Implementation of an integrated instrument control and data management system for point of care blood gas testing.

Critical care testing in a point of care (POC) setting can be very demanding for the laboratory. Lack of continuous monitoring of the normal functioning of POC instruments leads to late response to technical problems, and frequently results in more comprehensive interventions and reduced instrument availability. As most POC instruments lack adequate data transfer capabilities, manual data entry into the medical records with a high error rate is a component of POC programs. To reduce this data handling drawback of POC testing, our hospital opted for the replacement of the existing analysers with network-ready, expandable analysers (ABL700), linked to an integrated management system (RADIANCE). This new set-up enabled us to have continuous instrument control and maximal data management. The implementation of the integrated management system was well accepted by the operators. The network connectivity has led to real time technical support while reducing the workload by automating data collection.

Modulation of cell adhesion molecules on human endothelial cells by eosinophil-derived mediators.

Mechanisms that allow a selective eosinophil emigration in different eosinophilic lung diseases remain poorly understood. In this study, we tested the hypothesis that eosinophils might participate in their own recruitment, particularly through adhesion molecule expression on human endothelial cells (EC). Blood eosinophils from donors with blood eosinophilia were purified and maintained in culture medium for 1 and 18 h. The expression of ICAM-1, E-selectin, and VCAM-1 on human umbilical vein endothelial cells (HUVEC) was evaluated by ELISA and flow cytometry analysis after addition of eosinophil supernatants. Eosinophil supernatants collected after 1 h induced a weak increase of CAM expression on HUVEC. In contrast, supernatants from eosinophils cultured for 18 h considerably amplified ICAM-1, E-selectin, and VCAM-1 expression on the surface of EC. These levels of CAM expression (in optical density determined by ELISA) were about two- or threefold more important than those obtained with eosinophil supernatants collected after a 1-h culture. The characterization of the implicated molecules showed that anti-IL-1beta antibodies significantly inhibited ICAM-1, E-selectin, and VCAM-1 expression, whereas anti-TNF-alpha antibodies only induced a moderate inhibition. Our data support the hypothesis that eosinophils, through the release of at least IL-1beta and TNF-alpha, might participate in the amplification of the inflammatory reaction by activating the vascular endothelium.

Functional and phenotypic differences of monocyte-derived dendritic cells from allergic and nonallergic patients.

BACKGROUND: Several studies suggest a role for dendritic cell in the pathogenesis of allergic disease. OBJECTIVE: The purpose of this study was to compare function and phenotype of monocyte-derived dendritic cells (MoDC) from allergic asthmatic patients and healthy control subjects. METHODS: MoDCs were developed by incubating adherent monocytes for 5 days with IL-4 and granulocyte-macrophage colony-stimulating factor. Phenotype was assessed with flow cytometry, and the antigen-presenting function was assessed with the allogeneic mixed leukocyte reaction and an autologous specific antigen presentation. RESULTS: The morphology of the MoDCs was characteristic for immature dendritic cells. MoDCs from allergic asthmatic patients showed phenotypic differences in the expression of HLA-DR, CD11b, and the high-affinity receptor for IgE. A clearly enhanced accessory potential of MoDCs from atopic asthmatic patients in the mixed leukocyte reaction was also shown. Moreover, house dust mite-specific T-cell proliferation was increased. CONCLUSION: This study suggests the involvement of dendritic cells in the pathogenesis of atopic asthma by an increased immunostimulatory capacity of MoDCs.

MCP-1 secretion in lung from nonsmoking patients with coal worker's pneumoconiosis.

