Elastase-alpha 1-proteinase Inhibitor Research Articles

The effects of non-surgical periodontal therapy on neutrophil elastase and elastase alpha-1 proteinase inhibitor levels in GCF in periodontitis patients with or without acute coronary syndrome.

Periodontitis may contribute to vascular damage, resulting in the destabilization of atherosclerotic plaque leading to acute coronary syndrome (ACS). In this study, we explored the effect of non-surgical periodontal treatment (NSPT) on cardiovascular blood biomarkers and gingival crevicular fluid (GCF) neutrophil elastase (NE) and α1-proteinase inhibitor (α-1PI) levels in periodontitis (P) participants with and without ACS. Medical and dental examinations were performed to diagnose ACS and periodontitis, respectively. Seventeen patients with diagnosis of ACS and periodontitis were included in this study, as a test group (group ACS). Twenty-six age and sex-matched control patients with periodontitis (group P) were otherwise systemically healthy. Both groups received NSPT. Plasma levels of cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), C-reactive protein (CRP), GCF NE activity, GCF α1-PI levels, and GCF NE/α1-PI rates were measured at baseline, at1st and 3rd months after NSPT. GCF NE activity/time (μU/30s) decreased significantly at 3rd month compared to baseline values in the Group P after NSPT. First and 3rd months after NSPT, in the Group P GCF α1-PI activity/time (pg/30s) was significantly higher than the Group ACS. Moreover GCF NE/α1-PI rates decreased significantly compared to baseline values at 1st and 3rd months after NSPT in the group P. NSPT yields decrease in NE/α1-PI rates. NE and its possible interactions with α1-PI may play a crucial role in both periodontitis and ACS. GCF α1PI activity/time (U/30s) can be a potential biomarker in management of periodontitis associated with ACS. The GCF α1-PI reduction may alter the immune-inflammatory response in patients with periodontitis and thus increase the risk of ACS. Thai Clinical Trials.gov (NCT04785235).

Human Atherosclerotic Plaque Contains Viable Invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis

To the Editor: Because epidemiological evidence supports an association between cardiovascular and periodontal disease, we assessed whether periodontal pathogens were present in atherosclerotic lesions. To detect invasive bacteria, the natural tropism of the bacteria toward human tissues was exploited. Further, bacterial presence was demonstrated using quantitative polymerase chain reaction (Q-PCR). This confirms the presence of periodontal pathogens in atherosclerotic lesions, whereby the bacteria could contribute to the vascular pathology either directly through their cytotoxicity or indirectly by inducing or exacerbating inflammation. Cardiovascular disease (CVD) is the leading cause of death in the in the United States.1 According to the American Heart Association’s statistics from 2003, there were no previous symptoms in 50% of men and 63% of women who died suddenly from CHD. In a 10-year follow-up study, ≈25% of coronary deaths in males and 15% in females occurred in persons in the lowest two quintiles of the multivariate Framingham Heart Study risk scores.2 This and other data have led to an emerging paradigm shift from coronary heart disease having a purely hereditary/nutritional causation to possibly having an infectious component.3 Many epidemiological studies strongly suggest that periodontitis may be a risk factor for coronary heart disease (CHD).4 Serologically, edentulousness and serum IgG-antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in 1163 men were recently shown to be associated with CHD.5 In a larger prospective study of 6950 subjects, the same authors provide serological evidence that an infection caused by major periodontal pathogens is associated with future stroke.6 Previous studies have identified 16S rRNA of oral microbial pathogens, including P gingivalis and A actinomycetemcomitans , …

Stimulation of inflammatory markers after blunt trauma.

