Treatment with lithium has been shown to enhance behavioural and neurophysiological effects of se... more Treatment with lithium has been shown to enhance behavioural and neurophysiological effects of serotonin (Buus Lassen, 1976: Sangdee & Franz, 1978: Friedman et al., 1979) through mechanisms as yet not well known. The stimulation of the high-affinity uptake system for tryptophan by lithium by maintaining adequate membrane potentials across the membrane could lead to stabilization serotonin production, as has currently been demonstrated in long-term treatment with lithium.
Sulfur mustard is a blister agent that can cause death by pulmonary damage. There is currently no... more Sulfur mustard is a blister agent that can cause death by pulmonary damage. There is currently no effective treatment. N-acetyl-L-cysteine (NAC) has mucolytic and antioxidant actions and is an important pre-cursor of cellular glutathione synthesis. These actions may have potential to reduce mustard-induced lung injury. Evaluate the effect of nebulised NAC as a post-exposure treatment for inhaled sulfur mustard in a large animal model. Fourteen anesthetized, surgically prepared pigs were exposed to sulfur mustard vapor (100 μg.kg⁻¹), 10 min) and monitored, spontaneously breathing, to 12 h. Control animals had no further intervention (n = 6). Animals in the treatment group were administered multiple inhaled doses of NAC (1 ml of 200 mg.ml⁻¹ Mucomyst™ at + 30 min, 2, 4, 6, 8, and 10 h post-exposure, n = 8). Cardiovascular and respiratory parameters were recorded. Arterial blood was collected for blood gas analysis while blood and bronchoalveolar lavage fluid were collected for hematology and inflammatory cell analysis. Urine was collected to detect a sulfur mustard breakdown product. Lung tissue samples were taken for histopathological and post-experimental analyses. Five of six sulfur mustard-exposed animals survived to 12 h. Arterial blood oxygenation (PaO₂) and saturation levels were significantly decreased at 12 h. Arterial blood carbon dioxide (PaCO₂) significantly increased, and arterial blood pH and bicarbonate (HCO₃⁻) significantly decreased at 12 h. Shunt fraction was significantly increased at 12 h. In the NAC-treated group all animals survived to 12 h (n = 8). There was significantly improved arterial blood oxygen saturation, HCO₃⁻ levels, and shunt fraction compared to those of the sulfur mustard controls. There were significantly fewer neutrophils and lower concentrations of protein in lavage compared to sulfur mustard controls. NAC's mucolytic and antioxidant properties may be responsible for the beneficial effects seen, improving clinically relevant physiological indices affected by sulfur mustard exposure. Beneficial effects of nebulized NAC were apparent following inhaled sulfur mustard exposure. Further therapeutic benefit may result from a combination therapy approach.
... R. DJUKANOVIC, JK SHUTE, MK CHURCH, ST HOLGATE and DE DAVIESj ... Although the inflammatory r... more ... R. DJUKANOVIC, JK SHUTE, MK CHURCH, ST HOLGATE and DE DAVIESj ... Although the inflammatory response in asthma is characterized by an eosinophilic infiltrate, increased numbers of neutrophils have been found in the bronchoalveolar lavage (BAL) fluid fll and ...