Exposure to coal dust leads to the development of coal worker's pneumoconiosis (CWP), a disease associated with an accumulation of macrophages in the lower respiratory tract. Mechanisms controlling monocyte recruitment are still poorly understood. Since monocyte chemoattractant protein-1 (MCP-1) is recognized as a potent chemotactic factor for blood monocytes, we analysed the presence of MCP-1 in the pulmonary compartment of patients with CWP. Bronchoalveolar lavage fluid (BALF) from 16 nonsmoking control subjects and 27 nonsmoking CWP patients (16 with simple pneumoconiosis (SP) and 11 with progressive massive fibrosis (PMF)) was analysed. Alveolar macrophages (AMs) were purified by adherence and BALF was concentrated tenfold by lyophilization. MCP-1 was measured in BALF and in 3 h AM supernatants using a sandwich enzyme-linked immunosorbent assay (ELISA). The localization of MCP-1 in lung tissue was determined by immunohistochemistry on tissue sections from three patients with CWP and two control subjects. MCP-1 levels were significantly higher in concentrated BALF from patients with SP or PMF (median 370 and 555 pg x mL-1, respectively) than in those from control subjects (median 11 pg x mL-1) (p<0.001). Released MCP-1 in AM supernatants was enhanced in patients with CWP (median 83 pg x mL-1) but compared to controls (median 41 pg x mL-1) this level did not reach significance. Although significantly increased, AM counts in BALF from patients with CWP did not correlate with MCP-1 levels. MCP-1 levels in BALF correlated with MCP-1 levels in AM supernatants (p=0.47; p<0.02). In control lung specimens, MCP-1 was expressed by a few AMs, type II pneumocytes and perivascular smooth muscle cells. CWP sections were characterized by an increased number of AMs and mainly by the presence of fibroblasts (in the myogenic area of fibrotic lesions) and hyperplastic type II pneumocytes, which were strongly immunostained for MCP-1. Our data demonstrate that: 1) patients with coal worker's pneumoconiosis have a marked pulmonary overproduction of monocyte chemoattractant protein-1; and 2) in addition to alveolar macrophages, fibroblasts (probably myofibroblasts) and hyperplastic type II pneumocytes may also be responsible for this increased level of monocyte chemoattractant protein-1 in coal worker's pneumoconiosis.

Expression of leucocyte-endothelial adhesion molecules is limited to intercellular adhesion molecule-1 (ICAM-1) in the lung of pneumoconiotic patients: role of tumour necrosis factor-alpha (TNF-alpha).

Coal workers' pneumoconiosis (CWP) is characterized by a chronic inflammatory lung reaction associated with macrophage accumulation in alveolar spaces. In this study, we investigated in CWP the implication of adhesion molecules such as E-selectin, ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) and the role of TNF-alpha which is one of the cytokines inducing their expression. Adhesion molecule expression was analysed by immunohistochemistry on lung biopsies from patients with CWP and from healthy subjects. In parallel, soluble adhesion molecules were detected in bronchoalveolar lavage fluids (BALF) from patients by specific ELISA. The involvement of TNF in the induction of these adhesion molecules was measured (i) by immunohistochemistry on sections from lung fragments, and (ii) by evaluating in vitro the expression of adhesion molecules on endothelial cells and on alveolar epithelial cells in the presence of alveolar macrophage supernatants. In control subjects, a weak staining of ICAM-1 was detected only in alveolar walls, while E-selectin and VCAM-1 were undetectable. In pneumoconiotic patients, ICAM-1 was expressed at a high level by endothelium, by alveolar and bronchial epithelial cells and by alveolar macrophages. E-selectin and VCAM-1 expression remained undetectable. Measurement of soluble adhesion molecule showed that only the concentration of sICAM-1 was significantly increased in BALF from patients with CWP compared with controls. The involvement of TNF in this ICAM-1 expression was shown by the in vitro effect of alveolar macrophage supernatants on adhesion molecule expression by endothelial cells and epithelial cells (this effect was neutralized by anti-TNF antibodies) and by the increased production of TNF in the lung of pneumoconiotic patients. These data provide evidence for the involvement of ICAM-1, induced at least in part by alveolar macrophage-derived TNF, in the development of the inflammatory reaction in CWP.

Ozone stimulates synthesis of inflammatory cytokines by alveolar macrophages in vitro.