Inflammatory mediators are released after trauma and may be related to the pathogenesis of sepsis. A prospective combined study of the pattern of release of an inflammatory mediator, interleukin (IL) 6, leucocyte activation (polymorphonuclear leucocyte (PMN) CD11b receptor expression and plasma elastase-alpha1 proteinase inhibitor complex (E-alpha1PI)) and soluble endothelial adhesion molecule expression (soluble E-selectin (sE-selectin) and soluble intracellular adhesion molecule 1 (sICAM-1)) was performed in patients suffering blunt trauma without adult respiratory distress syndrome (ARDS) or multiple organ failure syndrome (MOFS). Thirty-one patients with a mean Injury Severity Score (ISS) of 14 (range 9-57) were studied. Venous blood samples were collected within 6 h of injury and then at 1, 3, 5 and 7 days. Leucocyte CD11b expression was quantified by flow cytometry. Serum IL-6, plasma E-alpha1PI, sE-selectin and sICAM-1 were measured by enzyme-linked immunosorbent assay. Serum IL-6, CD11b expression and E-alpha1PI levels were significantly raised above control values (P < 0.0001) on admission, slowly returning towards control values over the study period (median IL-6, 140 pg/ml versus undetectable; CD11b, 14.8 versus 6.4 mean channel fluorescence units; E-alpha1 PI, 208 versus 52 microg/l). The sICAM-1 level rose to a median of 539 ng/ml at 5 days (control 243 ng/ml). The median sE-selectin level also progressively increased to a maximum level of 80 ng/ml at 5 days (control 49 ng/ml). Eleven patients developed postoperative sepsis. Significant differences in CD11b expression were seen at days 3, 5 and 7 and in E-alpha1 PI at 6 h, 24 h and 3 days in patients who subsequently developed sepsis (P < 0.05). Severe injury (ISS 16 or greater) was associated with significantly greater responses in these measurements. These data show that markers of inflammation are specifically stimulated by trauma even when ARDS and MOFS do not occur. The CD11b receptor on PMNs may be useful in screening patients destined to develop post-traumatic sepsis.

Diagnostic potential of neutrophil elastase inhibitor complex in the routine care of critically ill newborn infants

It has been suggested that determination of the neutrophil elastase alpha1-proteinase inhibitor complex (E-alpha1PI) improves the diagnosis of bacterial infection in newborns. We evaluated the use of E-alpha1PI measurements in 143 newborns, consecutively admitted to a tertiary intensive care unit, employing a new random access assay and a sampling procedure that minimises post-collection artefacts. The 95% range for noninfected newborns was 20-110 microg/l up to the 5th day of life and 20-85 microg/l thereafter. The sensitivity as to the diagnosis of culture-proven bloodstream infection was 80% for E-alpha1PI, 86% for the immature to total neutrophil ratio, 64% for C-reactive protein and 37% for the total white blood cell count. The corresponding specificity amounted to 97%, 85%, 85% and 86%, respectively. E-alpha1PI increases preceded elevations of C-reactive protein by 18 h. Like C-reactive protein, E-alpha1PI levels did not distinguish between bloodstream infection and non-bacterial inflammatory responses. Results of E-alpha1PI became available within 1 h of collection and usually 2-3 h before manual leucocyte counts. Determination of neutrophil elastase alpha1-proteinase inhibitor levels yields diagnostic advantages comparable to those of manual differential counts but provide faster turnaround times.

Effect of collection and preprocessing methods on neutrophil elastase plasma concentrations.