Increased expression of interleukin-8 (IL-8), a potent neutrophil chemoattractant, is associated ... more Increased expression of interleukin-8 (IL-8), a potent neutrophil chemoattractant, is associated with a number of inflammatory diseases. Interleukin-8 binds to the glycosaminoglycan (GAG) heparin and the protease inhibitor alpha2-macroglobulin, molecules which regulate the function of a number of cytokines. Heparan sulphate was previously shown to enhance neutrophil chemotactic responses to IL-8. The purpose of this study was to investigate the effect of heparin, heparan sulphate and alpha2-macroglobulin on IL-8 binding to neutrophils and subsequent functional effects in vitro. The binding of 125I-IL-8 to normal neutrophils at 4 degrees C was studied and the IL-8 induced neutrophil chemotactic response was investigated using micro-Boyden chambers. Complexation of IL-8 with alpha2-macroglobulin was confirmed using gel filtration chromatography. Heparin, but not heparan sulphate, inhibited the binding of 125I-IL-8 to neutrophils (IC50=26 microg/mL) and IL-8 induced neutrophil chemotactic responses (IC50=4 microg/mL). The specific inhibitory effect of heparin was apparently due to an interaction with IL-8 which was charge-dependent, since dextran sulphate had a greater inhibitory effect on chemotactic responses (IC50=2 microg/mL) and FITC-heparin did not bind to neutrophils. The heparin-induced inhibition of IL-8 binding and chemotactic responses was reversed in a dose-dependent manner in the presence of alpha2-macroglobulin. The binding of 125I-IL-8 to neutrophils in the presence of alpha2-macroglobulin appears to be, in part, through the specific IL-8 receptor. These results point to an anti-inflammatory role for heparin and a novel, potentially, pro-inflammatory role for alpha2-macroglobulin which together indicate the importance of cytokine-binding macromolecules in determining net cytokine function.
Consideration of the evolutionary significance of IgE might provide insight into the immunologica... more Consideration of the evolutionary significance of IgE might provide insight into the immunological interactions occurring in utero and during early post-natal life that regulate later atopic disease. We postulated that the fetal gut is exposed to intact amniotic fluid IgE that might interact with local IgE receptors. IgE levels in matched maternal blood and amniotic fluid (n = 47) or breast milk (n = 15) collected from pregnant women in the UK (Southampton) and Brazil (Sao Paulo) were studied. Expression of IgE receptors, Fc epsilon RI and Fc epsilon RII (CD23), in fetal gastrointestinal tract (n = 19) and skin (n = 11) was examined immunohistochemically. Human amniotic fluid at 16-18 weeks' gestation contained intact IgE at levels that increased as maternal circulating levels increased (Spearman's rho = 0.897; P < 0.001). Circulating IgE levels from women in Sao Paulo, Brazil, associated positively not only with term (> 37 weeks' gestation) amniotic fluid (rho = 0.993; P < 0.001) but also breast milk IgE levels (rho = 0.785; P = 0.001). Maternal levels of IgE did not change significantly over pregnancy and fetal circulating levels of IgE were very low (< 0.6 IU/mL). Low-affinity IgE receptors (CD23) were expressed in lymphoid follicles of the fetal gut from 16 weeks of gestation (6/8), but not from 11 to 16 weeks (0/11) or in the skin. Amniotic fluid contains intact IgE that might bind to CD23+ cells within the lymphoid follicles of the fetal gastrointestinal tract. The evolutionary significance of these interactions might be to prepare the immune system for helminthic parasite exposure at birth via IgE-mediated antigen focusing, or "education" of the developing immune system about the prevailing extrauterine environment. However, at present in societies where helminthosis is not a significant health issue, this pathway may still be operational and associated with the development of atopic disease.
Background: There is evidence of activation of the extrinsic coagulation cascade in the asthmatic... more Background: There is evidence of activation of the extrinsic coagulation cascade in the asthmatic airway, and both plasma and locally derived factors may be involved. The hypothesis that the normal haemostatic balance of healthy airways sampled by sputum induction favours fibrin formation in asthmatic airways, and that inhaled corticosteroids (ICS) and plasma exudation influence this balance, was tested. Methods: ELISA and activity assays were used to measure a 2-macroglobulin (an index of plasma leakage) and coagulation factors in hypertonic saline-induced sputum of 30 stable subjects (10 controls, 10 with moderate asthma and 10 with severe asthma). Additionally, the moderate cohort were weaned off their ICS, followed by further sputum induction 5 days after cessation of steroids. Results: ICS wean induced a significant rise in plasminogen (median (interquartile range (IQR)): 13.92 (6.12-16.17) vs 4.82 (2.14-13.32) ng/ml; 95% CI 0.003 to 8.596, p = 0.0499) and tissue plasminogen activator (tPA; 5.57 (3.57-14.35) vs 3.88 (1.74-4.05) ng/ml; 95% CI 0.828 to 9.972, p = 0.0261) levels in sputum, such that tPA in untreated moderate asthma was significantly (p = 0.0029) higher than normal (2.14 (0.0-2.53) ng/ml). Subjects with severe asthma had significantly more a 2macroglobulin (p = 0.0003), tissue factor (p = 0.023), plasminogen activator inhibitor (p = 0.0091), thrombinactivatable fibrinolysis inhibitor (p = 0.0031) and fibrin degradation products (p = 0.0293) in their sputum than control subjects. Conclusion: Untreated moderate asthma is associated with increased fibrinolysis that is corrected by ICS. Severe asthma and high dose corticosteroid therapy is associated with a profibrinogenic, antifibrinolytic environment in the airways. This study suggests that inhibition of fibrin deposition in severe asthma may be a therapeutic approach.
