Effect of inhaled leukotriene D4 on human airways.
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Adult↗
Biomedical subjects
L J Smith
Publications and source records attributed to L J Smith.
Production of a monoclonal antibody directed against an interferon-induced 56,000-dalton protein and its use in the study of this protein.
Interferon (IFN) treatment of cells induces the synthesis of several new proteins. A hybridoma cell line producing monoclonal antibody to the IFN-induced 56,000-dalton protein has been developed. The IFN-induced 56,000-dalton protein is synthesized by a variety of different cells and in response to IFN-alpha, IFN-beta, and IFN-gamma. The induction of this protein is dependent on de novo RNA synthesis, since its induction is inhibited if actinomycin D and the IFNs are added to the cells simultaneously. Labeling of IFN-treated cells at 4-h intervals at various times after the addition of the IFNs reveals that the synthesis of the 56,000-dalton protein in IFN-alpha-treated cells peaks within 12 h after the addition of the IFN and is no longer enhanced 20 h after exposure to the IFN. In contrast, IFN-gamma-treated cells continue to show an enhanced synthesis of this IFN-induced protein even after 20 h of exposure to the IFN. Thus, the synthesis of the IFN-induced 56,000-dalton protein is regulated differently by the different IFNs. When cells are treated with IFN-alpha or IFN-gamma in the presence of cycloheximide, and actinomycin D is added prior to the removal of the cycloheximide, the cells produce the IFN-induced 56,000-dalton protein and develop an antiviral state in response to both IFN-alpha and IFN-gamma. These results demonstrate that the synthesis of the 56,000-dalton protein is not dependent on the synthesis of an intermediary protein and that the establishment of an antiviral state occurs in the absence of multiple transcriptional events.
Adaptor Proteins, Signal Transducing↗
Introduction of new genetic material into human myeloid leukemic blast stem cells by retroviral infection.
An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.
Blast Crisis↗
Hyperoxic lung damage in mice: appearance and bioconversion of peptide leukotrienes.
High concentrations of oxygen damage the lung and increase bronchoalveolar lavage (BAL) fluid levels of leukotrienes. We sought to identify the specific leukotrienes produced and their relationship to the severity of the lung damage and the inflammatory cell populations by exposing mice to 100% oxygen for up to 4 days. Leukotrienes were not detected in BAL fluid from air-exposed mice. Leukotriene D4 (LTD4) was found after 2 days of exposure to 100% oxygen, increased with longer periods of exposure, and then decreased while LTE4 appeared when the lung damage became severe. LTB4 and LTC4 were not found at any time. Neutropenic mice had identical results, indicating that neutrophils were not the source of the leukotrienes. To determine why LTC4 was not found and why LTD4 decreased and LTE4 increased on day 4, we measured the metabolic capacity of BAL supernatant for leukotrienes. Incubation of LTD4 in BAL supernatant from air-exposed mice resulted in the conversion of LTD4 to LTE4, which was blocked by L-cysteine, a dipeptidase inhibitor. Faster conversion occurred after exposure to 100% oxygen for 3 and 4 days. The rate of bioconversion correlated with the BAL protein concentration (r = 0.756, P less than 0.001), and it was similar in neutropenic and nonneutropenic mice. Little LTC4 and no LTE4 were converted in BAL supernatant from air- or oxygen-exposed mice. The early and progressive increase in LTD4 suggests that sulfidopeptide leukotrienes may play a role in the pathogenesis of hyperoxic lung damage. The increased dipeptidase activity during hyperoxic exposure may serve a protective role by converting the more potent LTD4 to the less potent LTE4.
Animals↗
A serial immunologic and histopathologic study of lung injury induced by trimellitic anhydride.