Elevated plasma levels of the elastase alpha 1-proteinase inhibitor complex (E-alpha 1 PI) have been proposed as a marker of bacterial infection and neutrophil activation. Liberation of elastase from neutrophils after collection of blood may cause falsely elevated results. Collection methods have not been validated for critically ill neonates and children. We evaluated the influence of preanalytical methods on E-alpha 1 PI results including the recommended collection into EDTA tubes. First, we compared varying acceleration speeds and centrifugation times. Centrifugation at 1550 g for 3 min resulted in reliable preparation of leukocyte free plasma. Second, we evaluated all collection tubes under consideration for absorption of E-alpha 1 PI. Finally, 12 sets of samples from healthy adults and 42 sets obtained from critically ill neonates and children were distributed into the various sampling tubes. Samples were centrifuged within 15 min of collection and analyzed with a new turbidimetric assay adapted to routine laboratory analyzers. One of the two tubes containing a plasma-cell separation gel absorbed 22.1% of the E-alpha 1 PI content. In the remaining tubes without absorption of E-alpha 1 PI no differences were observed for samples from healthy adult patients. However, in samples from critically ill neonates or children, significantly higher results were obtained for plain Li-heparin tubes (mean = 183 micrograms/L), EDTA tubes (mean = 93 micrograms/L), and citrate tubes (mean = 88.5 micrograms/L) than for the Li-hep tube with cell-plasma separation gel and no absorption of E-alpha 1 PI (mean = 62.4 micrograms/L, p < 0.01). Contrary to healthy adults, E-alpha 1 PI results in plasma samples from critically ill neonates and children depend on the type of collection tube.

Efficacy of systemically administered acetylsalicylic acid plus scaling on periodontal health and elastase‐α1‐proteinase inhibitor in gingival crevicular fluid

The purpose of this proof of principle trial was to assess whether conventional periodontal therapy and systemically administrated acetylsalicylic acid (ASA) are functionally synergistic when combined in the treatment of periodontitis. A total of 30 patients with untreated moderate to severe adult periodontitis were enrolled into the study and were given placebo q.i.d. between the baseline and 6-week examination, and acetylsalicylic acid (ASA) 500 mg q.i.d. between the 6-week and 12-week examinations. In addition, they received supra- and subgingival scaling in 1 quadrant after baseline examination and in 2 further randomly selected quadrants after the 6-week examination. The study design resulted in the following 4 therapies: (1) scaling plus ASA 500 mg q.i.d.; (2) scaling plus placebo q.i.d.; (3) ASA 500 mg q.i.d. alone; (4) placebo q.i.d. alone. Two-way analysis of variance showed functional synergism of ASA and scaling, resulting in a therapeutic efficacy approximately equivalent to the sum of each individual therapeutic efficacy (i.e., ASA alone and scaling alone) in reducing gingival inflammation and pocket probing depth over the 6-week observation period (interaction: p > 0.05). Only the effect of ASA was significant in reducing the concentration of elastase-alpha 1-proteinase inhibitor in gingival crevicular fluid (GCF E-alpha 1-PI) (p < 0.001), reduction in GCF E-alpha 1-PI concentrations by ASA may indicate a decreased risk in periodontal disease progression. The results suggest that the combination of therapies and their different mechanisms of action, i.e., reduction of bacterial plaque and inhibition of destructive components of the immune responses, may result in functionally synergistic therapeutic efficacies in patients with untreated adult periodontitis.

Inflammatory bronchopulmonary response of preterm infants with microbial colonisation of the airways at birth.

The inflammatory indicators in the tracheobronchial aspirate (TA) of 81 ventilated preterm infants with microbial colonisation of the airways and in non-colonised neonates were analysed on the first day of life. TA was assessed for chemotactic activity, neutrophil cell count, and concentrations of leukotriene B4, C5a, interleukin-1, interleukin-8, elastase-alpha 1-proteinase inhibitor, free elastase and albumin. Concentrations of mediators were related to concentrations of the secretory component of IgA. The infants' gestational age was mean (SD) 27.9 (2.0) weeks, birthweight 945 (179) g. In 12 infants (15%) microbial colonisation of the airways was present (Ureaplasma urealyticum n = 7; bacteria n = 5). Compared with non-colonised neonates (n = 69), chemotactic activity, neutrophil count, and concentrations of interleukin-1, leukotriene B4 and elastase-alpha 1-proteinase inhibitor were significantly higher in the colonised group. The difference was most pronounced for IL-1 concentrations, both with and without correction for secretory component. There was also a trend towards increased concentrations of interleukin-8 in the latter group. There were no differences for concentrations of C5a and albumin in the TA of both groups. It is concluded that airway colonisation with U urealyticum or bacteria at birth is associated with a clinically relevant bronchopulmonary inflammatory response. Increased concentrations of interleukin-1 in TA on the first day of life may be a marker of perinatal colonisation of the airways.