Lithium-sensitive inositol monophosphatase from bovine brain was purified from brain and from a r... more Lithium-sensitive inositol monophosphatase from bovine brain was purified from brain and from a recombinant strain of Escherichia coli BL21-DE3. The natural and recombinant enzymes displayed identical physical and kinetic properties. At low [Li'], Li' inhibited the hydrolysis of racemic myo-inositol 1-phosphate, myo-inositol4-phosphate and adenosine 2'-phosphate in a linear uncompetitive manner with apparent K, values of 1.1, 0.11 and 1.52mM, respectively. At Li' concentrations higher than 4 mM, Li' acted as a non-linear noncompetitive inhibitor for myo-inositol 1-phosphate, K, greater than 1.5 mM. The enzyme was unable to catalyze the transesterification of ['4C]inositol in the presence of inositol 1-phosphate or adenosine 2'-phosphate and attempts to trap a phosphorylated enzyme intermediate directly, were unsuccessful. In the presence of Li+, the enzyme was able to release inositol from inositol 1-phosphate, in a burst, faster than the rate of steady-state substrate turnover suggesting that Li' binds after P-0 bond cleavage in the substrate has occurred. The possibility that a free phosphorylated enzyme intermediate might exist was discounted when the exchange of "0 from water into phosphate was shown to be completely dependent upon inositol. The K, for inositol for "0 exchange was 190 mM and in the presence of saturating phosphate, VEX was at least 60% of V,, for the hydrolysis reaction. Thus, the enzyme operates via a ternary-complex mechanism, and Li' exerts its action by binding to enzyme/product complexes. At low concentration, Li' inhibition with respect to the cofactor, Mg2+ was non-competitive. Mg2+ acted as a non-competitive activator for substrate hydrolysis at pH 8.0, but as the second substrate in an equilibrium-ordered mechanism at pH 6.5. Cooperativity effects were observed for Mg2+ with inositol I-phosphate and 2'AMP as the substrate but not with inositol4-phosphate. The combined results indicate that Mgz' and substrate binding is ordered with substrate adding first. Inositol, the first product off, was a poor non-competitive inhibitor for inositol 1-phosphate whereas the other product, phosphate, was a competitive inhibitor. Phosphate inhibition was markedly pH dependent (K, = 8 mM at pH 6.5 and 0.32 mM at pH 8.0). In the presence of Li' and phosphate,
American Journal of Respiratory and Critical Care Medicine, Aug 1, 1997
Nitrogen dioxide (NO 2) is a free radical and a common oxidant in polluted air. Here we present d... more Nitrogen dioxide (NO 2) is a free radical and a common oxidant in polluted air. Here we present data on the time course of inflammation after NO 2 exposure, as reflected in bronchial biopsy and airway lavage specimens. Healthy, nonsmoking subjects were exposed to air or 2 ppm NO 2 for 4 h in random order on separate occasions. Endobronchial biopsies, bronchial washing (BW), and bronchoalveolar lavage (BAL) were done at 1.5 h (n ϭ 15) or 6 h (n ϭ 15) after exposure. In BW, exposure to NO 2 induced a 1.5-fold increase in interleukin-8 (IL-8) (p Ͻ 0.05) at 1.5 h and a 2.5-fold increase in neutrophils (p Ͻ 0.01) at 6 h. In BAL fluid (BALF), small increases were observed in CD45RO ϩ lymphocytes, B-cells, and natural killer (NK) cells only. Immunohistologic examination of bronchial biopsy specimens showed no signs of upregulation of adhesion molecules, and failed to reveal any significant changes in inflammatory cells at either time point after NO 2 exposure. In summary, NO 2 induced a neutrophilic inflammation in the airways that was detectable in BW at 6 h after NO 2 exposure. The increase in neutrophils could be related to the enhanced IL-8 secretion observed at 1.5 h after exposure. The absence of adhesion-molecule upregulation or cellular inflammation in mucosal biopsy specimens indicates that the major site of inflammation following exposure to NO 2 may be in the smaller airways and not in the alveoli.
