Proteinase inhibitory activities of antileukoprotease are represented by its second COOH-terminal domain.

Antileukoprotease or secretory leukocyte proteinase inhibitor is a potent serine proteinase inhibitor produced by exocrine glands of the human body. This monomeric protein (107 amino acids) comprises two homologous domains. It is generally thought that Leu19-Arg20-Tyr21 in the NH2-terminal domain represent the trypsin inhibitory activity, whereas Leu72-Met73-Leu74 in the COOH-domain represent the chymotrypsin and elastase inhibitory activity. Besides Met73, antileukoprotease contains three additional methionine residues all located in the COOH-terminal domain. Treatment of antileukoprotease with different amounts of methionine-selective reagents such as myeloperoxidase in the presence of H2O2 and Cl-, or cis-platinumdiammine dichloride resulted in a dose-dependent inactivation of all inhibitory activities, suggesting that methionine residues are involved in these activities. By using specific synthetic substrates, it was observed that elastase is able to displace trypsin from the inhibitor molecule, indicating that the trypsin and elastase inhibitory sites are located close to each other or at the same site. Incubation of antileukoprotease or its recombinant COOH-terminal domain with an antileukoprotease-specific monoclonal antibody (MoAb15) resulted in a strong selective increase of the trypsin inhibitory activity. The results presented reveal strong evidence that the inhibitory activities of antileukoprotease against trypsin, chymotrypsin and elastase are represented by its COOH-terminal domain, and that methionine residues are involved in interactions with these proteinases.

Use of automatic counting of basophil leukocytes: correlation of basophil degranulation with histamine release.

The sensitivity of leukocytes from allergic donors towards specific allergen was studied using the basophil degranulation test. In this test, basophil leukocytes were counted with the Technicon Hemalog D. A good correlation was found between the results of the basophil degranulation test and the results of the histamine release test on leukocytes of the same patient. The basophil degranulation test, using the Hemalog D, was found to be a simple method for in vitro assessment of the sensitivity of allergic patients to specific allergen.

Type-specific serologic diagnosis of respiratory syncytial virus infection, based on a synthetic peptide of the attachment protein G.

Peptides deduced from the central hydrophobic region (residues 158-189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA. Specificity and sensitivity, relative to a routine diagnostic ELISA that detects antibodies against the RSV F-protein in bovine sera, were 98% and 92% respectively for the indirect peptide-based ELISA, and 98% and 98% for the blocking peptide-based ELISA. In paired serum samples, rises in antibody titer were detected more frequently with the indirect peptide-based ELISA than with the routine F-ELISA. Furthermore, the peptide-based G-ELISAs were able to differentiate between antibodies against BRSV and HRSV, and between those against BRSV and ORSV. In addition, the indirect peptide-based ELISA was selective for HRSV subtype A and B antibodies. This study shows that peptides, corresponding to the central hydrophobic region of the attachment protein G of several RSVs, can be used successfully as antigens in highly specific and sensitive immunoassays.

Localization of low molecular weight protease inhibitor in serous secretory cells of the respiratory tract.

We prepared in rabbits an antiserum against low molecular weight protease inhibitor (LMI) purified from the sputum of patients with purulent bronchitis. Using this antiserum in an immunoperoxidase staining method we found that this inhibitor was located exclusively in the serous cells of the submucosal glands of human upper and lower airways. The inhibitor was localized also in serous cells of the sublingual and submandibular glands. In contrast, LMI could not be demonstrated in the serous cells of the parotid gland. In the tissues investigated a strong association between the localization of the protease inhibitor and lysozyme was observed. Our observations indicate that the inhibitor may be present together with lysozyme as a secretory product in the serous cell granules. The possible consequences of the coexistence of these two proteins in the defense mechanism of the respiratory tract is discussed.

Ultrastructural localization of the low molecular weight protease inhibitor in human bronchial glands.

Light microscopically the low molecular weight protease inhibitor (LMI) and lysozyme have both been demonstrated in the cytoplasm of serous cells of bronchial glands. By immunoelectron microscopy LMI was demonstrated in secretory granules of these serous cells. Many granules showed a peripheral or bean-shaped staining pattern, other granules were uniformly stained or not stained at all. In contrast to this latter finding, virtually all granules were found positive for the presence of lysozyme, suggesting that there is no association of LMI with lysozyme at the ultrastructural level.

Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein.

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.

