Bronchial wheezing predicts inflammation and respiratory failure in fire smoke victims.

BACKGROUND: Acute fire smoke inhalation injury involves inflammatory mediators whose roles are poorly understood. We carried out a prospective observational study of fire smoke victims to identify clinical and biochemical markers that may predict pulmonary dysfunction and investigated possible correlations between dysfunction and cytokines in bronchoalveolar lavage (BAL) fluid and blood. METHODS: Forty patients with respiratory and/or neurological symptoms following acute fire smoke inhalation had pulmonary function tests and blood gas analyses performed on admission, at discharge, and after 3 months. Cytokines were measured using BioPlex/XMap technology. RESULTS: On admission, 30 (75%) patients had dyspnea. Patients presenting with bronchial wheezing (n = 14) had significantly lower PEF (201 l/min, 82-360) than non-wheezing patients (406 l/min, 100-683) (n = 16, P = 0.03). Bronchial wheezing predicted need for ICU treatment with OR = 93.3 at 95% CI (P < 0.001) and was associated with gas exchange impairment, with mean pa O2 /FiO2 ratio 34.4 (11.8-49.8) kPa on admission and 21.3 (8.3-44.5) kPa 48 h later. Blood HbCO also predicted ICU treatment, with OR = 1.58 at 95% CI (P < 0.001). Serum CRP, IL-6, IL-8, and MCP-1 were significantly higher in wheezing patients after 12-24 h compared with non-wheezing patients and study controls. Cytokine levels were still elevated after 3 months. BAL fluid had significantly higher levels of IL-8, MCP-1, IL-1ß, and G-CSF compared with healthy controls. CONCLUSION: In victims of fire smoke inhalation, pulmonary wheezing predicts inflammation, pulmonary dysfunction, respiratory failure, and need for intensive care.

High-intensity training following lung cancer surgery: a randomised controlled trial.

BACKGROUND: Many patients with lung cancer are deconditioned with poor physical fitness. Lung resection reduces physical fitness further, impairing the patient's ability to function in daily life. METHODS: We conducted a single-blind randomised controlled trial of high-intensity endurance and strength training (60 min, three times a week, 20 weeks), starting 5-7 weeks after surgery. The control group received standard postoperative care. The primary outcome was the change in peak oxygen uptake measured directly during walking until exhaustion. Other outcomes included changes in pulmonary function, muscular strength by one-repetition maximum (1RM), total muscle mass measured by dual energy X-ray absorptiometry, daily physical functioning and quality of life (QoL). RESULTS: The intention-to-treat analysis of the 61 randomised patients showed that the exercise group had a greater increase in peak oxygen uptake (3.4 mL/kg/min between-group difference, p=0.002), carbon monoxide transfer factor (Tlco) (5.2% predicted, p=0.007), 1RM leg press (29.5 kg, p<0.001), chair stand (2.1 times p<0.001), stair run (4.3 steps, p=0.002) and total muscle mass (1.36 kg, p=0.012) compared with the controls. The mean±SD QoL (SF-36) physical component summary score was 51.8±5.5 and 43.3±11.3 (p=0.006), and the mental component summary score was 55.5±5.3 and 46.6±14.0 (p=0.015) in the exercise and control groups, respectively. CONCLUSIONS: In patients recently operated for lung cancer, high-intensity endurance and strength training was well tolerated and induced clinically significant improvements in peak oxygen uptake, Tlco, muscular strength, total muscle mass, functional fitness and QoL. This study may provide a basis for exercise therapy after lung cancer surgery. TRIAL REGISTRATION NUMBER: NCT01748981.

IL-18 neutralization during alveolar hypoxia improves left ventricular diastolic function in mice.

