Jörg Bläser

A 25 kDa α2-microglobulin-related protein is a component of the 125 kDa form of human gelatinase

Besides the monomeric mammalian 95 kDa progelatinase, two additional forms, a disulfide‐bridged 220 kDa dimer and a 125 kDa form were isolated from human PMN leukocytes. The 125 kDa progelatinase was identified as a covalently linked, disulfide‐bridged heterodimer formed of the monomer with a 23 kDa protein. This 25 kDa protein was isolated from gelatinase bound to the affinity support of gelatin‐Sepharose and eluted by DTE‐containing buffer. The amino acid sequence of tryptic peptides of this protein revealed homology with an α2‐microglobulin‐related protein from rats, a protein so far unknown in humans.

Interleukin-8 synthesis and the onset of labor

Objective To examine the role of cytokines in cervical dilation. Methods In 55 patients undergoing cesarean delivery, we took samples from the lower uterine segment, the decidua, and the membranes. We determined the concentrations of interleukin (IL)-2, IL-8, tumor necrosis factor-α, matrix metalloproteinase (MMP)-8, and MMP-9 in the different tissues. Results Depending on the state of labor, we observed a significant increase (P < .001) in IL-8 concentrations in the lower uterine segment. The leukocyte enzymes MMP-8 and MMP-9 were highly significantly correlated with the IL-8 concentrations. Conclusion Interleukin-8 is critically involved in the process of parturition in humans. Interleukin-8 concentrations in the myometrium, decidua, and membranes correlated strongly with the observed MMP-8 and MMP-9 concentrations.

Proteases and their inhibitors are indicative in gestational disease

OBJECTIVE To assess whether various proteolytic factors which are involved in trophoblast invasion show different concentrations in plasma and placenta of patients with HELLP syndrome, pre-/eclampsia and highly pathological Doppler flow measurements but without additional complications (hpD). DESIGN Case control and observational study; 18 women with HELLP syndrome, 21 with pre-/eclampsia, 13 with hpD, as well as healthy pregnant women (matched pairs); statistical analysis: sign test and Wilcoxon test. RESULTS Urokinase-type plasminogen activator (uPA), uPA receptor, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), matrix metalloproteinases MMP-8, MMP-9 and tissue inhibitor of metalloproteinases TIMP-1 were measured by ELISA. PAI-1 plasma levels are significantly elevated in all three groups studied. In HELLP syndrome, tPA and TIMP-1 are also elevated, and in patients with hpD, MMP-8 is increased, whereas MMP-9, and TIMP-1 are lower. In placenta extract, only pre-/eclampsia shows reduced MMP-9 concentrations. CONCLUSIONS The increased frequency of small-for-gestational-age infants observed in all three study groups is an expression of impaired placental implantation and remodelling processes. These disturbances manifest themselves in the form of changes in some of the factors in plasma and placenta extract that are involved in these processes.

Expression of Matrix-Metalloproteinases and their Inhibitors in Human Cholesteatomas

The proteolytic erosion of the temporal bone is the key event in the pathognomonic course of cholesteatoma progression. The molecular mechanisms of bone resorption, endangering the ossicles, the inner ear, the facial nerve, large vessels or the brain, are not understood. Recently, a new family of proteolytic enzymes, the matrix-metalloproteinases (MMPs) has been described and identified, which seems to play a pivotal role in matrix- and bone homeostasis and inflammatory osteolytic diseases, e.g. osteoarthritis and periodontitis. These enzymes are sophisticatedly controlled by specific inhibitors and activation cascades. We investigated whether human cholesteatoma tissue expresses MMPs and MMP-inhibitors. By immunocytochemistry of cholesteatoma-cryosections, the expression of MMP-2 (72 kD collagenase), MMP-9 (92 kD collagenase), and MMP-3 (stromelysin-1) could be seen to be strictly confined to the basal and suprabasal cell layer of the cholesteatoma epithelium. The neutrophil collagenase (MMP-8) showed a more disseminated expression in the epithelium and the granulation tissue as well. The tissue inhibitor of metalloproteases, TIMP-1, could be detected only in very limited areas of the granulation tissue in a quite randomized manner. Therefore, a derailment in favor of proteolysis of the normally tightly controlled MMP-system might be postulated. The results indicate that members of the MMP-family could play an active role in the molecular mechanisms of cholesteatoma invasion into the temporal bone. This offers new insights into the pathophysiology of the disease and of potential therapeutic approaches.

Determination of metalloproteinases, plasminogen-activators and their inhibitors in the synovial fluids of patients with rheumatoid arthritis during chemical synoviorthesis.