Trimellitic anhydride (TMA) can induce immunologic lung disease in exposed workers. We have developed a rat model of TMA lung injury characterized by lung hemorrhage and an immune response to trimellityl (TM) haptenized lung proteins. The model is similar to the pulmonary disease-anemia syndrome (PDA) seen in workers exposed to TMA fumes. Sprague-Dawley rats, 15 per exposure period, inhaled micronized TMA powder, 100 micrograms/m3, 6 h/day, for 2,6, or 10 days and were sacrificed. At each time period, total, IgG, IgA, and IgM antibody to TM-rat serum albumin (TM-RSA) were measured by radiolabeled antigen binding and enzyme-linked immunosorbent assay (ELISA) in serum and bronchoalveolar lavage fluid (BAL). Hemorrhagic lung foci, weight, and displacement volume were determined, and lungs were examined by light and electron microscopy. There was no lung injury or antibody response at 2 days. There was minimal lung injury at 6 days with low levels of antibody in BAL and serum. At 10 days, there was a marked increase in hemorrhagic foci and in BAL and serum antibody levels. BAL antibody levels at 6 and 10 days had higher correlations with measures of lung injury than corresponding serum levels. There was minimal ultrastructural change at 6 days. By Day 10, there was marked intraalveolar hemorrhage, alveolar septal inflammatory nodules, abundant alveolar macrophages, and evidence of endothelial and epithelial cell injury. These results indicate that the immune response to inhaled TMA occurs parallel with the development of lung lesions, and antibody levels in BAL and serum are highly correlated with lung injury.
Animals↗
Mechanism of platelet activating factor-induced bronchoconstriction in humans.
The inhalation of platelet activating factor (PAF) produces bronchoconstriction in normal and asthmatic subjects. To identify the mechanism by which PAF-induced bronchoconstriction occurs in humans, bronchoprovocation testing was performed in 7 subjects (3 normal, 4 with mild asthma) after pretreatment with phosphate-buffered saline (PBS), atropine, chlorpheniramine, or indomethacin. We determined the nebulizer concentration of PAF which reduced specific airway conductance (SGaw) 35% (PC35 SGaw) and the slope of the PAF dose-response curve. Atropine produced baseline bronchodilatation (SGaw increased 50%), while chlorpheniramine and indomethacin had no effect on baseline pulmonary function. Atropine increased airway responsiveness to PAF: the PC35 SGaw decreased 40% (p less than 0.05) and the slope of the PAF dose-response curve increased 86% (p less than 0.05). In contrast, chlorpheniramine inhibited the airway response to PAF: the PC35 SGaw increased 87% (p less than 0.05), while the slope of the PAF dose-response curve decreased an insignificant 37%. Indomethacin did not affect either measurement. Chlorpheniramine also prevented the PAF-induced facial flushing and feeling of warmth; atropine and indomethacin did not. These results suggest that PAF-induced bronchoconstriction in humans is mediated at least in part by histamine release, not by cholinergic or cyclooxygenase-dependent mechanisms. Other indirect effects, such as the release of sulfidopeptide leukotrienes, or a direct effect on airway smooth muscle may also contribute to PAF-induced bronchoconstriction. Why atropine heightened the airway response to PAF is unclear.
Adolescent↗
Human gastric lipase. Effects of fatty acid and bovine serum albumin on in vitro activity.
Lipase activity was determined in gastric juice obtained from newborn babies who were fed formula milk (180 ml/kg/day). Samples of gastric juice collected 3 h after a feed were pooled and incubated with bovine serum albumin (BSA). The incubation mixtures containing milk fatty acids (750 nmol/tube) and BSA (0-5%) showed maximal lipase activity (as measured by the liberation of radiolabelled fatty acid from radiolabelled triglyceride) with 1.87% BSA in the first 60 min, but at subsequent time intervals higher enzyme activities were observed with 2.5 and 5% BSA. After extracting milk fatty acids from gastric juice the effects of BSA and sodium oleate on lipase activity were studied. The addition of BSA appeared to inhibit the enzyme, but when sodium oleate was included in the incubating system there was either stimulation or inhibition of lipase activity. A dual effect of BSA (i.e. stimulatory or inhibitory) was observed depending on the relative amounts of BSA and fatty acid present in the incubation tube. It is suggested that 1.87-5% is the optimum concentration range of BSA for the determination of lipase activity in gastric juice, in the presence of 250-500 nmol of fatty acid.
Animals↗
Failure of non-selective inhibition of arachidonic acid metabolism to ameliorate hyperoxic lung injury.
