Search

OBJECTIVE: To investigate the effects of doxycycline on disease activity and joint destruction in patients with rheumatoid arthritis (RA). METHODS: A 36 week double blind, placebo controlled crossover trial was conducted. Patients (n = 66) received 50 mg doxycycline or placebo twice a day during 12, 24, or 36 weeks. Patient assessments were performed before the treatment was administered, at 6, 12, 24 and 36 weeks of treatment, and finally at 4 weeks after cessation of treatment. Patient assessments, swollen and tender joint counts, duration of morning stiffness, erythrocyte sedimentation rate, and Modified Disease Activity Score were used as measures of disease activity. Effects on joint destruction were assessed by urinary excretion of the pyridinolines hydroxylysylpyridinoline and lysylpyridinoline and by scoring radiographic damage of hands and feet before and after treatment. RESULTS: The changes of clinical or laboratory disease activity measures, pyridinoline excretion, or progression of radiographic joint damage during doxycycline or placebo treatment did not differ significantly. CONCLUSION: The results indicate that 50 mg doxycycline twice a day provided no therapeutic benefit for patients with RA.

Objective. To investigate the cartilage‐degrading capacity of granzyme B and the presence of granzyme B‐positive cells at sites of erosion in the rheumatoid synovium.Methods. Granzyme B was added to [3H]proline/[35S]sulphate‐labelled cartilage matrices and to cartilage explants. Proteoglycan degradation was assessed by the release of 35S and glycosaminoglycans into the medium and collagen degradation was assessed by the release of 3H and hydroxyproline and by measuring the fraction of denatured collagen. Granzyme B expression was studied at the invasive front of the synovium by immunohistochemistry.Results. Granzyme B induced loss of both newly synthesized, radiolabelled proteoglycans in cartilage matrices and resident proteoglycans of the cartilage explants. No effect on collagen degradation was found. Granzyme B‐positive cells were present throughout the synovium and at the invasive front.Conclusion. The presence of granzyme B‐positive cells at the invasive front of the synovium together with its ability to degrade articular proteoglycans supports the view that granzyme B may contribute to joint destruction in rheumatoid arthritis.

Joint destruction in rheumatoid arthritis (RA) is a result of degradation and invasion of the articular cartilage by the pannus tissue. The present study was undertaken to examine the role of the plasminogen activation system in cartilage degradation and invasion by synovial fibroblasts and investigate a novel gene therapeutic approach using a cell surface–targeted plasmin inhibitor (ATF.BPTI). Adenoviral vectors were used for gene transfer. The effects of ATF.BPTI gene transfer on RA synovial fibroblast–dependent cartilage degradation were studied in vitro, and cartilage invasion was studied in vivo in the SCID mouse coimplantation model. The results indicate that cartilage matrix degradation by rheumatoid synovial fibroblasts is plasmin mediated and depends on urokinase-type plasminogen activator for activation. Targeting plasmin inhibition to the cell surface of the fibroblasts by gene transfer of a cell surface–binding plasmin inhibitor resulted in a significant reduction of cartilage matrix degradation in vitro and of cartilage invasion in vivo. Compared with uninfected rheumatoid synovial fibroblasts, the mean ± SEM cartilage degradation in vitro was reduced to 87.9 ± 0.9% after LacZ gene transfer versus a reduction to 24.0 ± 1.6% after ATF.BPTI gene transfer (P < 0.0001). The mean ± SEM in vivo cartilage invasion score was 3.1 ± 0.4 in the control-transduced fibroblasts and 1.8 ± 0.4 in the ATF.BPTI-transduced fibroblasts (P < 0.05). These results indicate a role of the plasminogen activation system in synovial fibroblast–dependent cartilage degradation and invasion in RA, and demonstrate an effective way to inhibit this by gene transfer of a cell surface–targeted plasmin inhibitor.