AIM: In patients, an association exists between pulmonary diseases and diastolic dysfunction of the left ventricle (LV). We have previously shown that alveolar hypoxia in mice induces LV diastolic dysfunction and that mice exposed to hypoxia have increased levels of circulating interleukin-18 (IL-18), suggesting involvement of IL-18 in development of diastolic dysfunction. IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18. In this study, we hypothesized that neutralization of IL-18 during alveolar hypoxia would improve LV diastolic function. METHODS: Mice were exposed to 10% oxygen for 2 weeks while treated with IL-18BP or vehicle. Cardiac function and morphology were measured using echocardiography, intraventricular pressure measurements and magnetic resonance imaging (MRI). For characterization of molecular changes in the heart, both real-time PCR and Western blotting were performed. ELISA technique was used to measure levels of circulating cytokines. RESULTS: As expected, exposure to hypoxia-induced LV diastolic dysfunction, as shown by prolonged time constant of isovolumic relaxation (τ). Improved relaxation with IL-18BP treatment was demonstrated by a significant reduction towards control τ values. Decreased levels of phosphorylated phospholamban (P-PLB) in hypoxia, but normalization by IL-18BP treatment suggest a role for IL-18 in regulation of calcium-handling proteins in hypoxia-induced diastolic dysfunction. In addition, MRI showed less increase in right ventricular (RV) wall thickness in IL-18BP-treated animals exposed to hypoxia, indicating an effect on RV hypertrophy. CONCLUSION: Neutralization of IL-18 during alveolar hypoxia improves LV diastolic function and partly prevents RV hypertrophy.

The stimulatory effect of soluble fibrin on plasminogen activation by tissue plasminogen activator as studied by the Coa-set Fibrin Monomer test.

The stimulatory effect of various fibrin preparations on plasminogen activation by tissue plasminogen activator, was studied by the Coa-set Fibrin Monomer test (Kabi). Fibrin obtained by complete conversion of purified fibrinogen demonstrated a greater stimulatory effect on plasminogen activation than did equal amounts of fibrin obtained by partial conversion of fibrinogen. Soluble fibrin generated by treating human plasma with minute amounts of thrombin or bathroxobin, resembled partially converted purified fibrinogen. The plasminogen activating effect of completely converted fibrinogen was similar in thrombin and bathroxobin incubated samples. In preparations of partially converted fibrinogen and in plasma samples, bathroxobin digested fibrinogen expressed a more pronounced stimulatory effect on plasminogen activation than did thrombin digested specimens. The underlying mechanism for these differences are discussed.

Thrombin treated plasma employed as a standard for determination of soluble fibrin.

The Coa-set Fibrin Monomer test (CFM-test) is a quantitative method for determination of soluble fibrin in plasma. The fibrin standard of the CFM-test is produced by bathroxobin conversion of purified fibrinogen to fibrin. Plasma treated with minute amounts of thrombin may be considered as a more physiological way of preparing fibrin monomers. Therefore, we have employed such thrombin treated plasma (TTP) as an alternative fibrin standard for the CFM-test. The fibrin concentration of the TTP was determined indirectly by quantitation of released fibrinopeptide A. The TTP was stable during freezing, thawing and storage for 3 months at -70 degrees C. The standard curve obtained using TTP as a standard was linear in the range of 0-275 nmol/l fibrin in plasma, but the slope of the line was less steep than the original standard curve. This difference was probably due to the greater plasminogen activating effect of bathroxobin digested fibrinogen compared to soluble fibrin generated in plasma by thrombin, as observed in a previous study. Because of the less steep slope of the alternative standard curve, fibrin levels in plasma samples from 20 healthy volunteers and 25 patients were found to be higher employing TTP as a standard. Preparation of a fibrin standard by incubation of plasma with minute amounts of thrombin will to some extent mimic the process of fibrin generation in vivo. Since we have found a satisfactory stability of such a standard during freezing, thawing and storage, we think the TTP standard might be useful for quantitation of soluble fibrin in plasma.

Comparison of methods for detecting soluble fibrin in plasma. An in vitro study.

