Downregulation of serum epidermal growth factor in patients with inflammatory bowel disease. Is there a link with mucosal damage?

Background: Epidermal growth factor (EGF) is a multipotent peptide which contributes to epithelial development, inhibition of gastric acid secretion, acceleration of wound healing, and promotion of angiogenesis. The aim of this study is to evaluate serum EGF concentrations in inflammatory bowel disease (IBD) patients, with regard to disease and patients’ characteristics.

Methods: EGF determination was performed by a commercially available enzyme-linked immunosorbent assay. Fifty-two patients with ulcerative colitis (UC), 59 with Crohn's disease (CD), and 55 healthy controls (HC) were included in the study.

Results: Mean ( ± SEM) serum EGF levels were 217.2 ( ± 30.40) pg/mL in UC patients, 324.6 ( ± 37.29) pg/mL in CD patients, and 453.1 ( ± 39.44) pg/mL in HC. Serum EGF levels were significantly lower in UC and CD patients compared to HC (P < 0.0001 and P = 0.0199, respectively). Lower serum EGF levels were observed in UC compared to CD patients (P = 0.0277). Extent of the disease was found to affect serum EGF levels in UC, demonstrating significant reduction in patients with left-sided colitis and pancolitis in comparison with those with proctitis (P = 0.0190 and P = 0.0024, respectively). EGF concentration was not influenced by other characteristics of patients and disease.

Conclusions: Significantly, lower levels of serum EGF are observed in IBD patients compared to HC, while disease extent plays a key role in regulation of serum EGF in UC. Downregulation of serum EGF may be correlated with different patterns of bowel inflammation, epithelial development, and wound healing in IBD.     

Complex formation between protein c inhibitor and tissue plasminogen activator during thrombolytic therapy: Evidence of activation of protein C pathway

Protein C inhibitor (PCI) is a heparin-dependent serine protease inhibitor which, in vitro, inhibits activated protein C (APC, urokinase (u-PA) and tissue plasminogen activator (t-PA), plasma kallikrein (KK), thrombin and factors Xa and Ma. We have previously shown in vivo complexes of PCI with APC, u-PA and KK. Here we study the contribution of PCI to the inhibition of t-PA both in vitro and in vivo. PCI complexed to t-PA (t-PA:PCI) was measured by a sandwich ELISA using antibodies directed against each protein in the complex. The formation of t-PA:PCI complexes paralleled the inhibition of t-PA amidolytic activity by PCI in a purified system. In the plasma milieu, after incubation of 8 μg/mL t-PA (final concentration) with human plasma for 2 h at 37°C, in the presence of heparin, the residual t-PA activity was about 38% and plasma contained 0.56 μg/mL t-PA:PCI complexes. To study complex formation in vivo, human plasma samples from patients (n=5) with myocardial infarction were analyzed following infusion of 100 mg recombinant t-PA for 2.5h (10% initial bolus; 40% by a 20min infusion, and 50% by a 2-h infusion). Maximum peak values of t-PA:PCI complexes were measured immediately after the second and third t-PA doses, ranging from 16 to 135 ng/mL. In contrast, during t-PA infusion the level of complexes of t-PA with plasminogen activator inhibitor-1 (t-PA:PAI-1) increased only slightly, from 2.4±1.5 to 7.2±4.6 ng/mL, and reached a maximum mean value of 17.2±20.6 ng/mL 2h post-infusion. PCI antigen decreased during t-PA infusion from 80%±16% to 71%±7%. Protein C antigen levels decreased during t-PA infusion, with the subsequent appearance of complexes between APC and PCI (APC:PCI), and free protein S gradually declined during infusion, reaching levels of 69% of the basal value 2h post-infusion.These data show that in vivo interaction of PCI and t-PA exists, which in turn support the view that PCI may play a role in the regulation of the fibrinolysis pathway. They also show that thrombolytic therapy with t-PA produces activation and a subsequent decrease in protein C antigen levels as well as in free protein S levels.     