The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrop... more The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.
Patients suffering from chronic obstructive pulmonary disease (COPD) clinically manifest airway m... more Patients suffering from chronic obstructive pulmonary disease (COPD) clinically manifest airway mucus hypersecretion as sputum expectoration and cough. Evidence accumulated in the past decade has shown that the cholinergic system not only regulates airway smooth muscle contraction but also the activity of inflammatory and airway epithelial cells, including goblet cells, and submucosal gland activity. Long-acting muscarinic antagonists (LAMAs) with the most favourable M3/M2 muscarinic acetylcholine (ACh) receptors residency properties are not only excellent bronchodilators but potentially also mucus-modifying agents, able to positively impact on mucus hypersecretion and cough. The aim of this systematic review was to investigate the impact of LAMAs on mucus hypersecretion and cough in COPD patients. The evidence confirmed that LAMAs, mainly tiotropium and aclidinium, improved sputum production and cough in moderate to severe COPD. Thus, LAMAs not only antagonise the ACh-induced bronc...
IntroductionCurrent guidelines recommend an initial pleural aspiration in the investigation and m... more IntroductionCurrent guidelines recommend an initial pleural aspiration in the investigation and management of suspected malignant pleural effusions (MPEs) with the aim of establishing a diagnosis, identifying non-expansile lung (NEL) and, at times, providing a therapeutic procedure. A wealth of research has been published since the guidelines suggesting that results and outcomes from an aspiration may not always provide sufficient information to guide management. It is important to establish the validity of these findings in a ‘real world’ population.MethodsA retrospective analysis was conducted of all patients who underwent pleural fluid (PF) sampling, in a single centre, over 3 years to determine the utility of the initial aspiration.ResultsA diagnosis of MPE was confirmed in 230/998 (23%) cases, a further 95/998 (9.5%) were presumed to represent MPE. Transudative biochemistry was found in 3% of cases of confirmed MPE. Positive PF cytology was only sufficient to guide management i...
Advances in Experimental Medicine and Biology, 1993
Acute peritonitis in the rabbit induced by intraperitoneal injection of zymosan is characterised ... more Acute peritonitis in the rabbit induced by intraperitoneal injection of zymosan is characterised by the sequential generation of at least three neutrophil chemoattractants (I). An initial phase of C5a generation (0-2 hours) is followed (2 hours onwards) by the appearance of two structurally related chemoattractant cytokines, rabbit IL-8 and rabbit MGSA (2). The time course of cytokine generation is similar to that of neutrophil accumulation, and phagocytosing neutrophils release IL-8 in vitro. We have therefore investigated whether the infiltrating neutrophils are the source of the IL-8 observed in the cavity at 6 hours. Depletion of circulating neutrophils (>98%) did not inhibit the generation of inflammatory mediators in the peritoneal cavity measured by both in vivo bioassay in skin and using a rabbit IL-8 specific RIA. In addition, HPLC separation of the mediators in exudate from depleted animals revealed that the inflammatory activity comprised both IL-8 and MGSA. Thus, it appears that infiltrating neutrophils are not necessary for production of these cytokines in this model, implicating other cells such as macrophages or mesothelial cells as the source.