We have previously reported that bronchoalveolar lavage fluid cyclo-oxygenase products of arachidonic acid (AA) metabolism increase prior to the development of significant hyperoxic lung injury. To further assess the role of AA metabolites in the development of hyperoxic lung injury, we have utilized this same model of hyperoxic lung injury and administered either indomethacin (an inhibitor of the cyclo-oxygenase pathway of AA metabolism) or dexamethasone (inhibitor of AA release). A total of 46 adult rabbits were exposed to greater than 95% oxygen for 65 hours. Fourteen animals were given either 2 or 3 mg/kg/day indomethacin, 7 served as controls: 18 animals were given either 0.5 or 1.0 mg/kg/day of dexamethasone, 7 served as controls. The surviving animals were sacrificed after 65 hours of hyperoxia and bronchoalveolar lavage of the left lung was done; the right lung was examined by light microscopy. Treatment with indomethacin or dexamethasone failed to ameliorate the hyperoxic lung injury process. However, in both the indomethacin and dexamethasone treatment groups, significant suppression of 6-keto-PGF1 alpha, a PGI2 metabolite, was observed. Some suppression of TXB2 production was observed, but there was no evidence of any decrease in leukotriene production. We postulate that failure to ameliorate hyperoxic lung injury with either indomethacin or dexamethasone therapy was related to significant suppression of PGI2, a potentially protective AA metabolite, and/or to failure to significantly decrease production of potential pathogenic participants, such as TXA2 or LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)
6-Ketoprostaglandin F1 alpha↗
Hyperoxic lung injury in mice: effect of neutrophil depletion and food deprivation.
Conflicting data exist on the role of neutrophils (PMNs) in the pathogenesis of hyperoxic lung damage. We examined the contribution of PMNs and the contribution of food deprivation, a frequent complication of the methods used to produced neutropenia, to the lung damage that results when mice are exposed to high concentrations of oxygen. Mice were exposed to either 100% oxygen or air for up to 4 days. Neutropenia was induced by a single tail vein injection of nitrogen mustard (NM) given 1 day before the oxygen exposure. Food deprivation, which induced the same weight loss as that found in NM-treated mice, was achieved by withholding food (fasted) during the oxygen exposure. We examined mortality; weight loss; bronchoalveolar lavage (BAL fluid) protein concentration, cell count, and differential count; the number of PMNs in blood; and lung histologic conditions by light and electron microscopy. NM-treated mice lost approximately 25% of their body weight when exposed to either air or oxygen. They also had more severe lung damage than the saline-treated mice during hyperoxic exposure, despite a marked reduction in the number of PMNs in blood, BAL fluid, and lung tissue. Although a correlation was found between the number of blood PMNs and the BAL protein concentration in the nonneutropenic mice (r = 0.69; P less than 0.001), no correlation was seen in the neutropenic mice (r = 0.26). Fasted, oxygen-exposed mice had the same weight loss as the NM mice, but they had more severe lung damage at an earlier time (day 3 vs. day 4) and greater mortality than the saline-treated and the NM-treated mice. These results indicate that PMNs are not required for either the development or progression of hyperoxic lung damage in mice; fasting increases susceptibility to the lung damage; and differences in nutritional status may explain, in part, the controversial role of PMNs in oxygen-induced lung damage.
Animals↗
In vitro transformation of human B-cell follicular lymphoma cells by Epstein-Barr virus.
Human low-grade B-cell lymphoma cells cannot be readily maintained in long-term tissue culture. In an effort to obtain low-grade B-cell lymphoma cell lines for in vitro study, we used Epstein-Barr virus (EBV) as a transforming agent. T-cells were removed prior to EBV transformation by rosetting with sheep erythrocytes, followed by treatment with anti-T11 monoclonal antibody plus complement. The resulting cell population was cocultured with EBV prepared from tissue culture supernatants of marmoset cell line B95-8. Identical immunoglobulin gene rearrangements of tumor cells and EBV-transformed cells were the criteria used to determine that the transformed cells were of tumor origin. DNA was prepared from both biopsy tissue and EBV cell lines and digested with restriction endonucleases, and Southern blots were prepared by standard methods. B-cells isolated from biopsies of four low-grade B-cell lymphomas of follicular, small cleaved cell type and one of follicular, mixed cell type were transformed by EBV into rapidly growing in vitro tissue culture lines. Two of the five transformed cell lines had immunoglobulin heavy chain and light chain gene rearrangements which were present in cells from the original tumor biopsy, indicating that these EBV-transformed cell lines are of tumor origin.