Decreased Proinflammatory Cytokines Production in Children with Complicated Parapneumonic Pleural Effusion after Intrapleural Fibrinolytic Treatment

Intrapleural fibrinolytic therapy (IFT) provides clinical benefit in the treatment of complicated pleural parapneumonic effusion (CPE). Whether IFT influences the proinflammatory cytokines production and fibrinlytic activity is currently unclear. Therefore, we collected pleural effusion samples from CPE patients with IFT (study group) and patients without IFT (control group). A membrane human inflammatory cytokines array kit was used to compare the difference of targeted cytokine production between these two groups. Enzyme-linked immunosorbent assay (ELISA) methods were used for quantitative analysis of targeted cytokines and fibrinolytic enzymes. The results showed there were no significant differences between the study (n = 16) and control (n = 14) groups in patients’ demographic data. After fibrinolytic therapy, the patients in the study group had significant lower plasminogen activator inhibitor (PAI) level (732.36?±?254.09 ng/mL vs 1,509.36?±?1,340.11 ng/mL, p?<?0.05) and higher urokinase plasminogen activator (u-PA) level (75.56?±?41.70 ng/mL vs 6.87?±?5.07 ng/mL, p?<?0.05) than they did before treatment. Moreover, the tissue inhibitors of metalloproteinase-2 (TIMP-2) (1,560.03?±?403.49 pg/mL vs 3,686.45?±?1,263.83 pg/mL, p?<?0.05) and inflammatory chemokine, regulated on activation normal T-cell expressed and secreted/chemokine (C-C motif) ligand 5 (RANTES), (293.58?±?212.93 pg/mL vs 749.27?±?53.79 pg/mL, p?<?0.05), were also significantly lower in the study group after fibrinolytic therapy, but not in the control group. In conclusion, intrapleural fibrinolytic treatment with urokinase could enhance fibrinolytic activity and decrease TIMP-2 and RANTES production.     

Urokinase and its complex with plasminogen activator inhibitor-3/protein C inhibitor in urine

Urokinase-type plasminogen activator (u-PA) occurs either in a single-chain form or in a two chain form (scu-PA or tcu-PA, respectively). The occurrence of the different forms of u-PA and the formation of u-PA-inhibitor complexes was studied in urine samples from 10 healthy donors. Measurement by means of a bio-immunoassay showed that the urine samples contained 307±189ng/ml u-PA (mean±SD), of which 72±15% occurred in the active two chain form and 28±15% in the plasmin-activatable single chain form. SDS-polyacrylamide gel electrophoresis followed by fibrin zymography revealed that fresh urine contained no, or only traces of, u-PA-inhibitor complexes. In contrast to what has previously been published, the bulk of u-PA appeared to be in an uncomplexed 55 kD form. Incubation of urine at 37°C for 8 h did not lead to the formation of u-PA-inhibitor complexes. However, after rapid removal of the low molecular weight fraction of urine on a gel filtration column a 100kD complex, consisting of u-PA and plasminogen activator inhibitor-3/protein C inhibitor, was formed during incubation (t1/2≈45 min). Complex formation was dependent on stimulation by urinary glycosaminoglycans and only occurred at a low ionic strength, which explains why no complexes were formed in the untreated urine samples of healthy donors.     

Fibrinolytic changes during acute vascular damage induced by Mediterranean spotted fever

Hemostatic abnormalities and microvascular injury have been described in patients with Mediterranean spotted fever (MSF). Rickettsial vasculitis comprises endothelial injury and the immune and phagocytic host response. Only indirect evidence of activation of the fibrinolytic system has been reported in MSF, but no data on plasma levels of their main components, tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1), in this disease are available. To obtain quantitative information on the ‘in vivo’ activation of the fibrinolytic system during the acute and subacute phase of the endothelial damage, several fibrinolytic components were studied in 28 MSF patients. Upon admission (day 1), patients showed a significant increase both in t-PA (30±20 ng/ml) and in PAI-1 antigen levels (55±30 ng/ml) as compared with normal levels (9±4 ng/ml and 11±4 ng/ml, respectively). The levels of PAI-1 activity were also increased (median: 7U/ml, range: 0–24 U/ml), but not significantly (normal values, median: 3 U/ml, range: 0.5–5 U/ml). After remission of the disease (day 30) a significant decrease in t-PA and PAI-1 antigen levels was observed in comparison with day 1, but the PAI-1 antigen concentration remained increased in comparison with normal values. The decrease in the ratio VIII:C/vWF:Ag found in the acute phase of the disease and after 30 days could indicate that the endothelial damage remains after this period. Tissue necrosis factor α (TNFα), a potential modulator of fibrinolytic system during gram-negative sepsis, showed normal values during the acute phase of the disease and at day 30. The data found in this study indicate that the changes observed in the fibrinolytic system during MSF could be due to the endothelial damage found in the acute phase of the disease. The increased plasma PAI-1 levels could contribute to an increase in the risk of venous thrombosis in MSF patients.     

