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UNLABELLED: The comparison of endotoxin levels between study populations and countries is limited as a result of differences in sampling, extraction, and storage procedures. The objective of this study is to assess the levels and determinants of endotoxin in mattress and living room floor dust samples from three European countries, namely, Germany, the Netherlands, and Sweden, using a standardized sampling, storage, and analysis protocol. The mattress and living room floor dust was collected from the homes of 1065 German, Dutch, and Swedish (pre-)school children. All the samples were collected in the cool season and analyzed for endotoxin in a central laboratory. The determinants were assessed by a standardized questionnaire. The endotoxin concentrations in mattress and living room floor dust were found to be the highest in German homes and lowest in the Swedish ones. Differences between the geometric means were small (factor 1.1-1.7). Most of the associations between endotoxin concentrations and potential determinants were not statistically significant and heterogeneous across countries. However, keeping pets and having more than four persons living in the home were consistently associated with up to 1.7-fold higher endotoxin concentrations in mattress and floor dust. Furthermore, having carpets or rugs, and opening the windows frequently was associated with up to 3.4-fold and 1.3-fold higher endotoxin concentrations in living room floor dust, respectively. The proportion of variance explained by the questionnaire variables was generally low. In conclusion, the data on housing characteristics did not accurately predict the endotoxin concentrations in house dust, and could only partly explain the differences between countries. PRACTICAL IMPLICATIONS: The differences between the endotoxin concentrations in German, Dutch, and Swedish homes are small. House dust endotoxin concentrations are associated with a number of housing factors, such as pet-ownership, floor cover, number of persons living in the home, and ventilation. The variability of the endotoxin levels between homes and countries can only be partly explained by these factors.

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Radioligand binding studies have been developed to determine pharmacologic receptor characteristics in vitro. With this assay, not only the number and dissociation constant (KD) can be studied, but also the interaction of agonists with the receptor. We used this method to study a new beta 2-sympathicomimetic drug, tulobuterol (1-(0-chlorophenyl)-2-butylamino-ethanol hydrochloride). Two sets of experiments were performed. One set of experiments investigated the effects of tulobuterol and terbutaline in chronic administration, while the second set compared the beta-adrenoceptor-stimulating properties of tulobuterol with terbutaline and salbutamol. The effects of 10 days' administration of tulobuterol and terbutaline on beta-adrenergic characteristics in rats were assessed biochemically by means of radioligand binding studies on pulmonary membranes and functionally using isolated tracheal spirals. It was found that: (1) In vivo treatment with both drugs induced a reduction of the number of beta-adrenoceptors bound by 3H-dihydroalprenolol (3H-DHA); however, tulobuterol also induced an increased affinity for beta-adrenoceptor binding. (2) Tulobuterol induced a significant increase in the sensitization of tracheal smooth muscle, facilitating the relaxation of airway smooth muscle. The inhibition of 3H-DHA binding with the three drugs was best fit in a two-binding site model, showing high- and low-affinity binding sites. The high-affinity sites had similar KD values for terbutaline and tulobuterol (1.6 x 10(7) and 1.5 x 10(-7), respectively). The high-affinity sites for salbutamol had a higher KD value (9.4 x 10(-7), suggesting a lower affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

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1. In this study mast cells were found to comprise 2.1% of total cells recovered by enzymatic digestion of human lung tissue. 2. This mast cell population consisted of 79% formalin-sensitive, Alcian Blue-positive mast cells and 21% formalin-insensitive, Alcian Blue-positive mast cells. 3. By the use of centrifugal elutriation and subsequent Percoll gradient centrifugation, separate mixed cell populations could be obtained in which the mast cell constituents were either of the formalin-sensitive or -insensitive type. 4. Cell suspensions in which formalin-sensitive cells comprised 97% of mast cells contained approximately 1.34 pg of histamine per mast cell, whereas in preparations in which mast cells were 84% formalin-resistant the histamine content was approximately 4.17 pg of histamine per mast cell. 5. The histamine release upon anti-immunoglobulin E challenge of formalin-sensitive mast cells was greater than the release by formalin-insensitive mast cells. 6. After challenge with opsonized zymosan, only formalin-sensitive mast cells were able to release histamine. 7. Leukotriene C4 release was observed when formalin-sensitive mast cells were challenged with anti-immunoglobulin E. Formalin-insensitive mast cells showed no release of leukotriene C4. 8. Prostaglandin D2 release was observed when formalin-insensitive mast cells were challenged with anti-immunoglobulin E. Formalin-sensitive mast cells showed no release of prostaglandin D2.