Measurements of Plasma Elastase α1-Proteinase Inhibitor Complexes in Patients Receiving Cancer Chemotherapy With Granulocyte Colony-Stimulating Factor

We monitored the plasma elastase alpha 1-proteinase inhibitor complex levels in 21 patients with primary lung cancer who received combination chemotherapy with or without recombinant human granulocyte colony-stimulating factor (rhG-CSF), and 15 normal nonsmokers as controls. Of the 21 patients, 14 received combination chemotherapy without rhG-CSF (among them, 6 developed pneumonia) and 7 received combination chemotherapy with rhG-CSF (among them, 1 developed pneumonia). We measured peripheral WBC counts, C-reactive protein (CRP) levels, plasma elastase alpha 1-proteinase inhibitor complex (complex) levels, and complex/WBC values during cancer chemotherapy. In patients who received cancer chemotherapy without rhG-CSF and had no complications (n = 8), WBC values decreased after chemotherapy, and then gradually increased. Complex levels also decreased slightly after chemotherapy and gradually recovered. The value obtained from dividing the complex concentration by WBC count (complex/WBC value) remained stable during cancer chemotherapy. In patients who received cancer chemotherapy with rhG-CSF and had no complications (n = 6), WBC values decreased after chemotherapy, and then rapidly increased to abnormally high values. Complex levels also decreased slightly after chemotherapy and rapidly increased to abnormally high values together with the WBC counts. The complex/WBC values remained stable during cancer chemotherapy. In patients who developed pneumonia during cancer chemotherapy with or without rhG-CSF (n = 7), their complex levels, complex/WBC values, and CRP levels were elevated at the onset of pneumonia. The maximum complex levels (the highest levels during chemotherapy) were significantly higher in patients who received cancer chemotherapy with rhG-CSF and did not develop pneumonia (583.1 +/- 114.5 ng/mL) and in patients who developed pneumonia during cancer chemotherapy (516.7 +/- 113.2 ng/mL), compared with normal nonsmokers (130.2 +/- 5.5, p < 0.01) and patients who received cancer chemotherapy without rhG-CSF and did not develop complications (211.5 +/- 23.3, p < 0.01). The maximum complex/WBC values were not increased in patients who received cancer chemotherapy with rhG-CSF (0.08 +/- 0.01) and patients who received cancer chemotherapy without rhG-CSF (0.092 +/- 0.01, p < 0.01). The maximum complex/WBC values were significantly higher in patients with pneumonia (0.56 +/- 0.12) compared with normal nonsmokers (0.026 +/- 0.002, p < 0.01) and patients without complications. These findings suggest that although rhG-CSF increases total plasma elastase burden, increased release of neutrophil elastase from individual neutrophils does not take place in vivo in the absence of pneumonia.

Elastase Given Intracisternally Opens Blood-Brain Barrier in Newborn Piglets

The effect of elastase on the blood-brain barrier (BBB) permeability was intravitally studied by fluorescence photomacroscope using the open cranial window technique in newborn piglets. Eleven animals (group 1) were given intracisternal injection of porcine elastase (1.0 micrograms), while 7 piglets served as controls (group 2). Elastase administration resulted in spotty sodium fluorescein (MW 376 daltons) extravasations in pial venules in all animals of group 1 78 +/- 4 min (mean +/- SEM) after the challenge, and in a 2-fold increase (p < 0.05) in brain sodium fluorescein uptake both in occipital cortex and white matter. The concentration of elastase-alpha 1-proteinase inhibitor complex increased significantly (p < 0.05) in cerebrospinal fluid samples in group 1, 2 and 4 h after the injection, while it did not change in sera. A significant pleocytosis and leukocytosis was also seen in group 1, while there was no change in laboratory data and BBB remained tight in group 2. BBB permeability changes during neonatal meningitis may be caused, at least partially, by elastase.