B-Lymphocytes↗
Extraction of cellular DNA from human cells and tissues fixed in ethanol.
DNA can be extracted from ethanol-fixed lymphoid cells and tissues. The fixation procedure is simple and rapid, and the DNA extraction itself is the same as that normally used for fresh tissue or cells. DNA extracted from ethanol-fixed material is indistinguishable from DNA extracted from fresh samples based on its purity, its ability to be digested with restriction endonucleases, and its ability to specifically hybridize to DNA probes. The capability to extract DNA from ethanol-fixed cells and tissues eliminates the need for stringent handling and storage requirements of fresh or frozen specimens.
DNA↗
In vivo 31P NMR studies of the hepatic response to L-ethionine in anesthetized rats.
Phosphorus-31 surface coil spectroscopy has been used to study the effects of L-ethionine administration on the hepatic metabolism of the anesthetized Sprague-Dawley rat. ATP levels were found to decrease by approximately 30% 3 to 4 hr after administration of 1 mg/g body wt of L-ethionine to the anesthetized rat by gastric gavage. ATP levels returned to control values approximately 8 hr postadministration. The relatively small decrease in ATP level was confirmed by extraction and conventional enzyme assay and is a consequence of the mode of administration of the ethionine. Hepatic inorganic phosphate levels rose concomitantly with the ATP fall. There were no significant changes in either cellular pH or Mg2+ levels as monitored by the 31P shifts of sensitive metabolites. In vivo 31P NMR spectroscopy provides a promising approach to study the effects of hepatotoxicants on cellular ATP, pH, and Mg levels.
Adenosine Triphosphate↗
Mechanism of leukotriene D4-induced bronchoconstriction in normal subject.
Leukotriene (LT) D4 is a potent constrictor of human airways and a putative mediator of asthma. However, its mechanism of action in man has not been established. In the present study we sought to determine the role of upper airway reflexes, cholinergic pathways, and cyclooxygenase products of arachidonic acid metabolism in mediating LTD4-induced bronchoconstriction in man. Six normal subjects underwent bronchoprovocation testing with LTD4 after pretreatment with either aerosolized phosphate-buffered saline, aerosolized atropine (1.5 mg), aerosolized lidocaine (80 to 160 mg), or oral indomethacin (50 mg three times daily for 10 doses). Specific airway conductance (SGaw), the flow rate at 30% of vital capacity from a partial forced expiratory maneuver (V30P), and the FEV1 were measured at each concentration of LTD4. We calculated the provocative concentration of LTD4 required to produce a 35% fall in SGaw (PC35SGaw) or a 30% fall in V30P (PC30V30P) and the slope of the LTD4 dose-response curve. Atropine increased baseline SGaw 49% (p less than 0.01) and V30P 43% (p less than 0.005). Atropine also increased the PC35SGaw and PC30V30P and increased the slope of the LTD4 dose-response curve. However, neither of these opposing effects was significant. Lidocaine had no effect on either baseline function or the airway response to LTD4. Indomethacin produced small decreases in baseline V30P (12%) and FEV1 (3%) (p less than 0.05 for both), but it too had no effect on the airway response to LTD4. These results indicate that the bronchoconstriction produced by aerosolized LTD4 in normal subjects is not mediated by cyclooxygenase products of arachidonic acid metabolism or irritant receptors in the upper airways. Although cholinergic pathways may play some role, the data suggest that bronchoconstriction results, at least in part, from a direct effect of LTD4 on airway smooth muscle.
Adult↗
Examination of the utility of the rat as an animal model for human anticoagulation.