Exhaled carbon monoxide concentration is not elevated in patients with inflammatory bowel disease

The present study was initiated to examine whether the concentration of CO in the breath is elevated in patients with inflammatory bowel disease (IBD). Twenty-three clinically stable patients with IBD in the outpatient clinic (11 with Crohn’s disease, 12 with ulcerative colitis), who are non-smokers and non-passive smokers, were selected and the concentration of CO in their breath was measured using a breath gas analyser (TRI lyser mBA-3000). The concentration of CO in the breath of 23 patients with IBD was 2.5±0.9 (1.1–4.3) ppm. This concentration comes within the range of standard values in our previous reports (2.5±2.2 ppm). Any significant difference was not observed between 2.4±0.9 (1.5–4.3) ppm for the 11 Crohn’s disease patients and the 2.6±1.0 (1.1–3.9) ppm for the 12 ulcerative colitis patients. The results suggest that clinically stable patients with IBD do not show high values for concentration of CO in the breath.     

Biological properties of a kringleless tissue plasminogen activator (t-PA) mutant

We have previously described a recombinant t-PA mutant (mt-PA) which lacks the double kringle domains. Purified mt-PA (specific activity comparable to melanoma cell derived t-PA (nt-PA)) exhibits fibrin specificity similar to nt-PA but is inhibited 50 to 80% less than nt-PA by a human platelet PA inhibitor. The biological properties of mt-PA, nt-PA and urokinase (u-PA) were studied in a human plasma circulation system. [¹²⁵I] fibrinogen whole blood Chandler loop thrombi were suspended in a chamber perfused by plasma (50 ml) circulated at 120 ml/min. The plasminogen activators were given as a bolus (20% of the total dose) and followed by a 4 h constant rate infusion. Samples obtained were analysed for [¹²⁵I] fibrin degradation products, plasminogen and fibrinogen. At 5h, thrombolysis (% total thrombus [¹²⁵I]) released was: nt-PA (100u/ml) 51±710 (mean ±s.d.), mt-PA (100u/ml) 79±7%, u-PA (500u/ml) 88±12%. Plasminogen and fibrinogen values (% of control) were: nt-PA 63±90/ plasminogen, 68±19% fibrinogen; mt-PA 69±7% plasminogen, 72±23% fibrinogen; u-PA 34±8% plasminogen, 21±8% fibrinogen. In saline control studies, no thrombolysis and no plasminogen and fibrinogen changes occurred. Thus, mt-PA appears to be a more efficient thrombolysic agent than nt-PA; the two plasminogen activators cause comparable plasminogen and fibrinogen depletion. 100 u mt-PA is comparable to 500u u-PA/ml but as expected, u-PA extensively depletes both plasminogen and fibrinogen.     

On lipoprotein(a) and the coagulation/fibrinolysis balance in the acute phase of deep venous thrombosis