The ability of the COA-SET Fibrin monomer (COA-SET FM) test to detect soluble fibrin was evaluated by comparing the results of the COA-SET FM test with fibrinopeptide A (FPA) determinations following thrombin incubation of plasma or whole blood. In addition, two semiquantitative tests (erythrocytes-agglutination test (FM-test) and ethanol gelation test (EGT] were included in the study. Under the experimental conditions used, the COA-SET FM test proved less sensitive than the FPA-assay. There was a strong correlation between the results obtained by the two tests (r = 0.86, p = 0.0001). When solely regarding low levels of soluble fibrin, however, the correlation was weaker (r = 0.59, p = 0.0003). The FM-test was less sensitive than the COA-SET FM test, but more sensitive than EGT at normal and low fibrinogen concentrations. At high fibrinogen concentrations, however, EGT proved more sensitive than the FM-test. Knowing that 1-2 moles of FPA are released per mole of fibrin monomers formed, a discrepancy was observed between the FPA concentrations and the fibrin monomer concentrations as determined by the COA-SET FM test, the FPA levels being 2-25 times higher than the fibrin monomer levels. The discrepancy was greatest at incipient fibrinogen-fibrin transformation and at high plasma fibrinogen levels. This may suggest that fibrinogen in some way interfered with the stimulating effect of fibrin on the t-PA catalyzed activation of plasminogen, the principle upon which the COA-SET FM test is based.(ABSTRACT TRUNCATED AT 250 WORDS)

Local activation of the coagulation and fibrinolysis systems in lung disease.

Extravascular coagulation and fibrinolysis is an integral part of inflammatory reactions. Disordered expression of procoagulant and profibrinolytic factors by mononuclear phagocytes of the lung (i.e. lung alveolar macrophages (LAM) and interstitial macrophages) may have important bearings on inflammatory lung tissue destruction and repair. Based on this hypothesis we have measured the presence of trigger molecules and activation products of the coagulation and fibrinolytic system in cell-free bronchoalveolar lavage fluid and in bronchoalveolar cells. Patient groups with chronic obstructive disease (COLD) (n = 76), idiopathic pulmonary fibrosis (IPF) (n = 29), sarcoidosis (n = 22), lung cancer (n = 36), pneumonia (n = 39), acquired immunodeficiency syndrome (AIDS) (n = 17) and a control group (n = 60) were studied by bronchoalveolar lavage (BAL). In all patient groups tissue thromboplastin (TPL) and fibrinopeptide A (FPA) were significantly increased compared to controls. Plasminogen activator (PA) activity was significantly lower in patients than in normals, and usually associated with high levels of antifibrinolytic activity. The level of PA inhibitor (PAI-2) was not significantly higher in any patient group compared to controls. The sensitivity of the method for fibrin degradation products (FDP) analysis was not high enough to detect FDP in BAL fluid of control individuals, whereas such products could be demonstrated in 25-53% of patients in various categories. We conclude that disordered expression of procoagulant and plasminogen activator activities in bronchoalveolar lavage fluid may reflect a milieu that favours accumulation of fibrin in inflammatory lung tissue and form the basis for the development of pulmonary fibrosis.

Characteristics of a heat treated antihaemophilic cryoprecipitate.

In order to evaluate the influence of heat treatment (68 degrees C for 24 or 72 hours) on the essential components of antihaemophilic cryoprecipitate, i.e. factor VIII coagulant activity (VIII:C), von Willebrand factor (VIIIR:Ag and VIIIR:RCF) and fibrinogen, ordinary lyophilized cryoprecipitate was compared to heat treated, aminoacid-enriched specimens. The median reduction in factors VIII:C, VIIIR:Ag, VIIIR:RCF and fibrinogen during lyophilization of ordinary cryoprecipitate was 26 per cent, 11 per cent, 1 per cent and 8.5 per cent, respectively. Heat treatment of such cryoprecipitate resulted in 85 to 98.5 per cent reduction in these parameters, while the reduction following lyophilization and heat treatment (24 hours) of aminoacid-containing preparations was not significantly different from non-heated, ordinary cryoprecipitate. Following heating of aminoacid-enriched cryoprecipitate for 72 hours, only factor VIIIR:RCF was significantly reduced (32.5 per cent) compared to non-heated samples. Ordinary cryoprecipitate was almost insoluble following heat treatment. Enrichment with aminoacids, however, made the heat treated cryoprecipitate fully soluble, but the content of these vials were slightly slower in dissolving than non-heated preparations. Ultracentrifugation prior to lyophilization and heating did not improve the solubility. If heat treatment proves to be efficient in inactivating viral agents, we conclude that heated (68 degrees C for 24 hours), aminoacid-enriched cryoprecipitate may be a convenient product for treating haemophilia A, von Willebrand's disease and hypofibrinogenemia.

