potent stimulator of alpha1-antitrypsin secretion in lung epithelial cells: modulation by transforming growth factor-beta and interferon-gamma.

alpha1-Antitrypsin (alpha1-AT) plays a key role in lung homeostasis. Although the hepatocyte is considered as the primary source of alpha1-AT, we have previously demonstrated that rat alveolar epithelial type II cells as well as the human A549 cell line synthesize alpha1-AT, suggesting its local production within the lung. In the present study, we showed that oncostatin M, as opposed to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or IL-6, is a potent stimulator of alpha1-AT synthesis in the human A549 cell line. The oncostatin M-induced alpha1-AT secretion is modulated by interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) at both the protein and mRNA levels. IFN-gamma decreases oncostatin M-induced alpha1-AT secretion. By contrast, TGF-beta in combination with oncostatin M induces a dramatic and synergistic upregulation that is not observed in the HepG2 hepatocyte cell line. Our results suggest that during an inflammatory process, alveolar epithelial cells may contribute to the antiprotease defense within the lung.

Compartmentalized IL-8 and elastase release within the human lung in unilateral pneumonia.

Because interleukin 8 (IL-8) is a potent neutrophil chemotactic and activating cytokine, we investigated IL-8 production in relation to neutrophil migration and elastase release in the human lung during unilateral community-acquired pneumonia (CAP). In 17 patients, the local response in the involved lung was compared with that in the contralateral, noninvolved lung, and with the systemic response. Eight healthy volunteers served as controls. IL-8, total neutrophil elastase (NE), free elastase activity, alpha 1-antitrypsin (alpha 1-AT), and total leukocyte and neutrophil counts were evaluated in bronchoalveolar lavage fluids (BALF). Mean IL-8 concentrations in BALF from the involved lungs of the patients were significantly greater than those in BALF from the noninvolved lung or from controls (p < or = 0.001). By contrast, the serum IL-8 concentration was not different in patients and in controls. Total NE and alpha 1-AT concentrations were increased in BALF from the involved lung as compared with the noninvolved lung or controls (p < or = 0.001). The elastase-inhibitory capacity of alpha 1-AT in BALF was impaired in the involved lung of seven of the 14 patients as compared with the controls, leading to free elastase activity in the involved lung of all patients with CAP. Plasma total NE concentrations were significantly greater in the CAP patients than in the controls. IL-8 concentrations in BALF correlated positively with total leukocyte counts, absolute numbers and percentages of neutrophils, total NE concentrations, and free elastase activity. Our results suggest that during unilateral CAP, locally produced IL-8 may trigger neutrophil accumulation and activation, thus contributing to a local elastase/antielastase imbalance within the site of infection.

Toxicity of ferric oxide and benzo[a]pyrene alone or in combination in respiratory tract of Sprague Dawley rats.

The association of small quantities of ferric oxide with Benzo[a]Pyrene (BaP) appears to increase in vivo the toxic effect of BaP. The effect of Fe2O3 may be mediated by the recruitment of alveolar macrophages. These cells would contribute to the production of toxic and carcinogenic BaP metabolites and would stimulate development of tumors by producing cellular mediators of inflammation. In order to understand the mechanism of the synergic effect, we have instillated male Sprague Dawley rats 3 weeks of age with a single dose: Fe2O3 (3 mg) or BaP (3 mg)/combination Fe2O3-BaP (3 mg-3 mg) in 200 microliters of physiological saline solution. Control group of identical size (treated with physiological saline solutions and untreated) were used for this study. Animals were sacrificed 48 hours after instillation and a bronchoalveolar lavage (BAL) was performed. With each BAL we have obtained protein measurement, cells were stained with May-Grünwald-Giemsa method and slides were studied with polarised light. The malonaldehyde (MDA) was measured by High Performance Liquid Chromatography. The PMN elastase determination was performed by IMAC (immuno-activation) technology. An automated kinetic method for measuring cathepsins B and L was carried out using a fluorogenic substrate: Z-Phe-Arg-AMC, a specific inhibitor E64 and AMC as an internal standard. After a quantitative Dot-Blot of the samples of BAL, an immunodetection of alpha(1)-antitrypsin (alpha(1)AT) was performed. The inhibitory capacity of alpha(1)AT was determined by an enzymatic reaction with porcine pancreatic elastase. We have observed an increased MDA level for rats intoxicated with Fe2O3 (123%), BaP (31%) and Fe2O3 + BaP (56%). The levels of PMN elastase and cathepsin B and L were increased: Fe2O3 (51-58%), BaP (52-27%). This effect was not seen for rats intoxicated by Fe2O3 + BaP. The free alpha(1)AT was decreased with the three toxics (Fe2O3: 44%--BaP: 42%--Fe2O3: 41%). The inhibitory capacity of alpha(1)AT was lower in groups of rats instilled with toxics.