Matrix metalloproteinases (MMPs) play an important role in many pathologic processes, but their activities are difficult to determine since no simple specific andor chromogenic substrate exists. We have developed a novel MMP activity assay using a modified urokinase as a substrate. Protein engineering enabled the plasmin activation site in this urokinase to be substituted by a specific activation site recognized by MMPs. In this way the MMP activity can be monitored via urokinase activity as measured by a simple chromogenic assay. The assay was made specific for MMP-9 by a capture step using MMP-9-specific antibodies that do not interfere with MMP-activity. This assay monitors the amount of active enzyme as well as the latent, but potentially active proform. Using this assay the levels of MMP-9 were investigated in urine from patients with various kinds of carcinoma. High levels of both active and latent MMP-9 were detected in urine from patients with carcinoma of the bladder, whereas hardly any activity was observed in urine from healthy controls. MMP-9 in urine was present in its intact form. Surprisingly, MMP-9 was also increased in the urine of patients with nonurogenital carcinoma. Therefore, measurement of urinary MMP-9 activity levels may be a convenient diagnostic tool for various types of carcinoma.

New prodrugs of daunorubicin and doxorubicin designed for selective activation by the serine protease plasmin are described. The low toxic prodrugs 3, 4, and 5 are converted to the corresponding cytotoxic drugs upon proteolysis by the tumor-associated protease plasmin. Application of a self-eliminating spacer was essential for enzyme activation. A prodrug containing a chloro-substituted spacer was synthesized with the aim of enhancing the rate of conversion by plasmin. All prodrugs were highly stable in buffer solution and in serum and on the average 15-fold less cytotoxic than the parent drugs in seven human tumor cell lines. A marked in vitro selectivity was demonstrated by incubation of the doxorubicin prodrugs with a plasmin generating MCF-7 breast cancer cell line transfected with urokinase-type plasminogen activator (u-PA) in comparison with the nontransfected nonplasmin generating cell line. Prodrugs 4 and 5 showed the same cytotoxic effect as the free parent drug doxorubicin in the u-PA transfected cells, indicating complete conversion of the prodrug by plasmin. Addition of the plasmin inhibitor Trasylol drastically increased the ID50 values in the u-PA transfected MCF-7 cells for both prodrugs 4 and 5.

The structural and functional properties of the urokinase-type plasminogen activator (u-PA) that are involved in the mitogenic effect of this proteolytic enzyme on human melanoma cells M14 and IF6 and the role of the u-PA receptor (u-PAR) in transducing this signal were analyzed. Native u-PA purified from urine induced a mitogenic response in quiescent IF6 and M14 cells that ranged from 25 to 40% of the mitogenic response obtained by fetal calf serum. The half-maximum response in M14 and IF6 cells was reached at u-PA concentrations of approximately 35 and 60 nM, respectively. Blocking the proteolytic activity of u-PA resulted in a 30% decrease of the mitogenic effect, whereas inhibition of plasmin activity did not alter the mitogenic effect. No mitogenic response was elicited by low molecular weight u-PA, lacking the growth factor domain and the kringle domain. The ATF domain of u-PA induced a mitogenic response that was similar to complete u-PA. Defucosylated ATF and recombinant u-PA purified from Escherichia coli lacking all post-translational modifications did not induce a mitogenic response. Blocking the interaction of u-PA with u-PAR, using a specific monoclonal antibody, did not alter the mitogenic effect induced by u-PA. The binding of radiolabeled u-PA to M14 and IF6 cells was characterized by high affinity binding mediated by u-PAR and low affinity binding to an unknown binding site. These results demonstrate that proteolytically inactive u-PA is able to induce a mitogenic response in quiescent melanoma cells in vitro by a mechanism that involves the ATF domain but is independent of high affinity binding to u-PAR. Furthermore, it suggests that u-PA is able to bind with low affinity to a hitherto unidentified membrane associated protein that could be involved in u-PA-induced signal transduction.