Degradation of fibrinogen and cross-linked fibrin by human neutrophil elastase generates D-like fragments detected by ELISA but not latex D-dimer test.

Recently, we observed that D-dimers are degraded by human neutrophil elastase (HNE) into two D-like fragments, reacting with the monoclonal antibody in an ELISA D-dimer test but not reacting with the corresponding latex D-dimer test. To investigate this in more detail, we studied the degradation of cross-linked fibrin and fibrinogen by plasmin and HNE to see if this resulted in D-fragments or D-like fragments. Degradation of fibrinogen, both by plasmin and HNE, resulted in D- and D-like fragments, respectively, detected by the ELISA D-dimer test. Degradation of cross-linked fibrin by plasmin and HNE also resulted in D- and D-like fragments, which were detected by the ELISA method. Intact D-dimers detected by the latex D-dimer test were only observed after degradation of cross-linked fibrin with plasmin. We conclude that during lysis of cross-linked fibrin as well as fibrinogen by plasmin and HNE, D-fragments, and D-like fragments, detected by the ELISA D-dimer test, are formed. Only during degradation of cross-linked fibrin by plasmin, intact D-dimers, detected by latex D-dimer test, are formed. The ELISA D-dimer test may therefore be used to detect fibrin and fibrinogen degradation products generated by the combined action of plasmin and HNE in sepsis and other conditions with increased HNE activity, while the latex D-dimer test may be used to detect plasmin derived intact D-dimers.

Discrepancy between latex and ELISA D-dimer values in sepsis may be caused by human neutrophil elastase.

We have recently shown that D-dimers are degraded by human neutrophil elastase (HNE) in vitro, causing rapid decrease in the D-dimer levels measured by a Latex test, but not with an ELISA test employing the same monoclonal antibody against D-dimer. To see if such discrepant D-dimer concentrations occurred in patients with high HNE concentration, we examined 80 plasma samples from 8 patients with sepsis with a Latex and an ELISA test and calculated the ratio between the D-dimer values obtained with the two tests. Twenty healthy pregnant and twenty pre-eclamptic patients, who are known to have raised D-dimer but low HNE concentrations, were chosen as controls. HNE levels were estimated by determining the HNE-alpha 1-proteinase inhibitor complex (HNE-A1PI) concentration. HNE-A1PI concentration was increased in sepsis patients compared with pre-eclamptic patients (p < 0.0005) and healthy pregnant women (p < 0.0005). In sepsis patients, the D-dimer results were skewed towards lower ratios between Latex and ELISA values compared to pre-eclamptic patients (p = 0.008) and healthy pregnant women (p = 0.0001). In plasma samples from patients with the largest discrepancy between Latex and ELISA D-dimer values, Western blotting with immunostaining indicated degradation of D-dimers to D-like fragments similar to those observed following degradation of cross-linked fibrin by HNE in vitro. We conclude that in sepsis patients there is a marked discrepancy between Latex and ELISA D-dimer values that may be caused by HNE. In such patients Latex D-dimer assays may cause severe underestimation of fibrinolysis.

D-dimers are degraded by human neutrophil elastase.

To see if D-dimers were degraded by human neutrophil elastase (HNE), cross-linked fibrin was obtained by adding thrombin to purified fibrinogen in the presence of calcium ions and factor XIII, and the fibrin clot subsequently degraded by plasmin. Thereafter, the supernatant containing fibrin degradation products was removed and incubated with HNE. D-dimer levels were measured by two rapid semiquantitative tests, a latex agglutination test and the Nycocard immunofiltration test, and a quantitative ELISA-method. With increasing incubation time, D-dimer levels as measured by the latex and Nycocard tests rapidly decreased and subsequently became undetectable, while the ELISA D-dimer values remained essentially unchanged. By using SDS-electrophoresis and immunoblotting, the degradation of plasmic derivatives of cross-linked fibrin by fiNE was visualised. We conclude that in a purified system, D-dimers formed during plasmin mediated lysis of cross linked fibrin are further degraded by HNE. Such HNE degradation reduces the D-dimer concentration as measured by rapid semiquantitive tests, and may be partly responsible for discrepant results when using different D-dimer assays.