Elastase as a marker for neutrophilic myeloid cells.

The immunohistochemical results obtained with antibodies directed against human neutrophil elastase is described. It was found that neutrophil elastase can be used as a specific marker of cells of the neutrophilic lineage. In normal hematopoietic cell preparations, only promyelocytes and more differentiated myeloid cells stain positive for elastase. In acute or chronic myeloid and myelomonocytic leukemia, the same neutrophil myeloid cells stain positive, whereas neoplastic cells of the monocytoid line are negative. Using elastase in conjunction with other markers, it is possible to differentiate easily the involvement of different cell lines in myeloproliferative diseases.

Inhibition of polymorphonuclear leukocyte-mediated endothelial cell detachment by antileukoprotease: a comparison with other proteinase inhibitors.

The role of elastase and proteinase inhibitors in polymorphonuclear leukocyte(PMN)-mediated injury to human umbilical cord venous endothelial cells (HUVEC) was investigated. Both purified human neutrophil elastase and PMN that were stimulated with serum-treated zymosan (STZ) induced detachment, but not lysis of HUVEC. PMN-, but not purified elastase-mediated detachment was enhanced by the presence of methionine, which indicates a role for reactive oxygen metabolites in PMN-mediated HUVEC detachment. Detachment of HUVEC could be inhibited by secretory leukocyte proteinase inhibitor or antileukoprotease (ALP), alpha 1-proteinase inhibitor (alpha 1-PI) and N-methoxy-succinyl-ala-ala-pro-val-chloromethyl ketone (CMK). At concentrations at which elastase-mediated detachment was maximally inhibited, ALP and CMK, but not alpha 1-PI, were also able to inhibit maximally PMN-mediated detachment. An explanation for this difference could be that the larger size of alpha 1-PI reduces the access of alpha 1-PI to the interface between the PMN and the HUVEC.

Antileukoprotease in sputum during bronchial infections.

Production of sputum and concentrations of antileukoprotease (ALP) in sputum were serially measured in hypersecreting patients with recurrent or chronic bronchial infections. Patients with stable continuous mucoid (n = 4) or purulent (n = 5) secretions had constant sputum concentrations of ALP and a calculated daily production of 1.4 and 1.9 mg of ALP per 24 hours, respectively. In patients with overt recurrent bronchial infections, production of sputum and the ALP concentration were found to be negatively correlated during treatment with antibiotics (n = 14) and during the coming (n = 5) of an exacerbation. Daily production of ALP was rather constant in these groups (2.4 and 4.8 mg, respectively, per 24 hours). While ALP was not behaving like an acute-phase protein, bronchial infection appeared to be a determinant for production of ALP; however, between individual patients with comparable severity of disease, total production of ALP varied over tenfold. Therefore, bronchial infection is not the only factor in determining production of ALP.

Proteinase/proteinase inhibitor imbalance in sputum sol phases from patients with chronic obstructive pulmonary disease. Suggestions for a key role played by antileukoprotease.

In order to characterize the imbalance between proteinases and proteinase inhibitors in sputum sol phases, we studied 25 patients (mean age, 59 +/- 11 yr) with exacerbated chronic obstructive pulmonary disease (COPD). An aliquot of sputum was used for bacteriologic determinations, and the remainder was centrifuged in order to obtain gel and sol phases. On the basis of the bacteriologic data, patients were divided into colonized patients (14) and noncolonized patients (11). All of the major inhibitors were immunologically detectable in sol phases without a significant difference between colonized and noncolonized patients (alpha 1-proteinase inhibitor [alpha 1-PI], 2.56 microM +/- 0.53 microM and 2.39 microM +/- 0.72 microM; alpha 2-macroglobulin [alpha 2-MG], 0.21 microM +/- 0.07 microM and 0.16 microM +/- 0.05 microM; antileukoprotease (ALP), 1.78 microM +/- 0.57 microM and 1.53 microM +/- 0.6 microM, respectively [mean +/- SE]). With regard to proteinase activities, both free elastase-like and free chymotrypsin-like activities were detectable in the majority of patients (15/25) (0.59 microM +/- 0.15 microM and 0.74 microM +/- 0.15 microM for elastase-like activity [ELA], and 0.010 microM +/- 0.003 microM and 0.017 microM +/- 0.007 microM for chymotrypsin-like activity [CLA], respectively [mean +/- SE]). The inhibitory profile of proteinase activities, performed by means of a panel of inhibitors, allowed us to assign specific activities mainly to neutrophil elastase and cathepsin G (Cat G). Next we looked at the relationships between inhibitors and proteinase activities. We found a significant negative correlation between neutrophil elastase activity and ALP (r = -0.58; p < 0.01). In confirmation of this suggestion, sol phases were divided into samples (15) with detectable ELA (> 0.50 microM) and samples (10) with no detectable ELA (< 0.18 microM). Levels of alpha 1-PI and alpha 2-MG did not differ significantly between the two groups, whereas ALP values were higher in the group with no detectable ELA (3.12 microM +/- 0.69 microM) than in the other group (0.58 microM +/- 0.21 microM; p < 0.001). We conclude that most sputum sol phases from patients with exacerbated COPD have a high burden of free neutrophil elastase and Cat G. Antileukoprotease seems to be the major naturally occurring inhibitor effective in the modulation of proteinase activities in bronchial secretions under these conditions.