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Indicators of immune-mediated disease were studied in calves with severe natural bovine respiratory syncytial virus infection. Although antigen and antibody were detected concurrently in most calves, immune complexes were not detected by use of immunofluorescence, ELISA, and binding of the 1q component of complement. Complement component C3, however, was observed by immunofluorescence in the cranioventral, virus-infected portion of the lungs of 19 of 25 calves. Reductions in the amount of histamine and in the numbers of mast cells and mast cell granules in the virus-positive cranioventral and virus-negative caudodorsal portions of the lungs, indicated activation of mast cells and liberation of their granule contents. On the basis of these and previous findings, a model for the pathogenesis of bovine respiratory syncytial virus-induced disease was proposed.

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Chemotaxis (CT) of cells is an important factor in the defense of organisms. For the study of this process several assays are available using the Boyden chamber. Its popularity, however, has led to a use of the system of which the basic qualities are not always known and so may lead to erroneous interpretation of results. Experiments were performed with the purpose of elucidating the significance of parameters regularly used in CT: 1. the cell count at the bottom of the permeable filter, 2. a chemotactic index, and 3. the leading front method. Neutrophils and eosinophils were stimulated with FMLP (10(-13)-10(-3) M). It appeared that: 1. the parameters are indicative only for (small) parts of the cell population, 2. for the comparison of effects of compounds complete concentration-effect relationships are needed, 3. to avoid cell to cell interactions a maximal cell purity is desired, and 4. if possible, no combinations of chemotactic agents should be tested.

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Mast cells were isolated by enzymatic digestion from lung tissue obtained from patients with chronic obstructive lung disease and from normal subjects. Two mast cell subtypes could be demonstrated in human lung tissue. Mast cell subtypes were differentiated in formalin-sensitive and formalin-insensitive mast cells. It appeared that compared with normal individuals, patients suffering from chronic bronchitis had increased numbers of mast cells of the formalin-sensitive type, whereas patients with emphysema had reduced numbers, but the same ratio, of both mast cell subtypes.

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The contribution of mast cell subtypes and their different mediators to the pathogenesis of chronic obstructive lung diseases (COLD) has not yet been established. In the present study, enzymatic digestion, centrifugal elutriation and Percoll gradient centrifugation were used to obtain two populations of mast cell subtypes from human lung tissue. Mast cell subtypes were challenged with anti-human IgE, propranolol, compound 48/80, or opsonized zymosan. Both subtypes were able to release histamine, but differed in the amount of the amine release. Only the formalin-sensitive and alcian blue-positive type (FS-AB) released histamine on challenge with opsonized zymosan. The same subtype was able to release leukotriene C4 (LTC4) after challenge with anti-human IgE. The other subtype, the formalin-insensitive and alcian blue-positive type (FI-AB), did not respond to opsonized zymosan and did not release LTC4 after challenge with anti-human IgE. Stimulation with propranolol or compound 48/80 did not release histamine from the FS-AB mast cells while the FI-AB mast cells released only about 10% of their histamine content upon challenge with these secretagogues.

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A method of isolation has been developed to purify mast cells from human lung tissue. The purification steps are: (1) dispersion of human lung tissue in single-cell suspensions by enzymatic digestion, (2) partial purification by counterflow centrifugal elutriation, (3) Percoll gradient centrifugation, and (4) enrichment of the mast cells by affinity chromatography using anti-human IgE-Sepharose. Enzymatic dispersion yielded 0.6 +/- 0.2 x 10(6) mast cells per gram wet tissue with purities of 3.3 +/- 1.0% (mean +/- SEM n = 3). Elutriation and gradient centrifugation yielded 0.36 +/- 0.05 x 10(6) mast cells per gram lung tissue in fractions with purities of 30.8 +/- 10.7%. Enriched mast cell fractions were combined, and disposed of contaminating cells by affinity chromatography, thereby yielding 0.25 +/- 0.03 x 10(6) mast cells per gram lung tissue, and improving the purity to 75.3 +/- 8.3%. The purified mast cells were intact and vitality exceeded 95%. In this way from 1 g wet lung tissue 0.25 +/- 0.03 x 10(6) mast cells may be isolated with a mean recovery of 41.7 +/- 2.4% and a mean purity of 75.3 +/- 8.3%.

