A Sensitive and Robust Assay for Urokinase and Tissue-Type Plasminogen Activators (Upa and Tpa) and Their Inhibitor Type I (Pai-1) in Breast Tumor Cytosols

Grebenschikov, N.; Geurts-Moespot, Anneke; Witte, Hans De; Heuvel, J. J. T. M.; Leake, R E; Sweep, Fred C.G.J.; Benraad, Th. J.

doi:10.1177/172460089701200102

Cited by 85 publications

(80 citation statements)

References 19 publications

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“…After inclusion of RA patients, plasma was isolated and stored at Ϫ80°C at baseline, every 3 months during the first 2 years, and every 6 months thereafter. The plasma was then used in an ELISA for human MIF that we developed by using the 4-span approach, as described in detail by Grebenschikov et al (26).…”

Section: Methodsmentioning

confidence: 99%

Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor

Radstake

Sweep

Welsing

et al. 2005

Arthritis & Rheumatism

190

184

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Objective. To study whether genetic variants of macrophage migration inhibitory factor (MIF), the MIF ؊173G>C and CATT 5-8 alleles, are associated with disease severity and levels of circulating MIF in patients with rheumatoid arthritis (RA).Methods. Genotyping was performed in patients with early RA and in healthy controls. Demographic data, disease activity, and outcome measurements were compared between patients with and without the MIF variants. MIF ؊173G>C and CATT 5-8 polymorphisms were genotyped, and a newly developed enzyme-linked immunosorbent assay for human MIF was used. Allele and genotype distributions of the MIF ؊173G>C and CATT 5-8 polymorphisms were compared between patients and controls by chi-square test. Multiple regression analysis was performed to assess the independence of the MIF functional genetic variants as risk factors for radiologic joint damage.Results. Genotyping of the ؊173G>C and CATT 5-8 polymorphisms of MIF in RA patients and healthy individuals (n ‫؍‬ 277 each) revealed similar frequencies of genotypes and haplotypes in both groups. No significant differences in demographic or clinical features were observed between RA patients carrying the MIF ؊173C allele or the MIF CATT 7 allele or both and noncarrier RA patients. Radiologic joint damage was significantly higher in patients carrying risk alleles of the MIF ؊173G>C or the MIF CATT 5-8 functional variants. No synergistic effects between both genetic variants were observed. Multivariate regression analysis revealed that presence of the MIF ؊173C/C and MIF CATT 7/7 genotypes and having 1 MIF ؊173C allele were independent prognostic variables. Carriership of the MIF ؊173C allele (P ‫؍‬ 0.002) or MIF CATT 7 allele (P ‫؍‬ 0.004) was associated with significantly higher circulating MIF levels compared with those in subjects having none of the risk-conferring alleles, and greater circulating MIF levels correlated with more severe radiologic joint damage (r ‫؍‬ 0.64, P ‫؍‬ 0.001).Conclusion. The MIF polymorphisms are not associated with RA susceptibility but are associated with high levels of radiologic joint damage. High circulating MIF levels were shown to correlate strongly with radiologic joint damage, suggesting that MIF expression is genetically determined and can be used as a novel prognostic tool in RA.

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Section: Methodsmentioning

confidence: 99%

Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor

Radstake

Sweep

Welsing

et al. 2005

Arthritis & Rheumatism

190

184

View full text Add to dashboard Cite

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“…TNF␣ protein levels were measured in the same experimental setup as described previously for components of the plasminogen activator system (40). The structure of the assay involves a combination of 4 polyclonal antibodies (raised in 4 different animal species including duck, chicken, rabbit, and goat).…”

Section: Methodsmentioning

confidence: 99%

Increased expression of Fcγ receptors II and III on macrophages of rheumatoid arthritis patients results in higher production of tumor necrosis factor α and matrix metalloproteinase

Blom¹,

Radstake²,

Holthuysen³

et al. 2003

Arthritis & Rheumatism

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Objective. To evaluate Fc␥ receptor (Fc␥R) expression on synovial macrophages from rheumatoid arthritis (RA) patients and to determine whether this expression correlates with the production of the proinflammatory cytokines tumor necrosis factor ␣ (TNF␣), interleukin-1␤ (IL-1␤), IL-12, and matrix metalloproteinase 1 (MMP-1). We also sought to determine whether mature macrophages from RA patients express aberrant levels of Fc␥RI, Fc␥RII, and Fc␥RIII, and to determine the production of inflammatory mediators after immune complex (IC) stimulation.Methods. Immunohistochemistry was performed on cryostat sections of synovial biopsy specimens obtained from 27 RA patients and 5 controls. Fc␥R I, II, and III were detected, as well as the proinflammatory mediators IL-1, TNF␣, IL-12, and MMP-1. Monocytes were isolated from the blood of 10 RA patients and 10 healthy controls and cultured for 7 days with macrophage colony-stimulating factor to obtain macrophages. Using fluorescence-activated cell sorting, the expression of Fc␥RI, Fc␥RII, and Fc␥RIII was determined. On day 7, macrophages were stimulated with heat-aggregated gamma globulins (HAGGs) for 24 hours. Production of cytokines was measured using enzyme-linked immunosorbent assay, and production of gelatinases/ collagenases was measured by degradation of fluorescent gelatin.Results. Immunohistochemistry showed higher Fc␥RII and Fc␥RIII expression in RA synovium than in controls. Fc␥RII and Fc␥RIII, but not Fc␥RI, were highly correlated with the number of synovial macrophages. Consistent with this, TNF␣ expression correlated positively with Fc␥RIII expression. Moreover, MMP-1 expression strongly correlated with Fc␥R I, II, and III expression. Mature macrophages from RA patients showed significantly enhanced expression of Fc␥RII and Fc␥RIII compared with controls. Twentyfour hours after stimulation of RA macrophages with HAGGs, significantly higher production of TNF␣ and gelatinase/collagenase was measured.Conclusion. RA synovium and mature RA macrophages express significantly elevated levels of Fc␥RII and Fc␥RIII, resulting in much higher production of TNF␣ and gelatinase/collagenase after IC stimulation. These data suggest that disturbed expression of Fc␥R on mature synovial macrophages is involved in the pathology of RA.

