Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity.

Thrombomodulin (TM), recognized as an essential vessel wall cofactor of the antithrombotic mechanism, is also expressed by a wide range of tumor cells. Tumor cell lines subcloned from four patients with malignant melanoma displayed a negative correlation between TM expression and cell proliferation in vitro and in vivo. Overexpression of wild-type TM decreased cell proliferation in vitro and tumor growth in vivo. TM mutants with altered protein C activation capacity lead to a similar effect. In contrast, transfection of melanoma cells with mutant TM constructs, in which a portion of the cytoplasmic or lectin domain was deleted, abrogated the antiproliferative effect associated with overexpression of wild-type TM. Experiments performed with either peptide agonists/antagonists of the thrombin receptor, with hirudin, or with inhibitors of thrombin-TM interaction did not alter the growth inhibitory effect of TM overexpression. These data suggest that TM exerts an effect on cell proliferation independent of thrombin and the thrombin receptor, possibly related to the binding of novel ligands to determinants in the lectin domain which might trigger signal transduction pathways dependent on the cytoplasmic domain.

Fibrin-fibronectin compounds in human ovarian tumor ascites and their possible relation to the tumor stroma.

Covalently linked heterogeneous fibrin-fibronectin compounds were detected in ascitic fluid of 31 patients with advanced ovarian cystadenocarcinoma by means of enzyme-linked immunosorbent assay techniques, immunoaffinity chromatography, and Western blot analysis. Deposition of fibrin and fibronectin could also be demonstrated immunohistochemically in Carnoy-fixed tissue sections. Fibrin and fibronectin were found in the tumor stroma within tumor nests and more prominently in stroma surrounding the tumor nests. The association of fibrin and fibronectin was especially pronounced in the stroma surrounding the tumor islands. Fibronectin was also found to be associated with stroma cells. Areas within the tumor stroma showed superimposed staining for both fibrin and fibronectin supporting the assumption that the covalently linked fibrin-fibronectin conjugates found in ascitic fluid may stem from the provisional tumor stroma by proteolytic release.

Face and emotion expression processing and the serotonin transporter polymorphism 5-HTTLPR/rs22531.

Face cognition, including face identity and facial expression processing, is a crucial component of socio-emotional abilities, characterizing humans as highest developed social beings. However, for these trait domains molecular genetic studies investigating gene-behavior associations based on well-founded phenotype definitions are still rare. We examined the relationship between 5-HTTLPR/rs25531 polymorphisms - related to serotonin-reuptake - and the ability to perceive and recognize faces and emotional expressions in human faces. For this aim we conducted structural equation modeling on data from 230 young adults, obtained by using a comprehensive, multivariate task battery with maximal effort tasks. By additionally modeling fluid intelligence and immediate and delayed memory factors, we aimed to address the discriminant relationships of the 5-HTTLPR/rs25531 polymorphisms with socio-emotional abilities. We found a robust association between the 5-HTTLPR/rs25531 polymorphism and facial emotion perception. Carriers of two long (L) alleles outperformed carriers of one or two S alleles. Weaker associations were present for face identity perception and memory for emotional facial expressions. There was no association between the 5-HTTLPR/rs25531 polymorphism and non-social abilities, demonstrating discriminant validity of the relationships. We discuss the implications and possible neural mechanisms underlying these novel findings.

After coronary thrombolysis and reperfusion, what next?