Plasma levels of granulocyte elastase-alpha 1-proteinase inhibitor complex in patients with disseminated intravascular coagulation: pathophysiologic implications.

To investigate the role of neutrophil activation in the pathophysiology and sequelae of disseminated intravascular coagulation (DIC), we measured plasma levels of granulocyte elastase-alpha 1-proteinase inhibitor complex (GEPIC) in 41 patients with DIC and 27 patients with similar underlying conditions but without DIC. Mean GEPIC levels were significantly higher in patients with DIC (421.0 +/- 45.6 ng/ml) than in patients without DIC (246.1 +/- 41.9 ng/ml, P < 0.01). Significant differences were also noted in DIC patients with or without infection (474.7 +/- 61.2 ng/ml vs. 302.4 +/- 48.9 ng/ml, P < 0.04), with or without organ dysfunction (546.6 +/- 72.7 ng/ml vs. 305.6 +/- 42 ng/ml, P < 0.01), and with or without respiratory failure (640.0 +/- 91.2 ng/ml vs. 328.1 +/- 55.1 ng/ml, P < 0.01). No significant difference was found in mean GEPIC levels in DIC patients with or without renal failure, heart failure, hepatic failure, or gastrointestinal bleeding. The frequency of respiratory failure correlated with rising plasma levels of GEPIC. Mortality was higher in patients with GEPIC levels > 500 ng/ml (53.8%) than in patients with GEPIC levels < 500 ng/ml (28.6%). This correlation was particularly strong in patients with DIC, infection, and respiratory failure. Based on these data, we suggest that neutrophil activation, triggered by the coagulation cascade and perhaps augmented by endotoxin or cytokine release with infection, significantly contributes to respiratory failure and mortality in patients with DIC.

Demonstration of a two-step reaction mechanism for the inhibition of heparin-bound neutrophil elastase by alpha 1-proteinase inhibitor.

Heparin decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor as a result of its strong binding to the enzyme. Here, we used the slow-binding kinetic approach to decide whether the enzyme-inhibitor interaction proceeds via a two-step mechanism and to identify the step that is affected by heparin. The inhibition kinetics was assessed under pseudo-first-order conditions using conventional or stopped-flow spectrophotometry. In the absence of heparin, the pseudo-first-order rate constant of inhibition increased linearly with the inhibitor concentration indicating that within the experimental concentration range (< or = 6 microM) the enzyme-inhibitor association conforms either to a simple bimolecular reaction (E+I kass-->EI with kass = 10(7) M-1 s-1) or to a two-step reaction (E+I Ki*<==>EI* k2-->EI with Ki* > 0.4 microM and k2 > 4 s-1). In the presence of heparin, the rate constant of inhibition varied hyperbolically with the inhibitor concentration, indicating that the inhibition is a two-step process with Ki* = 80 nM and K2 = 0.15 s-1. Thus, heparin has two opposite effects on the elastase + alpha 1-proteinase inhibitor interaction: it favors the association by decreasing Ki* but impairs it by decreasing k2. This rationalizes the previously demonstrated rate-depressing effect of the sulfated polymer. Heparin does not significantly alter the stability of the irreversible elastase-alpha 1-proteinase inhibitor complex.

Peri-operative elastase-alpha-1 proteinase inhibitor in patients with postoperative intervertebral discitis.

In a prospective study of 251 patients operated upon for lumbar disc herniation it has been investigated whether the preoperative and early postoperative values of Elastase-alpha-1 Proteinase Inhibitor (EPI)-an indicator of inflammatory processes-and C-reactive Protein (CRP)-a well known predictor of some postoperative complications-were correlated to the later development of discitis. Postoperatively discitis developed in 14 patients. A randomly chosen group of 15 complication-free patients out of the total of 251 cases was used as control group. Elevated EPI plasma values, especially in the pre-operative and first postoperative days, turned out to be significantly related to the likelihood of later discitis development, but no such relation for the CRP plasma values could be established. Thus and early prediction of patients at risk for this complication seems to be possible by pre- and postoperative measurement of EPI. It could be justified-but its usefulness has yet to be proven-to give antibiotics prophylactically and other anti-inflammatory medication in patients with elevated pre- and postoperative EPI values.