Validation of a monoclonal antibody-based ELISA to detect antibodies directed against swine vesicular disease virus.

A simple, rapid and sensitive competitive monoclonal antibody-based ELISA for the detection of antibodies directed against swine vesicular disease virus (SVDV) was developed. The ELISA was validated using field sera originating from SVDV-infected and non-infected Dutch pig herds, reference sera obtained from the Community Reference Laboratory for Swine Vesicular Disease at the Institute for Animal Health, Pirbright Laboratory, UK, and sera from animals infected experimentally. When testing 4277 sera originating from non-infected Dutch pig herds and collected as part of the national screening program, this ELISA had only 0.6% false positive results, whereas approximately 2% of false positive results were obtained with a conventional blocking ELISA used until recently. A sensitivity relative to the virus neutralisation test of > 97% was achieved when testing sera collected from Dutch pig farms where an outbreak of SVDV had occurred. All international reference sera scored consistently correct. Sera collected sequentially from pigs experimentally infected with SVDV isolates representing all currently recognized antigenic groups, were scored positive slightly earlier by the ELISA compared to the virus neutralisation test. This monoclonal antibody-based competitive ELISA for SVDV antibodies designated the Ceditest ELISA for SVDV-Ab, is as sensitive but more specific than the ELISA used until recently. Because sera are tested at a single dilution (1:5), incubations are carried out at room temperature and test results are available within 3 h, this ELISA is simple, easy to automate and therefore very suitable for screening large numbers of serum samples.

The effect of assay conditions on the measurement of anti-elastase function in lung secretions.

We have determined the effect of altering assay conditions on the observed neutrophil elastase inhibitory capacity in lung secretions from emphysematous patients with normal serum alpha 1 PI. alpha 1 PI, ALP and alpha 2-macroglobulin were detected in all samples. Measurement at low enzyme concentration (less than 33.6 nmol/l) caused a 43% reduction in observed neutrophil elastase inhibitory capacity of sputum sol-phase, while inhibition by secretions in buffer without added NaCl was 20% greater than in the presence of 0.2 mol/l NaCl. Increasing the concentration of the synthetic substrate Suc-[Ala]3-pNA in the assay from 0.45 mmol/l to 7.2 mmol/l reduced the observed inhibitory capacity by 53% and the use of elastin-fluorescein gave lower results for inhibitory capacity than the Suc-[Ala]3-pNA (median 0.26 mol neutrophil elastase/mol measured inhibitors (range 0-0.72) with elastin; 1.40 mol neutrophil elastase/mol measured inhibitors (0.80-3.21) with Suc-[Ala]3-pNA). Assay conditions therefore greatly influence the results. In addition these findings suggest the presence of an additional inhibitor of neutrophil elastase in these secretions.

The involvement of ionic interactions during asbestos-induced enzyme release from polymorphonuclear leukocytes.

Chrysotile asbestos permeabilizes the plasma membrane of rabbit polymorphonuclear leukocytes (PMNs) which is evident from the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) from the cell. When Ca2+ is present in the medium exocytosis is observed, evident from the release of the granule associated enzyme lysozyme which is not liberated in the absence of Ca2+. Asbestos-induced enzyme release is inhibited by polyanions or by removal of positive charges on asbestos, and resembles enzyme release induced by synthetic polycations. Pretreatment of PMNs with neuraminidase does not affect the ability of asbestos to induce enzyme release from these cells. Asbestos induces release of glucose from glucose-loaded liposomes, and this effect can be inhibited by the polyanion poly-D-glutamic acid. The results are compatible with the view that positive charges play a decisive role in the interaction between PMNs and asbestos, and that the primary target of asbestos could be the lipid bilayer of the membrane. The interaction results in a permeabilized plasma membrane. When Ca2+ is present in the medium it moves into the cell and causes exocytosis of the granule enzyme lysozyme. Inhibition of cytotoxicity by polyanion may cause a diminished Ca2+-influx and hence inhibition of lysozyme release.