The influence of pH on heat denaturation of anti-haemophilic cryoprecipitate.

The influence of pH on heat denaturation of anti-haemophilic cryoprecipitate (CP) was studied by using phosphate and citrate buffers at three different pH levels for processing lyophilized, heat treated CP. The solubility and the content of FVIII:C and fibrinogen were determined following heating at 68 degrees C for 24 hours and compared to "ordinary", non-heated CP. Both the solubility and the biological activity were best preserved in the most acidic samples (pH 5.7-6.7), these batches equalled non-heated CP. At higher pH, heat treatment resulted in reduced solubility and a more pronounced loss of FVIII:C and fibrinogen. Amino acids (Syntamin 17) have previously been shown to stabilize CP during heat treatment. Some of the stabilizing effect seems to be due to a large buffering capacity, maintaining a low pH during lyophilization and heating. Raising the pH level in Syntamin-CP from 6.6 to 7.8 resulted in decreased solubility and FVIII:C content. The quality of heat treated, acidic Syntamin-CP was comparable to that of heated, acidic phosphate- and citrate-CP. We conclude that buffers used for processing heat treated CP should be of low pH, and that acidic buffers may replace Syntamin without lowering the quality of the heated product.

A novel principle for assessment of stimulated fibrinolysis.

After venous occlusion (VO), D-dimer levels were measured by means of an ELISA technique, in citrated plasma clotted by thrombin and in serum from whole blood. D-dimer levels increased with duration of incubation (30 min to 24 hours). D-dimer values, both in clotted plasma and in serum (n = 12), incubated 4 hours at room temperature, correlated well with euglobulin clot lysis time (ECLT) (r = -0.85 and -0.89, respectively, p less than 0.002) and tissue plasminogen activator (t-PA) activity, (r = 0.82 and 0.83, respectively, p less than 0.002). D-dimer concentrations from plasma and serum (n = 25) were compared (r = 0.90, p less than 0.001). Healthy volunteers (n = 65) were tested to establish reference values in serum from post-occlusive whole blood samples incubated 4 hours prior to centrifugation. Finally, a patient group (n = 62) was examined. For the whole material (n = 152) such D-dimer concentrations correlated well with both ECLT (r = -0.85, p less than 0.001) and t-PA activity (r = 0.81, p less than 0.001). D-dimer levels in serum were determined by a latex agglutination test as well. These semi-quantitative values also correlated significantly with both ECLT (r = -0.86, p less than 0.001) and t-PA activity (r = 0.87, p less than 0.001). We conclude that measurement of D-dimer as described above, represents a simple and accurate method for assessment of global fibrinolytic activity following VO. The latex agglutination test is particularly suitable as a screening procedure.

Comparison of thrombin-antithrombin complex (TAT) levels and fibrinopeptide A following thrombin incubation of human plasma using hirudin as an inhibitor of TAT formation.

Following incubation of citrated plasma with human thrombin, the interaction of thrombin with antithrombin III was measured as thrombin-antithrombin complex (TAT) concentration. Comparison was made to thrombin activity on fibrinogen, assayed as fibrinopeptide A (FPA). Light scattering studies were included to evaluate polymerization of fibrin. Hirudin, at a final concentration of 100 U/ml, effectively inhibited TAT generation at final thrombin concentrations below 0.4 NIH U/ml. Hirudin by itself did not affect the TAT ELISA analysis. TAT and FPA values correlated closely (r = 0.83, p less than 0.001) and may equally well represent in vitro thrombin activity.

Does Lp(a) lipoprotein inhibit the fibrinolytic system?