The feasibility of employing the rat as an experimental model for investigation of full-dose heparin anticoagulation was assessed. Striking similarities were found to exist between rats and humans regarding baseline-activated partial thromboplastin time (APTT) values, and dosage per kilogram of heparin required to produce an APTT value of 1 1/2-3 times normal, the clinical definition of full-dose heparinization. Based upon these similarities, it appears that the rat can effectively serve as an experimental model for investigating the effects of heparin in humans.
Animals↗
The bronchoconstrictor properties of platelet-activating factor in humans.
Platelet-activating factor (PAF) is an inflammatory mediator that causes bronchial smooth muscle contraction in vitro and in vivo in experimental animals. To characterize the effect of PAF on human airways, 6 normal subjects and 6 subjects with mild asthma inhaled PAF using a standard bronchoprovocation protocol and nebulizer concentrations ranging from 0.1 to 1000 micrograms/ml. Aerosolized PAF produced bronchoconstriction in 5 of 6 normal and 3 of 6 asthmatic subjects as defined by at least a 35% decrease in specific airway conductance (SGaw). However, in these same subjects, flow rates measured at 30% of vital capacity from a partial forced expiratory maneuver (V30P) did not decrease at least 30% nor did the FEV1 decrease by 20%. The 8 PAF responders were 5 to 836 times more sensitive to PAF than to methacholine when SGaw was used to assess the airway response. The relative airway sensitivity of the PAF nonresponders could not be assessed. Normal and asthmatic subjects could not be differentiated by their airway response to PAF, and there were no clinical features that differentiated PAF responders from nonresponders. The maximal airway response to PAF occurred within 2 to 3 min and lasted 15 to 45 min. There were no late reactions. Both normal (p less than 0.01) and asthmatic (p less than 0.05) subjects exhibited tachyphylaxis to PAF. Finally, PAF sensitized the airways of all normal subjects to methacholine, including the one PAF nonresponder (p less than 0.02), but it did not sensitize the airways of the asthmatic subjects.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
CT of the lung parenchyma and the pulmonary function test.
Computed tomography (CT) views of the lung parenchyma were compared with the corresponding pulmonary function tests (PFTs) in 39 patients. Scans were assigned a quantitative degree of involvement based on previously described CT findings. CT was fairly successful in confirming a normal appearance of the lungs in patients with normal PFTs (ten cases). CT was accurate in detecting the presence of a chronic interstitial pattern in patients with restrictive defects on PFTs (15 cases). CT was generally less successful in both the detection and quantitation in patients with PFT criteria of chronic obstructive lung disease (14 cases).
Humans↗
Expression of the p53 oncogene in acute myeloblastic leukemia.
We have investigated whether the p53 oncogene is expressed in the blast cells of patients with acute myeloblastic leukemia. p53 protein was detected in the blast cells of 19 out of 34 patients, but not in normal myelopoietic cells. We find a highly significant correlation between p53 protein synthesis in leukemic blast cells and the secondary plating efficiency of these cells (p = 0.0001). The latter provides an estimate for the self renewal capacity of progenitor cells in the blast population. These data indicate that p53 may be involved in leukemic stem cell renewal.
Cell Division↗
Phenotypic heterogeneity in lymphoblastic cell lines.
Four T-cell and two B-cell lines from patients with lymphoblastic leukemia were examined with a panel of monoclonal antibodies for a variety of lineage and differentiation stage-associated antigens during growth in liquid suspension. In five of the lines, markers normally associated with the granulopoietic lineage were found and the level of expression of these markers varied during culture. The sixth line, MOLT-3, was examined in more detail. Using clonal selection it was found that phenotypic heterogeneity could readily be obtained and that subclones could be acquired that expressed a wide range of markers, typically in reproducible kinetic patterns, that were not detected on the parent clone. Previous results were confirmed showing that treatment with the drug 5-azacytidine (5-aza) prior to selection promoted the expression of the granulopoietic lineage associated antigen 80H.5 on MOLT-3 subclones; however, treatment with 5-aza appeared to inhibit substantially the expression of other novel markers by subclones following a second selection compared to untreated controls. It appears that the expression of markers normally associated with other lineages on leukemic lymphoblasts (lineage infidelity) may be characteristic of such lines and that phenotypically variant subclones expressing lineage infidelity can readily be obtained by clonal selection.
Acute Disease↗