Thrombolytic therapy for the removal of intravascular thrombi was introduced when streptokinase was first given to humans 40 years ago, the same year the American College of Cardiology was founded. Streptokinase was first administered to patients with acute myocardial infarction in 1959. Today, thrombolytic therapy has been established to offer significant benefits to patients with acute myocardial infarction provided they are brought to medical attention early enough after the onset of symptoms. The two major agents, streptokinase and recombinant tissue-type plasminogen activator (rt-PA), have been shown to result in reperfusion of infarct-related arteries, to salvage ischemic myocardium, to improve myocardial performance and to reduce mortality. In spite of these impressive gains, this novel therapy has shortcomings. The interval from the start of thrombolytic treatment to coronary reperfusion varies significantly from patient to patient and may, at times, be too long to produce a real benefit in terms of salvage of ischemic myocardium. The rate of reocclusion lies somewhere between 10% and 20% and appears not to be influenced by concomitant heparin anticoagulation. The rate of bleeding complications even with the "fibrin-specific" rt-PA is higher than anticipated and may range from 10% to 30%. As a consequence, intensive efforts are being directed at the development of improved thrombolytic agents and for adjunctive therapy evaluating better anticoagulants than heparin and better antiplatelet agents than aspirin. This review is a status report summarizing where we are in thrombolytic therapy in acute myocardial infarction, where we need to improve treatment results and what is being done mainly at the preclinical level to bring about such improvements.

Recombinant soluble urokinase receptor as a scavenger for urokinase-type plasminogen activator (uPA). Inhibition of proliferation and invasion of human ovarian cancer cells.

A recombinant soluble human urokinase receptor comprising amino acids 1-277 was cloned and transfected into CHO cells. The mutant protein (rec-uPAR277), purified from the CHO cell supernatant by affinity chromatography on immobilized urokinase (uPA), in a four-fold excess, completely abolished the binding of FITC-labeled pro-uPA to the human ovarian cancer cell line, OV-MZ-6. This invasive and tumorigenic cancer cell line expresses uPA, its inhibitor PAI-1, and the high-affinity receptor for uPA, uPAR. Rec-uPAR277 significantly reduced the proliferation of OV-MZ-6 cells in a concentration-dependent manner without altering the viability of the cells. Invasion of OV-MZ-6 cells tested in an in vitro Matrigel invasion assay was inhibited by rec-uPAR277 up to 75%. In conclusion, these results demonstrate that rec-uPAR277 can function as a scavenger for uPA in vitro by inhibiting proliferation and invasion of human cancer cells.

Urokinase induces proliferation of human ovarian cancer cells: characterization of structural elements required for growth factor function.

Ovarian cancer metastasis is associated with an increase in the urokinase-type plasminogen activator (uPA) and its receptor uPAR. We present evidence that binding of uPA to uPAR provokes a mitogenic response in the human ovarian cancer cell line OV-MZ-6 in which endogenous uPA production had been significantly reduced by stable uPA 'antisense' transfection. High molecular weight (HMW) uPA, independent of its enzymatic activity, produced an up to 95% increase in cell number concomitant with 2-fold elevated [3H]thymidine incorporation as did the catalytically inactive but uPAR binding amino-terminal fragment of uPA, ATF. uPA-induced cell proliferation was significantly decreased by blocking uPA/uPAR interaction by the monoclonal antibody IIIF10 and by soluble uPAR. The efficiency of the uPAR binding synthetic peptide cyclo19,31 uPA19-31 to enhance OV-MZ-6 cell growth proved this molecular domain to be the minimal structural determinant for uPA mitogenic activity. Dependence of uPA-provoked cell proliferation on uPAR was further demonstrated in Raji cells which do not express uPAR and were thus not induced by uPA. However, upon transfection with full-length uPAR, Raji cells acquired a significant growth response to HMW uPA and ATF.

Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor (CD87).