Effects of dexamethasone on chemotactic activity and inflammatory mediators in tracheobronchial aspirates of preterm infants at risk for chronic lung disease

To evaluate the effects of dexamethasone on pulmonary inflammation and permeability in preterm infants at high risk for chronic lung disease (birth weight < 1200 gm), we assessed tracheobronchial aspirate fluid for chemotactic activity and concentrations of mediators of inflammation. In a prospective study, 21 infants still undergoing mechanical ventilation at day 10 of postnatal age who required a fraction of inspired oxygen > or = 0.3, a peak inspiratory pressure > or = 16 cm H2O, or both were randomly assigned to treatment with dexamethasone at day 10 (early treatment group, n = 10) or day 16 (late treatment group, n = 11). The groups were compared with respect to all measurements on day 15; the late treatment group served as a control group. Additionally, the effects of dexamethasone within both groups were evaluated. In the early treatment group, the chemotactic response of peripheral blood neutrophils exposed to tracheobronchial aspirate fluid was significantly reduced 5 days after initiation of dexamethasone treatment compared with pretreatment values of the late treatment group (median (25th to 75th percentile): migratory distance before dexamethasone, 149 microns (140 to 173 microns); after dexamethasone, 81 microns (68 to 114 microns); p < 0.01). In addition, the following values were decreased after dexamethasone therapy in the early treatment group: number of neutrophils in tracheobronchial aspirate fluid (p < 0.05), and concentrations of leukotriene B4 (p < 0.01), interleukin-1 (p < 0.01), elastase-alpha 1-proteinase inhibitor (p < 0.01), and albumin (p < 0.01). Free elastase activity was found in only two infants; detectable activity of protective alpha 1-proteinase inhibitor was present in the others. Analysis of dexamethasone effects within the groups showed that all measurements were significantly decreased after both the early and the late treatment regimens, with the exception of leukotriene B4 and interleukin-1, which declined only after early dexamethasone treatment. Our results indicate that the pulmonary inflammatory response and microvascular permeability are decreased by dexamethasone, which affects the release of inflammatory mediators and neutrophil influx into the airways of preterm infants who require mechanical ventilation.

Granulocyte elastase alpha 1-proteinase inhibitor complex measurement in very low birthweight infants with severe intraventricular hemorrhage.

We investigated the relationship between the levels of granulocyte elastase alpha 1-proteinase inhibitor complex (E-alpha 1-PI) in plasma and severe intraventricular hemorrhage (IVH) in very low birthweight infants. The concentrations of E-alpha 1-PI and the ratio of the concentrations of E-alpha 1-PI to polymorphonuclear leucocyte counts within 24 h of birth in infants with severe IVH were significantly higher compared with those in infants without severe IVH. E-alpha 1-PI seems to be a useful indicator of IVH.

Measurement of Elastase and Cysteine Proteinases in Synovial Fluid of Patients with Rheumatoid Arthritis, Sero-Negative Spondylarthropathies, and Osteoarthritis