Euglobulin lysis time (ELT), tissue plasminogen activator (tPA), and the fast-acting inhibitor of tPA, were measured pre-operatively in 128 patients who underwent elective major abdominal surgery. Deep venous thrombosis (DVT) was detected by 125I-labelled fibrinogen scan in 37 patients (29 per cent) after operation. Pre-operatively, there was diminished euglobulin lysis activity (332 ± 197 versus 255 ± 156 min, mean ± s.d.; P<0·025), and tissue plasminogen activator activity (4·2 ± 9·9 versus 7·7 ± 14·3 milliunits/ml, mean ± s.d.; P= 0·094) in patients who subsequently developed postoperative DVT compared with those who did not. There was no significant difference between the two groups in the level of inhibition of tissue plasminogen activator (160·6 ± 75·4 per cent versus 152·5 ± 77·5 per cent, mean ± s.d.; n= 47). Stepwise logistic discriminant analysis of the data obtained pre-operatively showed that tissue plasminogen activator, a more specific measure of fibrinolytic activity, was a weaker predictor of DVT than euglobulin lysis time. The results confirm other observations which indicate that lowered fibrinolytic activity is a risk factor for postoperative DVT. In addition, they suggest that this is not due entirely to low levels of activity of tissue plasminogen activator in plasma.

Using 1.2kb 3'-terminal Pst-I fragment of a full length tissue-type plasminogen activator (t-PA) cDNA clone (ptPA-8FL) and a set of rodent human somatic cell hybrids, the corresponding human gene PLAT was localized on chromosome 8.

The plasminogen activation system is one of the enzyme systems held responsible for bone and cartilage degradation in rheumatoid arthritis (RA). In this study, we evaluated the effect of tranexamic acid (TEA), an inhibitor of plasminogen activation, on urinary collagen cross-link excretion and radiological joint damage in rat adjuvant arthritis (AA) and on urinary collagen cross-link excretion in patients with RA. In the animal study, adjuvant arthritis was induced in male Lewis rats. From day 7 onward, high-dose TEA (500 mg/kg body weight, once daily) or placebo was administered orally. Study groups consisted of TEA-treated normal rats (C + TEA), placebo-treated normal rats (C + plac), AA rats treated with TEA (AA + TEA) or with placebo (AA + plac). To monitor joint destruction, urinary collagen cross-link excretion (pyridinoline, HP; deoxypyridinoline, LP) was measured by high-performance liquid chromatography at days 14 and 21. Radiological evaluation of joints was performed at day 21. In the patient study, TEA was administered to nine patients with RA as adjuvant medication ( 20 mg/kg body weight, three times daily) for 12 weeks. Urinary HP and LP excretion levels were measured before and during TEA treatment, and 14 weeks after the cessation of TEA treatment. In AA + TEA rats, a significant reduction of HP and a tendency towards a reduction of LP excretion was found compared with AA + plac rats (P &lt;0.05), at day 14, whereas the HP/LP ratio did not change. No difference was observed in HP, LP excretion, HP/LP ratio and radiological damage score between the TEA- and placebo-treated AA rats at day 21. In RA patients, a significant reduction of HP and LP excretion was found during the TEA treatment period (P &lt;0.05). After the cessation of TEA treatment, HP and LP excretion increased towards baseline levels. No effect on disease activity was observed. The plasmin antagonist TEA reduced the excretion of collagen pyridinoline cross-links in both experimental and rheumatoid arthritis. As such, this study not only supports the involvement of the plasminogen activation system in the destructive phase of arthritis, but also suggests a beneficial effect of therapeutic strategies against inhibition of matrix proteolysis.

Kluft C, Verheijen JH, Jie AFH, Rijken DC, Preston FE, Sue-Ling HM, Jespersen J, Aasen AO. The postoperative fibrinolytic shutdown: a rapidly reverting acute phase pattern for the fast-acting inhibitor of tissue-type plasminogen activator after trauma. Scand J Clin Lab Invest 1985; 45: 605-610.The plasma activity level of the recently discovered fast-acting inhibitor of tissue-type plasminogen activator (t-PA) was found to be temporarily increased after surgery, myocardial infarction and severe trauma. Detailed analysis of the postoperative period revealed simultaneously increased t-PA antigen and inhibition and decreased t-PA activity only on the first postoperative day. These changes were more rapid than those in fibrinogen and C-reactive protein. It is concluded that t-PA inhibition shows the most rapidly changing pattern observed so far in response to trauma. The postoperative fibrinolytic shutdown in blood fibrinolytic activity can be ascribed to a primary increase in t-PA inhibitor levels.