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“…In order to provide a further basis for comparison of our results with those obtained by other ELISAs, we analysed a reference sample designated '101094', kindly provided by Professor T Benraad, Department of Experimental and Chemical Endocrinology, Academic Hospital, University of Nijmegen, The Netherlands. The aliquots of freeze-dried powder provided (Grebenschikov et al, 1997) was reconstituted in 0.5 ml of a buffer of 0.01 M disodium hydrogen phosphate, pH 7.4, 0.15 M sodium chloride, 1% bovine serum albumin, 0.1S% Tween 20. Our ELISAs gave a uPA concentration of 0.68 ± 0.06 ng ml-' (four determinations) and a PAI-I concentration of 3.80 ± 0.17 ng ml-' (four determinations).…”

Section: Materials and Methods Patients And Tumoursmentioning

confidence: 99%

“…Our ELISAs gave a uPA concentration of 0.68 ± 0.06 ng ml-' (four determinations) and a PAI-I concentration of 3.80 ± 0.17 ng ml-' (four determinations). For comparison, Grebenschikov et al (1997) estimated a uPA concentration of 0.90 ng ml-' and a PAI-I concentration of 0.91 ng ml-'. Thus, our ELISAs give a slightly lower value for uPA and a fourfold higher value for PAI-I than that of Grebenschikov et al (1997).…”

Section: Materials and Methods Patients And Tumoursmentioning

confidence: 99%

Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer

Knoop

Andreasen²,

Hansen³

et al. 1998

Br J Cancer

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Summary The uPA-mediated pathway of plasminogen activation is central to cancer metastasis. Whether uPA and PAI-1 are related to local recurrence, metastatic spread or both is not clear. We present a retrospective study of 429 primary breast cancer patients with a median followup of 5.1 years, in which the levels of uPA and PAI-1 in tumour extracts were analysed by means of an enzyme-linked immunosorbent assay.The median values of uPA and PAI-1, which were used as cut-off points, were 4.5 and 11.1 ng mg-1 protein respectively. The levels of uPA and PAI-1 were correlated with tumour size, degree of anaplasia, steroid receptor status and number of positive nodes. Patients with high content of either uPA or PAI-1 had increased risk of relapse and death. We demonstrated an independent ability of PAI-1 to predict distant metastasis (relative risk 1.7, confidence limits 1.22 and 2.46) and that neither uPA nor PAI-1 provided any information regarding local recurrence.Keywords: urokinase; plasminogen; PAI-1; breast neoplasm mortality; prognosis Cancer cells undergo the following steps during metastasis: detachment from the primary tumour; migration; invasion of the blood and lymphatic vessels; adhesion to and penetration of the endothelium, allowing colonization at distant sites (Liotta et al, 1991). Tumour progression and metastasis also involve various processes that may be called cancer cell-directed tissue remodelling. Examples are angiogenesis (Folkman, 1995) and desmoplasia (Dvorak et al, 1995).Extracellular proteolysis has been implicated in cancer metastasis for many years, with the basic idea that release of proteolytic enzymes from a tumour leads to breakdown of basement membranes and extracellular matrix (ECM), thus allowing cancer cell invasion into the surrounding normal tissue. This is true for plasminogen activators and metalloproteinases (Dan0 et al, 1985;Liotta et al, 1991;Mignatti and Rifkin, 1993;Andreasen et al, 1997). There are two types of plasminogen activator, the urokinase-type uPA and the tissue-type tPA. Both are capable of catalysing the formation of the broad spectrum proteinase plasmin from the inactive zymogen plasminogen. There seems to be general agreement that uPA is most important for generation of plasmin in events involving degradation of ECM, while the primary role of tPA is to generate plasmin for thrombolysis. In relation to cancer metastasis, therefore, uPA is of main interest. There are two main types of inhibitors of plasminogen activator, PAI-1 and PAI-2. The urokinase receptor (uPAR) serves to localize plasminogen activation to cell surfaces (Dan0 et al, 1985;Andreasen et al, 1990Andreasen et al, , 1997Mignatti and Rifkin, 1993 Recent studies have shown that the levels of uPA and PAI-I in malignant tumours vary considerably and are related to the prognosis of the patient (Andreasen et al, 1997). Whether uPA and PAI-I are related to local recurrence, metastatic spread or both is not clear. None of the research groups has to our knowledge looked specifically into the levels of...

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A Sensitive and Robust Assay for Urokinase and Tissue-Type Plasminogen Activators (Upa and Tpa) and Their Inhibitor Type I (Pai-1) in Breast Tumor Cytosols

Cited by 85 publications

References 19 publications

Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor

Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor

Increased expression of Fcγ receptors II and III on macrophages of rheumatoid arthritis patients results in higher production of tumor necrosis factor α and matrix metalloproteinase

Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer

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