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An influx of polymorphonuclear and mononuclear cells into the lungs of smokers and patients with chronic obstructive lung disease (COLD) is thought to be an important factor in the development of pulmonary emphysema. Next to the synthesis and release of toxic oxygen radicals and mediators, an enhanced production and activity of proteolytic enzymes could play an important role in the pathogenesis of emphysema. In the present study changes were investigated in the broncho-alveolar lavage (BAL) fluid and BAL-cells, especially in alveolar macrophages. Pulmonary lavages were performed in the middle lobe with sterile saline of 37 degrees C in individuals, who could be divided on the basis of their history and lung function into normal/nonsmokers, normal/smokers, COLD-patients/nonsmokers and COLD-patients/smokers. Alveolar macrophages obtained by BAL were stained for different lysosomal enzymes. Isolated BAL-fluid and BAL-cells were assayed for elastolytic activity. In alveolar macrophages of smoking COLD-patients significantly more beta-glucuronidase could be demonstrated. Elastolytic activity changed with smoking habits, suggesting an enhanced release of elastolytic enzymes. No correlation was found between elastolytic activity and the amount of polymorphonuclear cells in the BAL-fluid. From these results it may be concluded that enzymes from alveolar macrophages play a more important role in the pathogenesis of emphysema than those from polymorphonuclear cells.

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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%.

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Direct contact of the cells normally present in the bronchial lumen, such as alveolar macrophages, with air pollutants (e.g. cigarette smoke) can lead to the activation of these cells. This activation is beneficial for the cleaning task these cells have in the bronchial tree, but also leads to the release of chemotactic substances, toxic oxygen radicals, enzymes and mediators responsible for bronchial obstruction. As a first step in these processes, an enhanced chemotactic activity can attract neutrophils to the bronchial lumen, where they help by cleaning the lungs from possible dangerous intruders, but can also cause damage to the normal lung architecture. In the present study concerned with the pathogenesis of chronic obstructive lung disease (COLD), broncho-alveolar lavage (BAL) was performed in 35 individuals, who could be divided on he basis of their history and lung function into normal/nonsmokers, normal/smokers, COLD-patients/nonsmokers and COLD-patients/smokers. Neutrophilic chemotactic activity was assayed using Boyden chambers where the patients' own neutrophils were tested. More neutrophils and more neutrophilic chemotactic activity were found in the BAL-fluid of the smokers and COLD-patients. A correlation was demonstrated between the amount of chemotactic activity released during the incubation of cells obtained by BAL and the airway resistance or the airway conductance. These data suggest an enhanced chemotactic activity as one of the initiating factors in the pathogenesis of chronic obstructive lung disease.

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Muscarinic cholinergic receptors have been identified and characterized by radioligand binding studies in human peripheral lung tissue. The tissue was obtained at thoracotomy of 12 patients, of whom four had chronic obstructive lung disease. The radioligand 1-quinuclidinyl [phenyl-4-3H]benzilate (3H-QNB) was used to label the muscarinic cholinergic receptors. Binding was saturable, protein dependent and showed a high affinity and stereospecificity. Specific binding could be inhibited by agonists and antagonists; molar inhibition constants determined for the agents used were of the same order of magnitude as those reported for 3H-QNB inhibition in various tissues of laboratory animals. Inhibition experiments with agonists resulted in Hill slopes which were significantly different from unity, indicating multiple binding sites. The stable GTP analogue guanyl-5'-imidodiphosphate had no effect on the Hill slopes of agonists or antagonists. The number of binding sites was significantly less in lung tissue from patients with chronic obstructive lung disease.

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Human granulocytes isolated from peripheral blood have been described to synthesize both LTB4 and LTC4 from arachidonic acid. We have observed that the amount of LTC4 produced by human granulocyte preparations is strongly dependent on the relative amount of eosinophils. To investigate a possibly significant difference in leukotriene synthesis of the eosinophilic and neutrophilic granulocytes, we developed a purification method to isolate both cell types from granulocytes obtained from the blood of healthy donors. Leukotrienes were generated by incubation of the purified cells with arachidonic acid, calcium ionophore A23187, calcium-chloride and reduced glutathione. Surprisingly, eosinophils were found to produce almost exclusively the spasmogenic LTC4. In contrast, neutrophils produce almost exclusively the chemotactic LTB4, its omega-hydroxylated metabolite 20-hydroxy-LTB4 and two non-enzymically formed LTB4 isomers.

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