Treatment with lithium has been shown to enhance behavioural and neurophysiological effects of se... more Treatment with lithium has been shown to enhance behavioural and neurophysiological effects of serotonin (Buus Lassen, 1976: Sangdee & Franz, 1978: Friedman et al., 1979) through mechanisms as yet not well known. The stimulation of the high-affinity uptake system for tryptophan by lithium by maintaining adequate membrane potentials across the membrane could lead to stabilization serotonin production, as has currently been demonstrated in long-term treatment with lithium.
Sulfur mustard is a blister agent that can cause death by pulmonary damage. There is currently no... more Sulfur mustard is a blister agent that can cause death by pulmonary damage. There is currently no effective treatment. N-acetyl-L-cysteine (NAC) has mucolytic and antioxidant actions and is an important pre-cursor of cellular glutathione synthesis. These actions may have potential to reduce mustard-induced lung injury. Evaluate the effect of nebulised NAC as a post-exposure treatment for inhaled sulfur mustard in a large animal model. Fourteen anesthetized, surgically prepared pigs were exposed to sulfur mustard vapor (100 μg.kg⁻¹), 10 min) and monitored, spontaneously breathing, to 12 h. Control animals had no further intervention (n = 6). Animals in the treatment group were administered multiple inhaled doses of NAC (1 ml of 200 mg.ml⁻¹ Mucomyst™ at + 30 min, 2, 4, 6, 8, and 10 h post-exposure, n = 8). Cardiovascular and respiratory parameters were recorded. Arterial blood was collected for blood gas analysis while blood and bronchoalveolar lavage fluid were collected for hematology and inflammatory cell analysis. Urine was collected to detect a sulfur mustard breakdown product. Lung tissue samples were taken for histopathological and post-experimental analyses. Five of six sulfur mustard-exposed animals survived to 12 h. Arterial blood oxygenation (PaO₂) and saturation levels were significantly decreased at 12 h. Arterial blood carbon dioxide (PaCO₂) significantly increased, and arterial blood pH and bicarbonate (HCO₃⁻) significantly decreased at 12 h. Shunt fraction was significantly increased at 12 h. In the NAC-treated group all animals survived to 12 h (n = 8). There was significantly improved arterial blood oxygen saturation, HCO₃⁻ levels, and shunt fraction compared to those of the sulfur mustard controls. There were significantly fewer neutrophils and lower concentrations of protein in lavage compared to sulfur mustard controls. NAC's mucolytic and antioxidant properties may be responsible for the beneficial effects seen, improving clinically relevant physiological indices affected by sulfur mustard exposure. Beneficial effects of nebulized NAC were apparent following inhaled sulfur mustard exposure. Further therapeutic benefit may result from a combination therapy approach.
... R. DJUKANOVIC, JK SHUTE, MK CHURCH, ST HOLGATE and DE DAVIESj ... Although the inflammatory r... more ... R. DJUKANOVIC, JK SHUTE, MK CHURCH, ST HOLGATE and DE DAVIESj ... Although the inflammatory response in asthma is characterized by an eosinophilic infiltrate, increased numbers of neutrophils have been found in the bronchoalveolar lavage (BAL) fluid fll and ...