Synovial fluid samples were collected from 45 patients with rheumatoid arthritis, spondylarthropathy, or osteoarthritis, to study their content of elastase (EC 3.4.21.37) and of cysteine proteinases (EC 3.4.22.1, 3.4.22.15). We measured both elastase complexed with alpha 1-proteinase inhibitor and elastase activity toward the substrate L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. Cysteine proteinase activities were measured with the substrates N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) and Z-Arg-Arg-AMC and the inhibitor E-64 [L-trans-epoxysuccinyl-leucyl-amido-(4-guanidino)-butane]. In all these enzyme assays, higher median values were obtained in inflammatory arthropathies than in osteoarthritis. The concentration of the elastase-alpha 1-proteinase inhibitor complex and of elastase and cysteine proteinase activities were statistically higher in patients with rheumatoid arthritis than in patients with osteoarthritis. The difference in results between patients with spondylarthropathy and patients with osteoarthritis was statistically significant only for the elastase-alpha 1-proteinase inhibitor complex. The median values of the complex and of both enzyme activities were higher in patients with rheumatoid arthritis than in patients with spondylarthropathy; however, the difference was statistically significant only for the cysteine proteinase activity measured with Z-Arg-Arg-AMC substrate. These results suggest that both elastase and cysteine proteinases, which are increased in patients with inflammatory arthritis, are involved in cartilage degradation in these arthropathies.

Capillary plasma elastase alpha 1-proteinase inhibitor in infected and non-infected neonates.

Capillary heel prick plasma elastase alpha 1-proteinase inhibitor (E alpha 1-PI) measured by an immunoassay (using commercially available reagents) was examined as an early indicator of neonatal sepsis. Fifty five infants were studied within 24 hours of birth; 60 (including 10 studied on the first day of life) were examined between two and 30 days after birth. Reference ranges for the neonatal period were developed. Raised E alpha 1-PI concentrations (range 440-2600 micrograms/l) were found at the outset of each of the 24 infectious episodes including five with concomitant neutropenia. On the first day of life, obstetric and neonatal complications were also associated with high concentrations (range 190-2400 micrograms/ml). In infants who survived infection, E alpha 1-PI normalised with antibiotic treatment. It is concluded that capillary heel prick plasma is suitable for E alpha 1-PI testing and raised concentrations provide a sensitive but non-specific index of infection in the first 24 hours after birth. Sequential testing may provide early warning of infectious complications and serve as a guide to the cessation of antibiotic treatment.

Increased levels of plasma thrombomodulin in chronic myelogenous leukemia.

Circulating blood plasma contains proteinase-degraded forms of thrombomodulin that are soluble. We quantitatively assayed the plasma levels of thrombomodulin in 15 patients with chronic myelogenous leukemia (CML) in chronic phase by method of an enzyme-linked immunosorbent assay using a monoclonal antibody to protease-degraded products of thrombomodulin. Plasma levels of thrombomodulin in patients with CML at diagnosis were significantly increased (19.5 +/- 6.2 ng/ml: means +/- SD) compared with the levels in normal controls (8.0 +/- 1.9 ng/ml, n = 20) (P less than 0.001). Fibrin degradation products (D-dimer), thrombin-antithrombin III complex, and plasmin alpha 2-antiplasmin complex were almost normal, suggesting that intravascular coagulation or plasmin-mediated fibrinolysis little occurred in these patients. On the other hand, the plasma levels of elastase-alpha 1-proteinase inhibitor (E-alpha 1PI) complex, which was the indicator of released leukocyte elastase, were significantly increased in CML (P less than 0.0001). The plasma levels of thrombomodulin and E-alpha 1PI complex were decreased in parallel with decline of leukocyte counts in 10 patients with CML following anti-leukemic therapy. Furthermore, a statistically significant correlation was observed between the plasma levels of thrombomodulin and E-alpha 1PI complex obtained at 39 time points in 15 patients with CML (r = 0.81, P less than 0.001). These results suggest that the increased plasma levels of thrombomodulin in CML may be partly caused by leukocyte elastase, which may split the surface thrombomodulin and release protease-degraded fragments of it into the circulation.