The serine protease urokinase-type plasminogen activator, uPA, when bound to its specific receptor, uPAR (CD87), plays a significant role in tumor cell invasion and metastasis. In breast cancer, enhanced uPA antigen in the primary tumor is correlated with poor prognosis of the patient. In an in vivo nude mouse model, we tested tumor growth and metastasis of human breast carcinoma cells that had been transfected with an expression plasmid encoding a soluble form of uPAR (suPAR). We explored, whether suPAR/uPA interaction reduces the binding of uPA to cell surface-associated uPAR, and, as a consequence, could suppress tumor growth and metastasis of the human breast cancer cell line MDA-MB-231 BAG. Overexpressed, secreted suPAR was shown to bind and thus scavenge the uPA secreted by the transfected lines suPAR3 and suPAR10. In vitro, an overexpression of suPAR did not alter the proliferation rate of the transfected tumor cells, nor did it affect the expression of uPA. Overexpression of suPAR led to a reduction in the plasminogen activation-related proteolytic activity of breast carcinoma cells. Primary tumor growth in the mammary fat pad of nude mice was followed up for 52 days. Overexpression of suPAR correlated with a reduction in tumor growth (from day 21, reaching 30% by day 34) as well as lung colonization (lung metastasis-positive mice in suPAR3: 4 of 17; suPAR10: 3 of 10; parental MDA-MB-231 BAG: 13 of 18). We conclude that suPAR overexpression leading to effective scavenge of uPA impairs proteolysis as well as the tumor growth and metastatic potential of breast carcinoma cells in vivo.

Antisense inhibition of urokinase reduces spread of human ovarian cancer in mice.

Urokinase-type plasminogen activator (uPA) is a protease involved in the process of tissue remodelling and cell migration in vitro. To explore whether uPA is a prerequisite for human ovarian cancer spread in vivo the expression of uPA was suppressed in human ovarian cancer cells by antisense phosphorothioate oligonucleotides (PS-ODN). The suppression of uPA expression was dependent on PS-ODN concentration and only observed in the presence of liposomes. This phenomenon seemed to be due to the fact that PS-ODNs were taken up by the cancer cells only in concert with liposomes as studied by fluorescently-labeled PS-ODNs using flow cytofluorometry and laser scanning microscopy. uPA-deprived cancer cells exhibited a significantly reduced invasive capacity in vitro compared with untreated cancer cells or cells treated with control PS-ODNs (P = 0.003). The intraperitoneal spread of the cancer cells in vivo was significantly diminished when nude mice were treated with uPA antisense PS-ODNs in comparison with control mice (P = 0.009). These results suggest that uPA expression may be required for spread of human ovarian cancer and that its inhibition could provide a therapeutic approach.

Serum cholesterol and triglyceride levels in progeria as a model of ageing.

Hutchinson-Gilford progeria was observed in two brothers. Their parents, sister and other relatives did not show any signs of this illness. Serum total cholesterol and total triglyceride levels were normal in the whole family. The serum high density lipoprotein cholesterol (HDL-C) level of parents was low and that of boys was extremely low. The serum HDL-C level of the healthy sister and other relatives was normal. These findings in homozygous children and heterozygous parents may explain the development of the very early fatal arteriosclerosis described in this disease. The connection between the disorder of the lipid metabolism and progeria can serve as a useful model in the study of the role of lipid metabolism in normal ageing.

Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy.

Extravasation and intravasation of solid malignant tumors is controlled by attachment of tumor cells to components of the basement membrane and the extracellular matrix, by local proteolysis and tumor cell migration. Strong clinical and experimental evidence has accumulated that the tumor-associated serine protease plasmin, its activator uPA (urokinase-type plasminogen activator), the receptor uPA-R (CD87), and the inhibitors PAI-1 and PAI-2 are linked to cancer invasion and metastasis. In cancer, increase of uPA, uPA-R, and/or PAI-1 is associated with tumor progression and with shortened disease-free and/or overall survival in patients afflicted with malignant solid tumors. uPA and/or its inhibitor PAI-1 appear to be one of the strongest prognostic markers so far described. Strong prognostic value to predict disease recurrence and overall survival has been documented for patients with cancer of the breast, ovary, cervix, endometrium, stomach, colon, lung, bladder, kidney, brain, and soft-tissue. Due to the strong correlation between elevated uPA and/or PAI-1 values in primary cancer tissues and the tumor invasion/ metastasis capacity of cancer cells, proteolytic factors have been selected as targets for therapy. Various very different approaches to interfere with the expression or reactivity of uPA or CD87 at the gene or protein level were successfully tested including antisense oligonucleotides, antibodies, enzyme inhibitors, and recombinant or synthetic uPA and uPA-R analogues.

Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis (review).

Tumor cell migration and invasion into the surrounding tissue depend on the invasive capacity of cells leading to the loosening of cell-cell and cell-substratum contacts via cell surface associated proteolytic enzyme systems. Plasmin is one of the enzymes involved in these complex events. It is generated by the cleavage of the proenzyme plasminogen upon the action of the urokinase-type plasminogen activator (uPA). uPA is synthesized and secreted by tumor cells and normal cells and interacts with a specific cell surface receptor (uPAR) thereby focalizing enzymatic activity to the cell surface. The activity of uPA is controlled by plasminogen activator inhibitors type-1 and type-2. A strong statistically independent prognostic impact has been attributed to uPA and its inhibitor PAI-1 in a variety of malignancies. Besides its proteolytic activity, uPA in concert with uPAR exert biological effects characteristic for molecules with signal transducing properties including chemotaxis, migration/invasion, adhesion, and mitogenesis.

Thrombomodulin, a receptor for the serine protease thrombin, is decreased in primary tumors and metastases but increased in ascitic fluids of patients with advanced ovarian cancer FIGO IIIc.

The human ovarian cancer cell line OV-MZ-19, established from a patient with cystadenocarcinoma of the ovary, expressing thrombomodulin (TM), a cell surface receptor for the serine protease thrombin, interacts with monoclonal and polyclonal antibodies having different specificity for TM. These antibodies detect TM antigen by means of flow cytofluorometry, laser scanning microscopy, immunocytochemistry, and ELISA. Therefore a highly sensitive ELISA for TM antigen was established using two different monoclonal antibodies to quantify TM in tissue extracts and biological fluids, e.g. peritoneal malignant ascites. Primary malignant ovarian tumors and metastases of the omentum and intestine contain TM antigen as determined by ELISA but in significantly lower concentrations than benign ovarian tumors (p=0.0056). In contrast, malignant ascitic fluid of patients with advanced ovarian cancer (FIGO IIIc) contain significantly elevated concentrations of soluble TM than benign peritoneal exudates (p=0.0003). Immunoaffinity purified ascites-derived TM efficiently activates protein C. Protein C activation of ascites-derived TM as well as TM expressed by the tumor cells is inhibited by the monoclonal antibodies. TM abrogates the procoagulant activity of thrombin, reduces pericellular thrombin via internalization, accelerates the thrombin-mediated inactivation of pro-uPA, and the EGF domains of TM exhibit mitogenic activity towards fibroblasts and tumor cells. Both, thrombin and pro-uPA play important roles in tumor invasion and metastasis. Therefore, downregulation and/or release of TM into ascitic fluid may play an important role in the malignant behavior of tumor cells.

Expression of urokinase-type plasminogen-activator (upa) and its receptor (upar) in human ovarian-cancer cells and in-vitro invasion capacity.

Expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA), their inhibitor PAI-1 and the uPA-receptor (uPAR) was characterized in six human tumor cell lines (OV-MZ-6, -10, -13, -15, -19 and OVCAR-3) established from patients with cystadenocarcinoma of the ovary. The invasive potential of the ovarian cancer cell lines determined in an in vitro invasion assay did neither correlate with the antigen level of uPA, t-PA, PAI-1 or uPAR nor with the cell surface uPA activity, however, did correlate with the cell surface-bound plasmin activity. The in vitro invasiveness of three cancer cell lines selected displaying a different pattern of uPA and uPAR expression was significantly inhibited by a recombinant soluble truncated form of the uPAR functioning as a scavenger for uPA. Our results suggest that the interference of the uPA/uPAR interaction leads to a reduced in vitro invasiveness of human ovarian cancer cells independent of the level of uPA and uPAR expression.