Increased expression of interleukin-8 (IL-8), a potent neutrophil chemoattractant, is associated ... more Increased expression of interleukin-8 (IL-8), a potent neutrophil chemoattractant, is associated with a number of inflammatory diseases. Interleukin-8 binds to the glycosaminoglycan (GAG) heparin and the protease inhibitor alpha2-macroglobulin, molecules which regulate the function of a number of cytokines. Heparan sulphate was previously shown to enhance neutrophil chemotactic responses to IL-8. The purpose of this study was to investigate the effect of heparin, heparan sulphate and alpha2-macroglobulin on IL-8 binding to neutrophils and subsequent functional effects in vitro. The binding of 125I-IL-8 to normal neutrophils at 4 degrees C was studied and the IL-8 induced neutrophil chemotactic response was investigated using micro-Boyden chambers. Complexation of IL-8 with alpha2-macroglobulin was confirmed using gel filtration chromatography. Heparin, but not heparan sulphate, inhibited the binding of 125I-IL-8 to neutrophils (IC50=26 microg/mL) and IL-8 induced neutrophil chemotactic responses (IC50=4 microg/mL). The specific inhibitory effect of heparin was apparently due to an interaction with IL-8 which was charge-dependent, since dextran sulphate had a greater inhibitory effect on chemotactic responses (IC50=2 microg/mL) and FITC-heparin did not bind to neutrophils. The heparin-induced inhibition of IL-8 binding and chemotactic responses was reversed in a dose-dependent manner in the presence of alpha2-macroglobulin. The binding of 125I-IL-8 to neutrophils in the presence of alpha2-macroglobulin appears to be, in part, through the specific IL-8 receptor. These results point to an anti-inflammatory role for heparin and a novel, potentially, pro-inflammatory role for alpha2-macroglobulin which together indicate the importance of cytokine-binding macromolecules in determining net cytokine function.
Consideration of the evolutionary significance of IgE might provide insight into the immunologica... more Consideration of the evolutionary significance of IgE might provide insight into the immunological interactions occurring in utero and during early post-natal life that regulate later atopic disease. We postulated that the fetal gut is exposed to intact amniotic fluid IgE that might interact with local IgE receptors. IgE levels in matched maternal blood and amniotic fluid (n = 47) or breast milk (n = 15) collected from pregnant women in the UK (Southampton) and Brazil (Sao Paulo) were studied. Expression of IgE receptors, Fc epsilon RI and Fc epsilon RII (CD23), in fetal gastrointestinal tract (n = 19) and skin (n = 11) was examined immunohistochemically. Human amniotic fluid at 16-18 weeks' gestation contained intact IgE at levels that increased as maternal circulating levels increased (Spearman's rho = 0.897; P < 0.001). Circulating IgE levels from women in Sao Paulo, Brazil, associated positively not only with term (> 37 weeks' gestation) amniotic fluid (rho = 0.993; P < 0.001) but also breast milk IgE levels (rho = 0.785; P = 0.001). Maternal levels of IgE did not change significantly over pregnancy and fetal circulating levels of IgE were very low (< 0.6 IU/mL). Low-affinity IgE receptors (CD23) were expressed in lymphoid follicles of the fetal gut from 16 weeks of gestation (6/8), but not from 11 to 16 weeks (0/11) or in the skin. Amniotic fluid contains intact IgE that might bind to CD23+ cells within the lymphoid follicles of the fetal gastrointestinal tract. The evolutionary significance of these interactions might be to prepare the immune system for helminthic parasite exposure at birth via IgE-mediated antigen focusing, or "education" of the developing immune system about the prevailing extrauterine environment. However, at present in societies where helminthosis is not a significant health issue, this pathway may still be operational and associated with the development of atopic disease.
Background: There is evidence of activation of the extrinsic coagulation cascade in the asthmatic... more Background: There is evidence of activation of the extrinsic coagulation cascade in the asthmatic airway, and both plasma and locally derived factors may be involved. The hypothesis that the normal haemostatic balance of healthy airways sampled by sputum induction favours fibrin formation in asthmatic airways, and that inhaled corticosteroids (ICS) and plasma exudation influence this balance, was tested. Methods: ELISA and activity assays were used to measure a 2-macroglobulin (an index of plasma leakage) and coagulation factors in hypertonic saline-induced sputum of 30 stable subjects (10 controls, 10 with moderate asthma and 10 with severe asthma). Additionally, the moderate cohort were weaned off their ICS, followed by further sputum induction 5 days after cessation of steroids. Results: ICS wean induced a significant rise in plasminogen (median (interquartile range (IQR)): 13.92 (6.12-16.17) vs 4.82 (2.14-13.32) ng/ml; 95% CI 0.003 to 8.596, p = 0.0499) and tissue plasminogen activator (tPA; 5.57 (3.57-14.35) vs 3.88 (1.74-4.05) ng/ml; 95% CI 0.828 to 9.972, p = 0.0261) levels in sputum, such that tPA in untreated moderate asthma was significantly (p = 0.0029) higher than normal (2.14 (0.0-2.53) ng/ml). Subjects with severe asthma had significantly more a 2macroglobulin (p = 0.0003), tissue factor (p = 0.023), plasminogen activator inhibitor (p = 0.0091), thrombinactivatable fibrinolysis inhibitor (p = 0.0031) and fibrin degradation products (p = 0.0293) in their sputum than control subjects. Conclusion: Untreated moderate asthma is associated with increased fibrinolysis that is corrected by ICS. Severe asthma and high dose corticosteroid therapy is associated with a profibrinogenic, antifibrinolytic environment in the airways. This study suggests that inhibition of fibrin deposition in severe asthma may be a therapeutic approach.