Ozone (O3) is one of the major irritant oxidant gases in photochemical smog. In the present study, the in vitro effect of low concentrations of O3 (0.1 to 1 ppm) was evaluated on cell viability and cytokine secretion by alveolar macrophages (AM) from guinea pigs and healthy subjects. Cell injury was estimated immediately after O3 exposure by evaluation of ATP cell content (measured by bioluminescence) and lactic dehydrogenase (LDH) release in the culture medium. No cytotoxic effect was found: the ATP cell content of both guinea pig AM and human AM did not significantly change after O3 exposure and similarly the LDH release in the culture medium was unchanged. AM-derived cytokines (tumor necrosis factor alpha [TNF alpha], interleukin-1 beta [IL-1 beta], interleukin-6 [IL-6], and interleukin-8 [IL-8]) were evaluated in AM supernatants. O3 exposure was associated with a significant increase in cytokine secretion, with a peak value at 0.4 ppm O3. The exposure of the guinea pig AM to 0.4 ppm O3 for 60 min increased the IL-6 activity by 252 +/- 60% and TNF activity by 202 +/- 35%. The increase in monokine production by the human AM was 443 +/- 208% for TNF alpha, 484 +/- 171% for IL-1 beta, 383 +/- 147% for IL-6, and 226 +/- 45% for IL-8 after a 60-min exposure to 0.4 ppm O3. Lowest O3 concentrations (0.1 and 0.2 ppm) only increased TNF alpha secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion and mRNA expression of TNF alpha and IL-6 in the lungs of pneumoconiosis patients.

Exposure to coal-mine dust leads to coal workers' pneumoconiosis (CWP), characterized by the development of a perifocal and progressive fibrotic reaction. In order to confirm their in vivo participation in the pathogenesis of CWP, the expression of tumor necrosis factor (TNF) and interleukin-6 (IL-6) was evaluated in bronchoalveolar lavage (BAL) specimens collected from 12 patients with simple pneumoconiosis (SP) and six with progressive massive fibrosis (PMF), and in pulmonary tissue from one patient with SP and three with PMF. Expression of TNF and IL-6 was assessed using both in situ hybridization and immunohistochemistry. The number of positive cells found in BALF was significantly higher for patients with PMF (TNF = 55 +/- 6%; IL-6 = 46 +/- 12.8%) than for those with SP (TNF = 34 +/- 11.6%; IL-6 = 26 +/- 10.2%) or normal controls (TNF = 15 +/- 5.5%; IL-6 = 13.3 +/- 6%), and was correlated with cytokine concentrations in supernatants from alveolar macrophages (AM). In lung biopsies, the expression of messenger ribonucleic acid (mRNA) for TNF was associated with the presence of coal dust and was limited to lung macrophages; mRNA for IL-6 was detected in mononuclear phagocytes but also in other types of cells such as endothelial cells. Monokine synthesis was confirmed by immunohistochemistry. These data confirm that TNF and IL-6 production is increased in the lungs of pneumoconiotic patients. Moreover TNF and IL-6 expression was associated with the presence of coalmine dust particles, suggesting a direct role of mineral particles in the cytokine production and development of pneumoconiotic lesions in CWP.

Cytokines and cytokine network in silicosis and coal workers' pneumoconiosis.

The alveolar macrophage (AM) is a critically important cell playing a prominent role in lung inflammation via the production of oxygen radicals, enzymes, arachidonic acid metabolites, and also a large panel of cytokines. Among interstitial lung disorders, silicosis and coal workers' pneumoconiosis (CWP) are the most widespread fibrotic lung diseases. Although their pathophysiology remains incompletely understood, several lines of evidence suggest the participation of cytokines produced by AMs at least in the initiation of the alveolitis. In vitro exposure of AMs (obtained from healthy subjects) to coal dust particles triggered a significant release of tumour necrosis factor (TNF) and interleukin-6, by comparison with titanium dioxide used as a biologically inert control dust. Moreover, it appeared that coal mine dust was more aggressive than similar concentrations of pure silica, suggesting that cytokine secretion induced by coal mine dust was not exclusively related to the presence of silica but resulted from a complex interaction between the different components. In silicosis and CWP, bronchoalveolar lavage showed a large influx of mononuclear phagocytes, with an increased spontaneous production of oxidants, fibronectin, neutrophil chemotactic factor, and also of interleukin-6 and TNF-alpha. This spontaneous cytokine release was associated with an increased cytokine messenger ribonucleic acid (mRNA) expression in the lungs of coal miners.(ABSTRACT TRUNCATED AT 250 WORDS)

E-selectin expression in salivary endothelial cells and sera from patients with systemic sclerosis. Role of resident mast cell-derived tumor necrosis factor alpha.