Respiratory syncytial virus infections in human beings and in cattle.

Respiratory syncytial virus (RSV) causes yearly outbreaks of respiratory disease in human beings and cattle all over the world. Most severe human respiratory syncytial virus (HRSV)-associated disease is observed in children less than 1 year of age while most severe bovine respiratory syncytial virus (BRSV)-associated disease is observed in calves less than 6 months of age. Two subgroups of HRSV have been identified. The existence of two subgroups of BRSV has been repeatedly suggested but is not yet well established. BRSV and HRSV are closely related antigenically but antigenic differences have been observed. Seasonal periodicity of RSV infection is usual with highest incidences in autumn and winter. Stress such as caused by movement, crowding and temperature changes are considered to play a role in bovine outbreaks. Human beings and cattle are the natural hosts of HRSV and BRSV, respectively. Primarily infected individuals are the most important source of RSV during outbreaks. The role of other species in the spread of HRSV and BRSV is unknown. Protective efficacy of maternally derived antibodies is considered to be incomplete. Such antibodies do not reduce shedding of virus after HRSV and BRSV infection. RSV is often transmitted by contact with nasal secretions but may also be transmitted by aerosols. Seroprevalence of HRSV and BRSV among adult human beings and cattle is over 70% and is always higher than it is among younger individuals. Both human beings and cattle of all ages may be reinfected with RSV. During BRSV reinfections, signs of respiratory tract disease and shedding of virus are seldom observed whereas these are common during HRSV reinfections. Persistent HRSV and BRSV infections in human beings and cattle have been suggested but have not so far been reported.

Comparative study on sixteen enzyme-linked immunosorbent assays for the detection of antibodies to bovine herpesvirus 1 in cattle.

Sixteen commercial or non-commercial enzyme-linked immunosorbent assays (ELISAs) for bovine herpesvirus 1 (BHV1)-specific antibody detection in serum were compared using 41 bovine sera of well defined origin. All ELISAs were able to detect correctly most of the antibody negative sera (specificity > or = 92%). The ability, however, to detect specific antibodies varied considerably between ELISAs. Sensitivity, estimated by testing 18 positive sera, ranged between 50% and 100%. Sera with titers of at least 64, as measured by the 24 h virus neutralisation test, were identified as being positive by all ELISAs. Most assays were unable to detect specific IgM antibodies present in sera collected 9 days after experimental infection. Only one assay, an indirect ELISA using undiluted test serum, showed a sensitivity of 100%. This ELISA was found to be 8 times more sensitive than the 24 h neutralisation test and had the unique property of showing a weak consistently positive response with some sera collected from breeding bulls. The findings of this study indicate the need for international standardisation of tests to detect BHV1-specific antibodies in cattle.

A European inter-laboratory trial to evaluate the reliability of serological diagnosis of bovine herpesvirus 1 infections.

The detection of cattle latently infected with bovine herpesvirus 1 (BHV1) is of importance in control programs and in international trade activities. Therefore, tests to detect specific antibodies in serum must be highly sensitive. To evaluate the reliability of serological diagnosis of BHV1 infections in Europe, seventeen laboratories in 15 European countries were asked to determine BHV1-specific antibodies in a panel of bovine serum samples using the serological tests available in their laboratory. Laboratory tests included virus neutralisation tests (1, 2 and 24 h), indirect immunofluorescence tests and ELISAs. The serum panel consisted of 12 duplicate lyophilised samples which were randomly coded from 1 to 24 and included negative, weak- and strong-positive samples as well as international reference sera. Virus neutralisation tests and ELISAs showed a high specificity. All participants using neutralisation tests (n = 13) scored the negative samples correctly. Twenty three of 25 ELISAs showed 100% specificity. A serum sample obtained at day 7 after infection was scored as negative by all tests except one home-made blocking ELISA. Samples obtained at day 9 and at day 11 after infection were scored as positive by approximately half and by all tests, respectively. To score the weak-positive European reference standard (EU2) correctly, 24 h neutralisation tests (positive by 8 of 9 laboratories) and home-made blocking ELISAs (positive by 5 of 6 laboratories) were the most reliable. The results indicate that standardisation is urgently needed to ensure that BHV1-infected animals with low antibody titres are recognised.