Lithium-sensitive inositol monophosphatase from bovine brain was purified from brain and from a r... more Lithium-sensitive inositol monophosphatase from bovine brain was purified from brain and from a recombinant strain of Escherichia coli BL21-DE3. The natural and recombinant enzymes displayed identical physical and kinetic properties. At low [Li'], Li' inhibited the hydrolysis of racemic myo-inositol 1-phosphate, myo-inositol4-phosphate and adenosine 2'-phosphate in a linear uncompetitive manner with apparent K, values of 1.1, 0.11 and 1.52mM, respectively. At Li' concentrations higher than 4 mM, Li' acted as a non-linear noncompetitive inhibitor for myo-inositol 1-phosphate, K, greater than 1.5 mM. The enzyme was unable to catalyze the transesterification of ['4C]inositol in the presence of inositol 1-phosphate or adenosine 2'-phosphate and attempts to trap a phosphorylated enzyme intermediate directly, were unsuccessful. In the presence of Li+, the enzyme was able to release inositol from inositol 1-phosphate, in a burst, faster than the rate of steady-state substrate turnover suggesting that Li' binds after P-0 bond cleavage in the substrate has occurred. The possibility that a free phosphorylated enzyme intermediate might exist was discounted when the exchange of "0 from water into phosphate was shown to be completely dependent upon inositol. The K, for inositol for "0 exchange was 190 mM and in the presence of saturating phosphate, VEX was at least 60% of V,, for the hydrolysis reaction. Thus, the enzyme operates via a ternary-complex mechanism, and Li' exerts its action by binding to enzyme/product complexes. At low concentration, Li' inhibition with respect to the cofactor, Mg2+ was non-competitive. Mg2+ acted as a non-competitive activator for substrate hydrolysis at pH 8.0, but as the second substrate in an equilibrium-ordered mechanism at pH 6.5. Cooperativity effects were observed for Mg2+ with inositol I-phosphate and 2'AMP as the substrate but not with inositol4-phosphate. The combined results indicate that Mgz' and substrate binding is ordered with substrate adding first. Inositol, the first product off, was a poor non-competitive inhibitor for inositol 1-phosphate whereas the other product, phosphate, was a competitive inhibitor. Phosphate inhibition was markedly pH dependent (K, = 8 mM at pH 6.5 and 0.32 mM at pH 8.0). In the presence of Li' and phosphate,
American Journal of Respiratory and Critical Care Medicine, Aug 1, 1997
Nitrogen dioxide (NO 2) is a free radical and a common oxidant in polluted air. Here we present d... more Nitrogen dioxide (NO 2) is a free radical and a common oxidant in polluted air. Here we present data on the time course of inflammation after NO 2 exposure, as reflected in bronchial biopsy and airway lavage specimens. Healthy, nonsmoking subjects were exposed to air or 2 ppm NO 2 for 4 h in random order on separate occasions. Endobronchial biopsies, bronchial washing (BW), and bronchoalveolar lavage (BAL) were done at 1.5 h (n ϭ 15) or 6 h (n ϭ 15) after exposure. In BW, exposure to NO 2 induced a 1.5-fold increase in interleukin-8 (IL-8) (p Ͻ 0.05) at 1.5 h and a 2.5-fold increase in neutrophils (p Ͻ 0.01) at 6 h. In BAL fluid (BALF), small increases were observed in CD45RO ϩ lymphocytes, B-cells, and natural killer (NK) cells only. Immunohistologic examination of bronchial biopsy specimens showed no signs of upregulation of adhesion molecules, and failed to reveal any significant changes in inflammatory cells at either time point after NO 2 exposure. In summary, NO 2 induced a neutrophilic inflammation in the airways that was detectable in BW at 6 h after NO 2 exposure. The increase in neutrophils could be related to the enhanced IL-8 secretion observed at 1.5 h after exposure. The absence of adhesion-molecule upregulation or cellular inflammation in mucosal biopsy specimens indicates that the major site of inflammation following exposure to NO 2 may be in the smaller airways and not in the alveoli.