Lp(a) lipoprotein contains a unique apolipoprotein, apolipoprotein (a), that has a striking homology with plasminogen. This homology has brought forward speculations as to an inhibitory effect of Lp(a) lipoproteins on fibrinolysis. The present investigation was undertaken to study the influence of Lp(a) lipoprotein on the fibrinolytic system. In an in vitro model, we have studied the influence of purified Lp(a) lipoprotein on plasminogen activation by tissue plasminogen activator (t-PA) in the presence of soluble fibrin. Increasing concentrations of Lp(a) lipoprotein (0-32 mg/dl) did not inhibit plasminogen activation by t-PA in the presence of thrombin or bathroxobin digested fibrinogen. When purified Lp(a) lipoprotein was added to whole blood, the degree of fibrin degradation obtained following standardized coagulation, as evaluated by the generation of D-dimer, was not reduced. D-dimer levels in plasma and in serum after standardized coagulation, as well as conventional parameters for evaluation of the fibrinolytic system, were determined in 10 individuals with high and 10 individuals with low levels of Lp(a) lipoprotein. No differences in the fibrinolytic parameters were observed between the groups. Thus, we found no evidence that Lp(a) lipoprotein interferes with the fibrinolytic process in the present experiments.

Fibrin generation during production of freeze dried antihaemophilic cryoprecipitate.

Thrombin activity during separation and cryoprecipitation of CPD-blood was monitored by fibrinopeptide A (FPA) determinations. After pooling and lyophilization of cryoprecipitate, the total amount of contaminating fibrin was estimated by N-terminal amino acid analyses. In addition, retention of fibrin in standard transfusion filters (170 micron) was examined by gamma counting of 125I des-AA fibrin monomer enriched cryoprecipitate prior and subsequent to filtration. Prior to pooling of cryoprecipitate, thrombin activity, as estimated by FPA levels, was most pronounced during collection of blood from the blood donors and during cryoprecipitation and thawing of the plasma bags. Comparison of these FPA concentrations to the total amount of fibrin in pooled, freeze dried cryoprecipitate, as estimated by N-terminal analyses, revealed a considerable generation of fibrin during the process of lyophilization. In freeze dried cryoprecipitate, 5.3 per cent (range 3.0-7.5 per cent) of the fibrinogen had been converted to fibrin, implying a fibrin content of 20.3-60.3 mg per bottle of 500 U factor VIII. The amount of fibrin in two bottles of a commercially available factor VIII concentrate, also containing 500 U of factor VIII, was 14.1 and 19.8 mg, respectively. Sham transfusions of 125I des-AA fibrin monomer enriched cryoprecipitate revealed that only 1.0 per cent (range 0-2.5 per cent) of the fibrin was retained in the standard transfusion filters. Thus, substantial amounts of fibrin may be transfused to patients upon treatment with freeze dried cryoprecipitate.

Contaminating fibrin in CPD-blood: solubility in plasma and distribution in blood components following separation.

In order to estimate the solubility of contaminating fibrin in CPD-blood, thrombin induced fibrin polymerzation in CPD-plasma was examined by light scattering and fibrinopeptide A (FPA) determinations. In addition, I125 fibrin monomer enriched CPD-blood was used to investigate fibrin monomer retention in blood bags and transfusion filters (170 microns) and fibrin distribution in blood components derived from CPD-blood. Initial fibrin polymerization in CPD-blood occurred after conversion of 15 per cent of the fibrinogen to fibrin, implying that substantial amounts of fibrin may be kept solubilized in CPD-blood bags. Only minor amounts of I125 fibrin monomers were retained in blood bags (2.4 per cent) and in transfusion filters (2.9 per cent) after sham transfusions. After separating I125-fibrin monomer enriched CPD-blood into its constituent components, the major part of fibrin (75.0 per cent) could be traced in the cryoprecipitate.

Degradation of the alpha-chain of fibrin by human neutrophil elastase reduces the stimulating effect of fibrin on plasminogen activation.