OBJECTIVE: To assess endothelial cell activation in patients with systemic sclerosis (SSc). METHODS: Concomitant study of salivary gland biopsy tissues and sera for expression of E-selectin and its potent activator tumor necrosis factor alpha (TNF alpha), using immunostaining and enzyme-linked immunosorbent essay. RESULTS: E-selectin was overexpressed in SSc patients, but not in controls. TNF alpha was detected in mast cells. CONCLUSION: Mast cell-derived TNF alpha may contribute to endothelial cell activation in SSc.

Mechanisms of fibrosis in coal workers' pneumoconiosis. Increased production of platelet-derived growth factor, insulin-like growth factor type I, and transforming growth factor beta and relationship to disease severity.

To identify the clinical relevance of cytokines involved in the development of lung fibrosis observed in patients with coal workers' pneumoconiosis (CWP), we investigated the BAL fluid contents and AM secretions of three mediators that modulate fibroblast growth: platelet-derived growth factor (PDGF), Type I insulin-like growth factor (IGF-I), and transforming growth factor Type beta (TGF-beta). Our study population consisted of 25 patients with CWP (16 simple pneumoconiosis, SP, 9 progressive massive fibrosis, PMF, 9 control subjects, and 6 patients with idiopathic pulmonary fibrosis (IPF). The fibrotic potency of AM supernatants was also tested for their ability to promote the growth of a human lung fibroblast cell line appreciated by [3H]-thymidine incorporation. PDGF and IGF-I concentrations were increased in BAL fluids of patients with PMF compared with SP and control subjects, whereas TGF-beta concentration was significantly higher in BAL fluid of patients with SP compared with PMF and control subjects. PDGF, IGF-I, and TGF-beta concentrations in AM supernatants followed the same profile observed in BAL fluids, suggesting that AM is one of the main cell sources of PDGF, IGF-I, and TGF-beta in the lung of pneumoconiotic patients. After treatment by acidification, which activated the latent form of TGF-beta, AM from patients with SP induced an inhibition of [3H]-thymidine incorporation and fibroblast growth was restored after neutralization of TGF-beta by specific antibodies. In contrast, AM supernatants from patients with PMF and IPF promoted the proliferation of fibroblasts and treatment by acidification did not modify this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of endothelial cell adhesion molecule expression in a situation of chronic inflammatory stimulation.

Different disorders affecting the respiratory tract are characterized by the development of a chronic inflammatory reaction with an accumulation of immune and inflammatory cells into the bronchial walls and in interstitial and alveolar spaces. By its ability to secrete a large panel of cytokines, in particular TNF-alpha, the alveolar macrophage (AM) is undoubtedly playing a key role in the complex interactions between inflammatory and structural cells potentially implicated in the inflammatory reaction of the lung. One aspect of these interactions is the capacity of AM-derived TNF-alpha to induce the expression of cellular adhesion molecules (CAM) on the surface of endothelial cells. The main information concerning the induction of CAM have been obtained under experimental conditions mimicking an acute inflammatory reaction. In the present study, we propose an in vitro model allowing the reproduction of the conditions of a chronic inflammation, namely, the endothelial cell behavior submitted to a chronic TNF-alpha stimulation. The endothelial cell activation parameters were the modulation of expression of adhesion molecules: endothelial-leukocyte adhesion molecule-1 (E-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular adhesion molecule-1 (VCAM-1). Their functional abilities was evaluated using U937 cell adhesion analysis. Results demonstrated an overexpression of ICAM-1 after chronic stimulation performed with low but repeated doses of TNF-alpha. The level of ICAM-1 expression persisted throughout the culture to reach a high stable level despite the absence of detectable TNF-alpha activity in culture supernatants. If the stimulation was stopped after 6 days, the expression of ICAM-1 was still observed at least until Day 15. E-selectin and VCAM-1 expression remained absent or not significantly increased. The expression of ICAM-1 on endothelial cells was directly related to an enhanced adhesion capacity as demonstrated by the adhesion of U937 cells to endothelial cells by an ICAM-1/LFA-1 adhesion pathway. Taken together, these elements could bring new approaches in the investigation of mechanisms by which inflammatory cells are recruited and participate in chronic inflammatory processes.