Plasminogen activators are involved in the growth, the local invasion, and the metastatic spreading of cancer. In particular, the urokinase type of plasminogen activator (u-PA) is produced and secreted by adenocarcinomas of the gastrointestinal tract. Adenomatous polyps of the colorectum, the precursor lesion to adenocarcinomas, express, to a lesser extent, a pattern of the plasminogen activator profile similar to that of the adenocarcinomas. The increased content of urokinase in neoplasms and its functional relation with invasiveness and metastasis formation are indicative of an important role in the development of malignancy. These plasminogen activator-related findings open new possibilities for both diagnostic evaluation and therapeutic intervention in gastrointestinal cancer.

We describe the construction of a recombinant DNA plasmid, consisting of the vector pBR322 and full-length tissue-type plasminogen-activator (t-PA) cDNA, by using polyadenylated RNA from cultured Bowes melanoma cells as substrate. A 1280-base-pair PstI restriction fragment, covering the 3′ untranslated region and part of the coding region for the t-PA L-chain, was used as a radiolabelled probe to determine the size and the number of t-PA mRNA molecules in cultured endothelial cells of different origin from the same individual. Northern blotting showed that in all these cells a t-PA mRNA is synthesized of about 2500 nucleotides, indicating that transcriptional initiation, splicing and polyadenylation is similar. The number of t-PA mRNA molecules per cell measured, by using a dot-blotting technique and t-PA mRNA made in vitro, with a plasmid DNA preparation harbouring a specific promotor of the Salmonella typhimurium bacteriophage SP6, t-PA cDNA and SP6 RNA polymerase as standard, is approx. 10,000 in all cultured endothelial cells from adult vessels. However, the amount of t-PA antigen synthesized and/or secreted differs by a factor of 6-20. Relatively large amounts of t-PA antigen secreted were detected in conditioned medium from vena-cava-derived cells, whereas low amounts were found in conditioned medium from arteria-iliaca-derived cells.

To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the absence and the presence of epsilon-amino caproic acid (EACA). Occupation of the lysyl binding site in the K2 domain with EACA has a pronounced effect on the binding of FGK1K2P to a fibrin clot (C50 = 77 +/- 11 nM versus 376 +/- 45 nM with EACA). Deleting the lysyl binding site in the K2 domain (substitution D236N) also impairs fibrin binding but to a lesser extent (C50 = 169 +/- 20 nM). Although the binding of K2P to a fibrin clot is weak (C50 = 1163 +/- 490 nM), it still is 2 orders of magnitude stronger than the binding of EACA to K2P. Therefore it was surprising to find that deletion of the lysyl binding site in K2P completely abolishes fibrin binding. Even when both the F domain and the lysyl binding site were deleted, considerable fibrin binding is still observed (C50 = 557 +/- 126 nM), suggesting other than F and K2-mediated interactions. The binding of FGK1K2P, FGK1K2P (D236N), GK1K2P, and GK1K2P (D236N) to fibrin could be competitively inhibited by FGK1K2P and K2P, indicating that all molecules recognize the same interaction sites on a fibrin clot. Based on these results, a new model for the interaction of t-PA with a forming fibrin clot is proposed. The fibrin binding sites in t-PA are not confined to the F and K2 domain. The main role of the lysyl binding site in the K2 domain of t-PA appears indirect rather than direct, most likely stabilizing a conformation favorable for fibrin binding.