The concentrations of matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-8 (MMP-8), matrix metalloproteinase-9 (MMP-9), lactoferrin and urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA) and the inhibitors, tissue inhibitor of metalloproteinase-1 (TIMP-1), plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor (PAI-2), and alpha2-macroglobulin in the synovial fluids of patients with rheumatoid arthritis was determined before and during chemical synoviorthesis with a sodium salt of the fatty acids from cod-liver oil (Varicocid). Synovial fluids were obtained before treatment from 37 patients with rheumatoid arthritis and, in most cases, at 8 and 24 h after injection of the agent. Well-established ELISAs were used to determine the amounts of all proteins. All patients with rheumatoid arthritis revealed very high levels of metalloproteinases (about 1-15 mu g/ml) in their synovial fluids. During the inflammation inducing treatment the granulocyte enzymes increased. In contrast to this, the level of MMP-1 decreased. All granulocyte-derived enzymes were strongly correlated with each other, whereas their dependence on the granulocyte count was only weak. uPA and PAI-2 showed good correlations with the granulocytes-derived enzymes, but were also only weakly correlating with the cell counts. t-PA was not detected by the ELISA used. The proteases, MMP-8, MMP-9 and uPA were increased 8 h after the treatment, whereas the specific inhibitors TIMP-1, PAI-1 and PAI-2 showed significant changes only 24 h after the injection. Matrix metalloproteinases are important factors in the pathogenesis of rheumatoid arthritis. The inflammatory activity in the joint could be better correlated to the granulocyte enzymes than to the granulocyte counts. The levels of uPA and PAI-2 are also parallel to the granulocyte enzyme levels and might underly the same regulatory mechanism.

A sandwich enzyme immunoassay for the determination of neutrophil lipocalin in body fluids

Human neutrophil lipocalin was purified from human buffycoat. A polyclonal antibody was obtained by immunisation of rabbits. The antibody reacted with the free lipocalin as well as with the PMNL-gelatinase bound protein. This antibody was used to establish a sensitive sandwich-ELISA for the determination of the protein in body fluids using the biotin/streptavidin system. The mean intra-assay C.V. was 2.3% and the mean inter-assay C.V. 6.7%. The recovery in human plasma was determined to be 98.8%. The ELISA allowed the determination of the protein in the concentration range 0.2-25 micrograms/l. Measurement of the neutrophil lipocalin concentration showed that human plasma of healthy donors contained 9.7 +/- 81 micrograms/l (n = 122) and that the concentrations in serum were significantly higher (P < 0.001) with 133 +/- 90 micrograms/l (n = 122). Neutrophil lipocalin was also found in the urine of healthy donors (8.1 micrograms/l; n = 9). Very high concentrations of this lipocalin were found in the synovial fluids of patients suffering from inflammatory rheumatoid arthritis (1.7 +/- 1.4 mg/l; n = 37).

Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase)

Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl2 and was investigated by kinetic analysis and N‐terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp15‐Leu16, Glu40‐Met41, Leu52‐Leu53 and Ala74‐Met75 peptide bonds. The PRCGVPD sequence motif (residues Pro78‐Asp84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C‐terminal cleavages. The Glu666‐Leu667, Ala506‐Glu507 and Ala398‐Leu399 bonds were hydrolysed sucessively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys497, Cys653, Cys683. Cleavage of the first C‐terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin‐like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.

Angiogenin plasma levels during pregnancy

OBJECTIVE Our purpose was to determine the levels in plasma of angiogenin in healthy pregnant women and to examine whether there are differences between uncomplicated pregnancies and patients with the syndrome of hemolysis, elevated liver enzymes, and low platelets, preeclampsia-eclampsia, and highly pathologic Doppler flow findings without additional complications. STUDY DESIGN Angiogenin was measured with a novel enzyme-linked immunosorbent assay. A case control and observational study was conducted in 68 healthy women from the tenth to fortieth weeks of pregnancy and in 18 patients with the syndrome of hemolysis, elevated liver enzymes, and low platelets, 21 with preeclampsia/eclampsia and 13 with highly pathologic Doppler flow findings at admission for delivery. RESULTS Between the tenth and fortieth weeks of uncomplicated pregnancy angiogenin plasma levels rose from 150 to 250 ng/ml (significant correlation). In patients with highly pathologic Doppler flow findings angiogenin is significantly reduced compared with healthy pregnant matched pairs (150 vs 219 ng/ml, p < 0.01). CONCLUSION Rising plasma angiogenin levels in pregnancy may reflect persisting placental transformation and remodeling processes: in patients with highly pathologic Doppler flow findings these processes are disturbed and thus placental function is impaired.

A highly sensitive immunoenzymometric assay for the determination of angiogenin.

A polyclonal antibody to human recombinant angiogenin was prepared in rabbits using a Pam3CysSerGly conjugate. The antibody was then used to develop the first highly sensitive enzyme-labelled immunometric assay for this vascularisation inducing and tumour associated protein. The assay was suitable for quantification of angiogenin in body fluids between 2.5 and 0.05 micrograms/l. The mean intra-assay imprecision was 6.0% and the inter-assay imprecision 7.9%. Angiogenin in human plasma was found to lie in the range of 0.38 to 0.11 mg/l with a mean of 0.25 +/- 0.07 mg/l.

Formation of a covalent Hg-Cys-bond during mercurial activation of PMNL procollagenase gives evidence of a cysteine-switch mechanism

A common method for the activation of mammalian matalloproteinases is the use of mercurial compounds. Activation of PMNL procollagenase by soluble mercurials takes place as a three‐step mechanism with a final intermolecular loss of the PRCGVPD autoinhibitor region. In this study covalently bound mercury in the form of mercurial agarose was chosen to probe activation of PMNL procollagenase. Activation was not achieved, since the final intermolecular cleavage with removal of the PRCGVPD motif could not take place. An intermediate form of the enzyme was bound to the column. Its N‐terminal sequence determination proved cleavage of the Asp64‐Met65 peptide bond leaving the cysteine of the propeptide domain for covalent attachment to the mercurial agarose. This gives further evidence of a cysteine‐switch mechanism involving Cys71.