Urokinase-type plasminogen-activator (upa), a protease with cytokine-like activity in human hl-60 leukemic-cell line.

HL-60, a cell line originating from a human myeloid leukemia, expresses urokinase-type plasminogen activator (uPA) on the mRNA level and secretes the protein into the culture supernatant. Additionally, uPA receptors (uPA-R) could be detected in HL-60 on both the mRNA and the protein level, whereas the lymphoblastic cell line Raji studied in parallel was uPA-R negative. The cell lines were further studied in their clonal growth in methylcellulose under the influence of rhuPA (rhpro-uPA). The growth of Raji cells was not influenced by rhuPA. Colony and cluster formation of HL-60 was not reproducibly affected by rhuPA in concentrations between 1-100 ng/ml. In some experiments however, there were higher numbers of colonies in the HL-60 cultures incubated with rhuPA which was due to a cluster-to-colony shift. Furthermore, the HL-60 colonies in the rhuPA incubated plates always showed morphological alterations including an adherent basis indicating functional differentiation. This assumption was further supported by the observation that the secondary plating efficiency (PE2) of HL-60 cells taken from single colonies of uPA-incubated plates decreased significantly when compared with PE2 of cells from colonies grown without the presence of uPA. In conclusion, the intact uPA molecule functions like an autocrine cytokine for a human leukemic cell line, which in addition to its effects in tumor invasion makes it an interesting target molecule for further studies on tumor suppression.

The uPA/uPA receptor system as a target for tumor therapy.

Invasiveness of a variety of tumors depends on the regulated expression of proteolytic enzymes that degrade the surrounding extracellular matrix and dissociate cell-cell and/or cell-matrix attachments. The tumor cell surface-associated urokinase-type plasminogen activator (uPA) system plays an especially important role in tumor cell invasion and metastasis. It consists of the serine protease uPA, its membrane-bound receptor (uPAR, CD87) and one of the natural inhibitors PAI-1 or PAI-2. There are strong indications based on animal experiments that interference with this system by inhibiting the enzymatic activity of uPA and/or antagonizing its binding to the receptor is of therapeutic relevance. With the recent solution of various X-ray structures of uPA/inhibitor complexes, structural information is available for optimizing existing lead compounds in their affinity and selectivity for uPA. Furthermore, peptide compounds capable of mimicking the structural epitope of uPA responsible for binding to the receptor efficiently antagonize this recognition process. Thus, both approaches prove to be well suited for the design of highly promising drugs in human medicine.

Fibrinolysis in ascitic fluid: isolation and characterization of fibrin derivatives, their interaction with albumin.

A method for the preparative isolation of high molecular weight fibrin degradation products (XDP) from ascitic fluid of patients with advanced ovarian cancer is described. By non-reduced and reduced PAA-Gel electrophoresis we could demonstrate that high molecular weight fibrin derivatives exist as E-complexes. A similarity of the polypeptide chain composition from in vitro samples and ascitic fluid could be shown. Immunoadsorption with anti-albumin followed by Westernblotting with anti-fibrinogen-demonstrated the existence of XDP/albumin-associates in ascitic fluid.

Epitope mapping of monoclonal antibodies directed to PAI-1 using PAI-1/PAI-2 chimera and PAI-1-derived synthetic peptides.