UNLABELLED: The degradation of fibrin by human neutrophil elastase (HNE) and the interference of such degradation on the stimulating effect of fibrin on plasminogen activation by tissue plasminogen activator (t-PA) was studied. By using SDS electrophoresis and Western blotting with subsequent immunostaining with monoclonal antibodies, degradation of the fibrin molecule was monitored. This degradation was related to the stimulating effect on plasminogen activation. Degradation of the alpha-chain was seen to occur before degradation of the beta- and gamma-chains. On the alpha-chain it was found that C-terminal degradation occurred prior to visible degradation of the N-terminal end. This C-terminal degradation was associated with a fall in the stimulation of plasminogen activation, coinciding with a corresponding reduction in the polymerization of fibrin. With further degradation, including N-terminal proteolysis of the alpha-chain, the stimulating effect of fibrin was reduced to that of fibrinogen. CONCLUSIONS: Our results indicate that HNE degradation of the alpha-chain of fibrin occurs initially from the C-terminal end, affecting the polymerization of fibrin. This impaired polymerization may be important for the observed reduction in the t-PA mediated plasminogen activation.

Impaired coagulation of fibrinogen due to digestion of the C-terminal end of the A alpha-chain by human neutrophil elastase.

Human neutrophil elastase (HNE) possesses fibrinogenolytic capacity, with a high susceptibility towards degradation of the A alpha-chain. To study the influence of HNE digestion of the A alpha-chain on the coagulation of fibrinogen by thrombin, fibrinogen was incubated with human neutrophil elastase (HNE). At intervals, thrombin clotting time (TCT) and clottability were determined and compared with the patterns obtained with SDS electrophoresis and Western blotting with subsequent immunostaining, using monoclonal antibodies against the N-terminal end and C-terminal half of the A alpha-chain. Apparently, initial HNE digestion of the fibrinogen molecule occurred from the C-terminal end of the A alpha-chain, and was associated with prolongation of the TCT. With further C-terminal degradation TCT became indefinite and the degradation products were no longer clottable. Finally, N-terminal degradation of the A alpha-chain was observed. The present observations suggest that initial HNE-digestion of fibrinogen occurs from the C-terminal end of the A alpha-chain, and that the C-terminal end is crucial for the coagulation of fibrinogen.

Leukotriene antagonism reduces the generation of endothelin-1 and interferon-gamma and inhibits eosinophilic airway inflammation.

The cysteinyl leukotrienes (cysLTs) and the peptide hormone endothelin (ET)-1 are potent bronchoconstrictor substances, and these mediators are also claimed to be implicated in the development of eosinophilic airway inflammation. In the present study, we have investigated the effect of the cysLT1 receptor antagonist montelukaston the development of an eosinophilic airway inflammation 24 h after intratracheal Sephadex (SDX) provocation in rats. Furthermore, the effect of montelukast treatment on the generation of ET-1 and other pro-inflammatory mediators has been studied. The inflammatory response was significantly reduced in the animals receiving SDX + montelukast compared to animals receiving solely SDX, as evaluated by a decrease in bronchoalveolar lavage fluid total cell count (10.3 +/- 1.2 vs. 18.5 +/- 1.8 x 10(4) ml(-1), P<0.001), number of eosinophils (299.7 +/- 43.8 vs. 577.6 +/- 46.6 x 10(2) ml(-1), P<0.001), and lymphocytes (116.8 +/- 20 vs. 222.0 +/- 34.8 x 10(2) ml(-1), P<0.05), as well as the degree of tissue inflammation (P<0.05). Montelukast also inhibited the increase in the concentration of the pro-inflammatory mediators ET-1 (28.5 +/- 75 vs. 40.9 +/- 7.3 x pg ml(-1), P<0.05) and interferon (IFN)-gamma (4.3 +/- 2.2 vs. 15.6+/-8.7 x pg ml(-1), P<0.05), but not tumor necrosis factor-gamma or interleukin-8. In summary, treatment with the cysLT1 receptor antagonist montelukast reduced the inflammatory response during development of an eosinophilic airway inflammation, possibly by inhibiting the release of pro-inflammatory mediators like ET-1 and IFN-gamma.