Effect of Dialyser Composition and Reuse on Neutrophil Count and Elastase α-1 Proteinase Inhibitor Complex Formation

To assess the inter-relationship of leucopenia and PMN elastase release we undertook a prospective crossover study of 6 patients dialysed with new and reused cuprophane, cellulose acetate and polysulfone membranes. Serial blood samples were analysed for PMN count, and elastase-alpha 1-proteinase inhibitor complex (E alpha 1PI) concentrations. After 15 min dialysis with new membranes median PMN counts fell by 72.2%, 25.3% and 22.1% with cuprophane, cellulose and polysulfone, respectively. With reuse the decreases were reduced to 6.4%, 8% and 13.6%. All membranes produced a gradual increase of E alpha 1PI. Median E alpha 1PI accumulation rates (ng ml-1 min-1) with new membranes were 175, 169 and 187 for cuprophane, cellulose acetate and polysulfone, respectively. With reuse of cuphrophane and cellulose acetate these rates fell to 99 and 109 (p less than 0.05 and p less than 0.05, respectively), however, with polysulfone it remained unchanged at 180 ng ml-1 min-1. This study highlights differences between two aspects of the neutrophil response to haemodialysis, and demonstrates that extrapolation from individual parameters to conclusions concerning biocompatibility may be inappropriate.

Towards an understanding of the mode of action of Bacillus Calmette-Guérin-therapy in bladder cancer treatment, especially with regard to the role of fibronectin.

The interaction between Bacillus Calmette-Guérin (BCG) and the host was investigated after repeated intravesicular BCG-therapy for superficial bladder cancer. Studies were performed on (a) the local reaction in the bladder, (b) the systemic reaction, and (c) short and long term interactions in both the bladder and the serum/plasma. The analytes measured included anti-BCG IgA and IgG, fibronectin, lactoferrin, elastase-alpha 1-proteinase inhibitor, myeloperoxidase and alpha 2-proteinase inhibitor. All analytes, with the exception of alpha 2-proteinase inhibitor, were measured in both serum/plasma and urine. An additional group of 94 patients undergoing bronchoalveolar lavage was used for comparison with other diseases affecting mucous membranes. In vitro studies on human bladder in culture were also carried out to study the relationship between BCG, elastase and fibronectin. The results revealed a normal defence reaction, in which IgA and IgG antibodies specific to BCG were produced by the host. Maximal concentrations of all analytes in urine were found about 4 h after BCG instillation. Immunoglobulins, soluble fibronectin, and granulocyte markers all appeared in urine after instillation and all showed a similar time course. The in vitro study showed the synergistic effect of elastase and BCG in stimulating the host defence reaction. The relationship between BCG and fibronectin can be seen as fortuitous but not indicative of the efficacy of BCG-therapy in patients with superficial bladder cancer.

Pseudomonas aeruginosa elastase does not inactivate alpha 1-proteinase inhibitor in the presence of leukocyte elastase

Pseudomonas aeruginosa elastase rapidly inactivates alpha 1-proteinase inhibitor by splitting its Pro-357-Met-358 peptide bond. The present study was aimed at testing whether this reaction takes place in the presence of leukocyte elastase. To this end was added alpha 1-proteinase inhibitor to a mixture of the two elastases, and we performed the following assays: (i) measurement of the residual leukocyte elastase activity, (ii) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) immunoassay of the leukocyte elastase-alpha 1-proteinase inhibitor complex. These experiments were done with various concentrations of the three proteins. All experiments gave the same result: leukocyte elastase was fully inhibited by alpha 1-proteinase inhibitor in the presence of P. aeruginosa elastase even when the bacterial enzyme was 10-fold more concentrated than the neutrophil enzyme. We also measured the initial rate of the P. aeruginosa elastase-catalyzed inactivation of alpha 1-proteinase inhibitor as a function of the inhibitor concentration. The kcat/Km value derived from this experiment was 9 x 10(4) M-1 s-1, a value much lower than the rate constant for the leukocyte elastase-inhibitor association (kass, 1.7 x 10(7) M-1 s-1). This rationalizes the above results. In conclusion, when alpha 1-proteinase inhibitor is faced with its target enzyme, leukocyte elastase, it will perform its physiologic antielastase function even if the bacterial elastase is present in excess.