Development of a classical swine fever subunit marker vaccine and companion diagnostic test.

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD(50) of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E(RNS) antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E(RNS) antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E(RNS) test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population.

A simple, rapid and reliable enzyme-linked immunosorbent assay for the detection of bovine virus diarrhoea virus (BVDV) specific antibodies in cattle serum, plasma and bulk milk.

To detect Bovine Virus Diarrhoea Virus (BVDV)-specific antibodies in cattle serum, plasma and bulk milk, a simple, reliable and rapid blocking ELISA ("Ceditest") has been developed using two monoclonal antibodies ("WB112" and "WB103") directed to different highly conserved epitopes on the non-structural peptide NS3 of pestiviruses. The test can be performed at high reproducibility using undiluted samples. In testing 1000 field serum samples, the ELISA showed a specificity and a sensitivity relative to the virus neutralization test of 99% and 98%, respectively. The blocking ELISA is able to detect specific antibodies in serum obtained 12 days after an acute infection and in serum of vaccinated and challenged animals. A frequency distribution diagram, obtained by testing almost 1800 random Dutch field serum samples, showed a clear separation between a negative population (maximum frequency of the % inhibition at -5%) and a positive population (maximum frequency of the % inhibition at 95%). Based on these data, the prevalence of seropositive animals was 65% (95% confidence interval: 63%-67%). By exchanging plasma- and bulk milk samples between two laboratories (The Netherlands and Denmark), a good overall agreement was found between results obtained with the Ceditest and those obtained with the Danish blocking ELISA as used in the Danish BVDV eradication programme. The results indicate that BVDV infections can reliably be diagnosed by the Ceditest ELISA and that the test is suitable for use in large scale screening and eradication programmes.

A bovine respiratory syncytial virus strain with mutations in subgroup-specific antigenic domains of the G protein induces partial heterologous protection in cattle.

Bovine respiratory syncytial virus (BRSV) strains are tentatively divided in subgroups A, AB and B, based on antigenic differences of the G protein. A Dutch BRSV strain (Waiboerhoeve: WBH), could not be assigned to one of the subgroups, because the strain did not react with any monoclonal antibody against the G protein. We describe here that the WBH strain has accumulated critical mutations in subgroup-specific domains of the G protein gene, which also occur but then independently in G protein genes of BRSV subgroup A or B strains. Although the comparison of nucleotide residues 256-792 of the G gene of the WBH strain with those of subgroup A and B strains showed that the G gene of the WBH strain is different from that of BRSV subgroup A and B strains, the sequence divergence was not more than observed within the G genes of human respiratory syncytial virus subgroup A or B strains. The WBH strain did not induce severe disease after experimental infection of calves, and induced partial protection against a heterologous challenge. Despite the dissimilarity of the conserved central regions of the G protein of the WBH strain and that of the challenge strain, a secondary antibody response against this region was induced in WBH-infected calves after challenge. We conclude that complete BRSV virus can partially protect against a BRSV infection with a strain that contains an antigenic dissimilar G protein.

Subgrouping of bovine respiratory syncytial virus strains detected in lung tissue.

Bovine respiratory syncytial virus is an important respiratory pathogen in cattle. Recently, subgroups of BRSV have been identified, based on antigenic differences. However, little is known about subgroups of BRSV that circulate in the cattle population. Therefore, we determined the reactivity of monoclonal antibodies (mAbs), directed against the G, F, or P protein of BRSV, with lung tissue from 47 calves, that suffered from severe respiratory disease. Fourteen animals (30%) proved to be infected with BRSV, because they all reacted with mAbs against the P or F protein, as detected by fluorescent antibody tests. Monoclonal antibodies against the G protein were able to discriminate between the BRSV-positive specimens: 7 strains were identified as subgroup A strains, and 5 strains as subgroup AB, which is introduced as BRSV subgroup in this paper. Two strains could not be identified unambiguously. It is concluded that BRSV subgroup A and AB were associated with severe respiratory disease, and that strains belonging to either subgroup circulated concurrently in the cattle population in the Netherlands.