Five out of six human melanoma cell lines tested were able to degrade in vitro a smooth muscle cell extracellular matrix in a plasmin-dependent way. In three of these five cell lines, this process was mediated by tissue-type plasminogen activator (t-PA) and in the other two cell lines by urokinase-type plasminogen activator (u-PA). All melanoma cell lines produced t-PA mRNA and protein, whereas only the two cell lines showing u-PA-mediated matrix degradation produced u-PA mRNA and protein. These latter cell lines also produced plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein. u-PA receptor (u-PA-R) mRNA and binding of radiolabeled u-PA was found in all melanoma cell lines. The metastatic capacity of these cell lines was studied in nude mice. All cell lines were able to develop primary tumors at the subcutaneous inoculation site. The production of plasminogen activators, their inhibitors and urokinase receptor by subcutaneous tumors corresponded with the production by the parental cell lines in vitro. The two u-PA and PAI-1 producing cell lines showed the highest frequency to form spontaneous lung metastases after subcutaneous inoculation, whereas five of the six cell lines formed lung colonies after intravenous inoculation. In conclusion, u-PA mediated matrix degradation in vitro and production of u-PA and PAI-1 by human melanoma cell lines correlated with their ability to form spontaneous lung metastasis in nude mice. No correlation was found with the ability to form lung colonies after intravenous injection. These findings suggest a role for u-PA and PAI-1 in a relatively early stage of melanoma metastasis.

This work describes the first automated solid-phase synthesis of metal derivatives of peptide nucleic acid (PNA) oligomers and their interaction with DNA and PNA. PNA constitutes a relatively young and very promising class of DNA analogues with excellent DNA and RNA binding properties. However, PNA lacks a suitable handle that would permit its sensitive detection on its own as well as when hybridized with complementary oligonucleotides. Metal complexes, on the other hand, offer high potential as markers for biomolecules. In this paper, we describe the synthesis of PNA heptamers (tggatcg-gly, where gly is a C-terminal glycine carboxylic acid amide) with two covalently attached metal complexes at the PNA N-terminus, namely a ferrocene carboxylic acid derivative and a tris(bipyridine)ruthenium(II) derivative. We show how all synthesis steps may be carried out with high yield on a DNA synthesizer, including attachment of the metal complexes. The conjugates were characterized by HPLC (>90% purity) and ESI-MS. Binding studies of the purified Ru-PNA heptamer to complementary DNA and PNA and comparison to the isosequential metal-free acetyl PNA heptamer proves that the attached metal complex has an influence on the stability (UV-Tm) and structure (CD spectroscopy) of the conjugates, possibly by disruption of the nearby A:T base pair.

Scythrops novaehollandiae:Sehr selten im westlichen Teil der Insel. Als Wirte ließen sich nachweisen:Corvus florensis(fütterte Junges vonScythrops) undCorvus m. macrorhynchos.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Protcolytic joint destruction in inflammatory and non-inflammatory arthropathy is believed to be mediated, at least in part, by the plasminogen activation (PA) system. To further investigate possible involvement of the PA system, we quantified immunoreactive urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), both plasminogen activator inhibitors (PAI-1 and PAI-2) and u-PA-receptor (u-PAR) in synovial tissue extracts of 14 patients with rheumatoid arthritis (RA) and 12 with osteoarthritis (OA). u-PA, PAI-1, PAI-2 and u-PAR concentrations were significantly higher in RA than in OA patients. t-PA antigen levels were significantly lower in RA than in OA synovial tissue extracts. Immunohistochemistry was performed to compare the distribution and staining intensity of these components in samples of RA and OA synovial tissue. Intense immunostaining of u-PA, u-PAR, PAI-1 and, to a lesser degree, PAI-2 was observed predominantly in the synovial lining of RA patients. In OA patients, u-PA, PAI-1, PAI-2 and u-PAR were barely detectable. t-PA immunostaining was restricted to the endothelial side of vascular walls in both groups. We conclude that the observed increase of u-PA, u-PAR and PAI expression, distributed mainly in the synovial lining area of proliferative and invasively growing synovial tissue in RA patients, supports a pathogenic role for the PA system in destructive arthritis. Depressed t-PA-mediated plasminogen activation might contribute to delayed intra-articular fibrin removal.