A colorimetric assay to evaluate the immune reactivity of blood platelets based on the reduction of a tetrazolium salt.

Since the first report in 1983, blood platelet reactivity from patients with immediate hypersensitivity reactions, and particularly allergic asthma and aspirin-sensitive asthma, has been evaluated by the release of cytotoxic mediators able to induce the death of parasite larvae, and by the generation of oxygen derived free radicals, measurable by chemiluminescence. We demonstrate here that platelets are able to reduce MTT tetrazolium salt proportionally to the stimulation level in two models of platelet triggering, one IgE-dependent and one aspirin-dependent. This method correlated significantly with the cytotoxicity assay of platelet stimulation (r = 0.965, p < 10(-5) for IgE-dependent stimulation; r = 0.723, p < 10(-4) for aspirin-dependent stimulation). The MTT colorimetric assay should complement or possibly replace previous methods of assessing platelet activation which were of limited use allowing broader investigations on the involvement of platelets in immune processes and in allergic or pseudoallergic reactions.

Separate induction of human blood platelet aggregation or cytotoxicity by different concentrations of PAF-acether and thrombin.

Using decreasing concentrations of PAF-acether or thrombin, it was possible to observe on human platelets, first, aggregation, classically associated to activation, then , below a threshold, cytotoxicity towards Schistosoma mansoni larvae, proposed here as stimulation. These two activities appeared as distinct and antithetic. However, their induction might be the consequence of triggering of the same receptors with different intensity, since PAF-induced, but not thrombin-induced, cytotoxicity could be inhibited with specific PAF-antagonists BN 52021 and BN 52024 also known to inhibit PAF-induced aggregation. These results give credit to the hypothesis that haemostatic and cytotoxic properties of platelets are two distinct functions of these blood elements.

Production of tumor necrosis factor-alpha and interleukin-6 by human alveolar macrophages exposed in vitro to coal mine dust.

Following our previous demonstration of cytokine secretion by alveolar macrophages (AM) from coal miners and from patients with coal workers' pneumoconiosis, we investigated the effect of in vitro exposure to coal dust and to its silica content on tumor necrosis factor-alpha (TNF), interleukin (IL)-1 beta, and IL-6 production by normal human AM. TNF and IL-1 beta concentrations were estimated by a specific radioimmunoassay, while IL-6 levels were evaluated by the proliferation of 7TD1 cells. After 24-h culture, coal dust triggered a significant release of TNF and IL-6 at the dose of 0.1 mg/ml and more obviously at 1 mg/ml in comparison with titanium dioxide (TiO2), used as a biologically inert control dust (with 1 mg/ml of dust: 3,526 +/- 3,509 versus 330 +/- 138 pg TNF/ml and 224 +/- 74 versus 72 +/- 34 U IL-6/ml, respectively; P less than 0.01 in both cases). After 3-h culture, a significant TNF secretion as well as an increased TNF mRNA expression were also detected for AM stimulated by coal dust at variance with TiO2. In contrast, no modification of IL-1 beta concentration could be evidenced in AM exposed to coal dust, although we detected an increased expression of specific mRNA expression. In order to define the role of silica among the main components of coal dust in AM activation, we evaluated the effect of silica (alpha-quartz, 30 micrograms/ml, which is the concentration and the type of silica present in our coal dust) alone or mixed with TiO2 (1 mg/ml) on monokine production.(ABSTRACT TRUNCATED AT 250 WORDS)