Plasminogen activator inhibitor type-1 is a key regulatory protein of the fibrinolytic system that is involved in a variety of physiological and pathophysiological processes. A panel of 14 monoclonal antibodies directed against plasminogen activator inhibitor type-1 was analyzed regarding epitope specificity on plasminogen activator inhibitor type-1. For this purpose, chimera consisting of plasminogen activator inhibitor type-1 and another plasminogen activator inhibitor, plasminogen activator inhibitor type-2, with different portions of the respective wild-type proteins, were generated and plasminogen activator inhibitor type-1-derived 20-mer and 10-mer linear peptides were synthesized. Nine of the 14 monoclonal antibodies recognized an epitope located in the region between amino acid 76-188 of plasminogen activator inhibitor type-1, which encompasses the binding sites for vitronectin, heparin, and part of the fibrin binding region. Of these nine monoclonal antibodies, six reacted with a quadruple plasminogen activator inhibitor type-1 mutant (N152H, K156T, Q321L, M356I), and seven detected a plasminogen activator inhibitor type-1 deletion mutant (DeltaF111-H114). Two of the remaining five monoclonal antibodies recognized epitopes located between amino acid 209-227 and amino acid 352-371, respectively, while the other three antibodies reacted with wild-type plasminogen activator inhibitor type-1, only. Additional experiments revealed that two of the 14 mAbs neutralized and one monoclonal antibodies increased plasminogen activator inhibitor type-1 activity toward urokinase-type plasminogen activator, one of its target proteases.

Effects of directionality in deductive reasoning: I. The comprehension of single relational premises.

Four experiments involving 123 university students tested directionality effects in the comprehension of spatial relations, quantified statements, and propositional connectives with a sentence-picture verification task. Presentation of the referents of terms in the statements was separated by 1 s, and presentation order was congruent or incongruent with the order of terms in the statement. Some relations showed faster verification times for congruent display order, others for incongruent display order, and still others showed no directionality effect. The authors proposed a 2-step process model for the construction of semantic representations of relational statements, in which a reference object is established first, and then the second object is attached in relation to it. This theory explains the various directionality effects as a general preference to process information about the reference object before information about the target object.

Incidence of adenoviruses in raw and treated water.

Adenoviruses are of major public health importance and are associated with a variety of clinical manifestations, i.e. gastroenteritis, eye infections and respiratory infections. The importance of water in the epidemiology of adenoviruses and the potential health risks constituted by adenoviruses in water sources and supplies are widely recognised. This study was conducted to assess the incidence of human adenoviruses in raw and treated water systems. Various raw and treated water were routinely monitored for the presence of adenoviruses, over a 1-year period (July 2000-June 2001). The supplies were derived from acceptable quality surface water sources using treatment processes, which conform to international standards for the production of safe drinking water. Adenoviruses were detected by firstly amplifying the viruses in cell cultures and then amplifying the extracted nucleic acids of these viruses using molecular techniques (nested PCR). The results indicated human adenoviruses present in 13 (12.75%) of the raw and 9 (4.41%) of the treated water samples tested. The combination of cell culture and nested PCR has proved to be a quick and reliable method for the detection of adenoviruses in water environments.

[Measuring the concentration of various plasma and placenta extract proteolytic and vascular factors in pregnant patients with HELLP syndrome, pre-/eclampsia and highly pathologic Doppler flow values]. / Konzentrationsmessungen verschiedener Proteolyse- und Gefässfaktoren im Plasma und Plazentaextrakt bei Schwangeren mit HELLP-Syndrom, Prä-/Eklampsie und hoch-pathologischen Dopplerflussbefunden.

OBJECTIVE: Impaired trophoblast invasion plays a major role in the development of preeclampsia. Therefore various factors that are involved in invasion were investigated in gestational disease. METHODS: In pregnant women with HELLP-syndrome (n = 18), pre-/eclampsia (n = 21) and highly pathological Doppler flow measurements (hpD) (n = 13), plasma and placental tissue extract concentrations of uPA, uPA-receptor, tPA, PAI-1, MMP-8, MMP-9, TIMP-1, thrombomodulin, and angiogenin were measured using ELISA. RESULTS: In all three collectives, PAI-1 plasma concentrations were significantly higher (p < 0,05) than in normal pregnancies, in patients with HELLP-syndrome, tPA and TIMP-1 plasma levels were also elevated. MMP-9 concentrations in placental tissue extracts were lower in pre-/eclampsia than in normal pregnancies. CONCLUSIONS: Impaired placental implantation and remodelling in gestational disease is reflected by changes in plasma and placental tissue extract concentrations of various factors that are involved in these processes.