The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrop... more The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.
Patients suffering from chronic obstructive pulmonary disease (COPD) clinically manifest airway m... more Patients suffering from chronic obstructive pulmonary disease (COPD) clinically manifest airway mucus hypersecretion as sputum expectoration and cough. Evidence accumulated in the past decade has shown that the cholinergic system not only regulates airway smooth muscle contraction but also the activity of inflammatory and airway epithelial cells, including goblet cells, and submucosal gland activity. Long-acting muscarinic antagonists (LAMAs) with the most favourable M3/M2 muscarinic acetylcholine (ACh) receptors residency properties are not only excellent bronchodilators but potentially also mucus-modifying agents, able to positively impact on mucus hypersecretion and cough. The aim of this systematic review was to investigate the impact of LAMAs on mucus hypersecretion and cough in COPD patients. The evidence confirmed that LAMAs, mainly tiotropium and aclidinium, improved sputum production and cough in moderate to severe COPD. Thus, LAMAs not only antagonise the ACh-induced bronc...
IntroductionCurrent guidelines recommend an initial pleural aspiration in the investigation and m... more IntroductionCurrent guidelines recommend an initial pleural aspiration in the investigation and management of suspected malignant pleural effusions (MPEs) with the aim of establishing a diagnosis, identifying non-expansile lung (NEL) and, at times, providing a therapeutic procedure. A wealth of research has been published since the guidelines suggesting that results and outcomes from an aspiration may not always provide sufficient information to guide management. It is important to establish the validity of these findings in a ‘real world’ population.MethodsA retrospective analysis was conducted of all patients who underwent pleural fluid (PF) sampling, in a single centre, over 3 years to determine the utility of the initial aspiration.ResultsA diagnosis of MPE was confirmed in 230/998 (23%) cases, a further 95/998 (9.5%) were presumed to represent MPE. Transudative biochemistry was found in 3% of cases of confirmed MPE. Positive PF cytology was only sufficient to guide management i...
Advances in Experimental Medicine and Biology, 1993
Acute peritonitis in the rabbit induced by intraperitoneal injection of zymosan is characterised ... more Acute peritonitis in the rabbit induced by intraperitoneal injection of zymosan is characterised by the sequential generation of at least three neutrophil chemoattractants (I). An initial phase of C5a generation (0-2 hours) is followed (2 hours onwards) by the appearance of two structurally related chemoattractant cytokines, rabbit IL-8 and rabbit MGSA (2). The time course of cytokine generation is similar to that of neutrophil accumulation, and phagocytosing neutrophils release IL-8 in vitro. We have therefore investigated whether the infiltrating neutrophils are the source of the IL-8 observed in the cavity at 6 hours. Depletion of circulating neutrophils (>98%) did not inhibit the generation of inflammatory mediators in the peritoneal cavity measured by both in vivo bioassay in skin and using a rabbit IL-8 specific RIA. In addition, HPLC separation of the mediators in exudate from depleted animals revealed that the inflammatory activity comprised both IL-8 and MGSA. Thus, it appears that infiltrating neutrophils are not necessary for production of these cytokines in this model, implicating other cells such as macrophages or mesothelial cells as the source.
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Papers by Janis Shute