Interleukin-8 and neutrophils in systemic sclerosis with lung involvement.

We investigated the mechanisms and consequences of neutrophil accumulation in the airspace in 11 patients with systemic sclerosis (SSc) and interstitial lung disease. Seven normal subjects served as controls. We measured total neutrophil elastase burden, elastase activity, and alpha-1-antitrypsin (alpha 1AT) in bronchoalveolar lavage (BAL) fluid and we evaluated the in vitro interleukin-8 (IL-8, a potent chemoattractant for neutrophils) secretion by alveolar macrophages (AM). A mild neutrophil alveolitis was observed in patients when compared with control subjects. Total BAL elastase burden was higher in patients than in control subjects and correlated positively with the percentage of neutrophils in BAL. BAL elastase activity was undetectable in control subjects, but it was detected in all patients but one (mean: 257 +/- 87 mU/L). Spontaneous IL-8 secretion by AM was higher in patients with SSc than in control subjects (518 +/- 115 versus 228 +/- 65 ng/ml, p = 0.04) and positively correlated with the percentage of neutrophils in BAL (r = 0.505). We conclude that (1) the neutrophil could participate in the pathogenesis of SSC lung disease through the release of elastase; (2) the AM could contribute to the influx of neutrophils in the alveolus through the release of IL-8.

Secretion of alpha 1-antitrypsin by alveolar epithelial cells.

We have investigated the ability of alveolar epithelial cells (human A549 cell line and rat type-II pneumocytes) to produce alpha 1-antitrypsin (AAT). Northern blot analysis demonstrated the presence of an AAT-specific mRNA transcript in A549 cells. Unstimulated A549 cells secreted immunoreactive AAT at a rate of 0.51 +/- 0.04 ng/10(6) cells/h, with a modified glycosylation compared to serum AAT. AAT formed a complex with neutrophil elastase. Rat type-II pneumocytes secreted immunoreactive AAT. Our results suggest that alveolar epithelial cells could participate in antiprotease defense within the lung through local AAT production.

Comparison of enhanced chemiluminescence and colorimetric techniques for the immuno-detection of alpha 1-antitrypsin.

In order to detect, characterize and quantify blotted proteins, such as human alpha 1-antitrypsin (AAT), there is a need for a specific, extremely sensitive, non-radioactive and uniform revelation system applicable to diluted biological fluids and to culture supernatants of cells isolated from such fluids. We compared two immunochemical revelation systems, enhanced chemiluminescence (ECL) and colorimetric procedures, applied to human ATT, after determining their optimal conditions of performance. ECL was the most sensitive method (down to 50 pg blotted AAT), but could not be used to quantify AAT. In contrast, the colorimetric method enables quantification of blotted AAT, either simply dotted or transferred after SDS-polyacrylamide gel electrophoresis, but is not as sensitive as ECL. Using these two complementary procedures, we have been able to detect AAT in the culture supernatant of a monocytic cell line (THP-1), to characterize the different forms of AAT present in the culture supernatant of blood monocytes and to quantify both.

Changes in the glycoforms of rat alpha-1-acid glycoprotein during experimental polyarthritis.

We analyzed the carbohydrate moiety of purified alpha-1-acid glycoprotein (AGP) from Lewis adult male rats that were healthy (AGPh) or had experimental polyarthritis (AGPi). Sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after N-glycanase treatment showed that AGPi had a slightly lower molecular mass (43 kDa vs. 45 kDa for AGPh) due to a lesser carbohydrate content. Carbohydrate analysis of purified AGP showed a slight decrease in the sialyl and galactosyl molar ratio in polyarthritis. However, the same difference in AGPh and AGPi (i.e. 0.6 residue) between the sialyl and galactosyl molar ratio indicated more than one sialyl residue per complex-type branch. Affinity for concanavalin A (ConA) of the whole glycoprotein and released oligosaccharides showed a progression during polyarthritis towards more reactive glycoforms or more ConA-bound oligosaccharides. Anion-exchange HPLC of the ConA-fractionated oligosaccharides corroborated the decreased sialylation in polyarthritis. Taken together, these results suggest a fall in branched and sialylated oligosaccharides during experimental polyarthritis. These structural changes might be related to an increase in Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase activity described elsewhere in inflammatory states.