In vitro studies have suggested that lipoprotein(a) (Lp(a)), an important risk factor for the development of atherosclerotic cardiovascular and cerebrovascular disease, might interfere with fibrinolysis. We studied the correlations in vivo between Lp(a) and various coagulation and fibrinolysis factors in acute deep venous thrombosis, a state associated with an activated coagulation system and increased reactive fibrinolysis. In 31 patients with established acute deep venous thrombosis of the lower limb, coagulation parameters (fibrin monomers, thrombin-antithrombin III) and fibrinolysis parameters (D-dimers, tissue plasminogen activator antigen, plasminogen) were studied in relation to Lp(a) concentrations. Elevated thrombin-antithrombin III and fibrin monomer levels were found together with enhanced D-dimers and tissue plasminogen activator concentrations, signs of an activated coagulation system and of increased reactive fibrinolysis. Significant correlations were obtained for fibrin monomers with thrombin-antithrombin III and D-dimers (r=0.56; p=0.002 and r=0.68; p=0.0002 respectively), between fibrin monomers and plasminogen (r=0.38; p=0.03) and for thrombin-antithrombin III with tissue plasminogen activator antigen (r=0.64; p=0.006). These data are indicative of a normally functioning coagulation/fibrinolysis axis in acute deep venous thrombosis. No correlation between Lp(a) concentrations and coagulation or fibrinolysis parameters was found in these patients with acute deep venous thrombosis, which suggests that variations in Lp(a) concentrations are not accompanied by parallel changes in coagulation and fibrinolysis parameters.     

肝癌患者组织中凝血及纤溶因子的基因表达检测与分析

目的:检测组织因子（TF）、尿激酶型纤溶酶原激活物（uPA）及其受体（uPAR ）mRNA在肝细胞癌组织中的表达并探讨其临床意义。 方法:利用RT-PCR法分别检测27例肝细胞癌、癌旁及27例正常肝组织中TF、uPA、uPA mRNA表达阳性率及相对表达强度，并结合临床病理资料进行分析。 结果:TF、uPA、uPAR在肝细胞癌组织中阳性率及相对表达强度分别为62.96%（17/27）、70.37%（19/27）、77.78%（21/27）；0.567±0.268、0.964±0.458、0.784±0.322,均显著高于癌旁组织及正常组织，差异显著(P<0.05)。3者在肝癌组织中相对表达强度与肿瘤大小及部分侵袭转移指标有关，其中TF mRNA表达强度在肝内及肝外转移及门脉癌栓组高于无肝内及肝外转移及无门脉癌栓组(P<0.05) ，uPA mRNA在有包膜侵润、肝内转移及门脉癌栓组高于无包膜侵润、无肝内转移及门脉癌栓组(P<0.05)；uPAR mRNA在有肝内转移及门脉癌栓组高于无肝内转移及门脉癌栓组(P<0.05)。经Pearson检验肝细胞癌患者TF、uPA和uPAR mRNA表达呈正相关［TF-uPA：r=0.373(P<0.01)，TF-uPAR：r=0.534(P<0.01)，uPA-uPAR：r=0.365 (P<0.01)］。 结论:肝细胞癌组织中TF、uPA及uPAR的mRNA显著升高并与部分侵袭转移指标有关，提示3者可能在肝细胞癌的发生及侵袭转移起协同作用。     

Interleukin-10 (IL-10) genotypes in inflammatory bowel disease

Interleukin-10 (IL-10) is an anti-inflammatory cytokine. Its production in humans is under genetic control, and genotype defines high or low producers of this cytokine. This study addresses the hypothesis that idiopathic inflammatory bowel disease (IBD) patients are more likely to have the low IL-10 producer genotype and phenotype. DNA was extracted from blood cells of patients with Crohn's disease (CD) or with ulcerative colitis (UC) for IL-10 genotyping. The frequency of the high IL-10 producer allele (-1082*G) was decreased in the whole IBD group (41% vs. 51%, P = 0.03) and in the UC patients compared with normal controls (37% vs. 51%; P = 0.04). Hence, there appears to be an association between the IL-10 genotypes and IBD. This suggests that individuals genetically predisposed to produce less IL-10 are at a higher risk of developing IBD, in particular, UC.     

Endothelium-fibrinolysis system interaction

The role of urokinase-type plasminogen activator (u-PA) in capillary growth was investigated using cultured bovine endothelial cells (BCE) on type I collagen gels and analyzed by morphometry for quantitative assessment of angiogenesis in vitro. BCE migrated into the gel matrix and formed capillary-like networks. The morphometrical analyses by measuring the length of tube formation enabled us to evaluate the effects of fibrinolytic proteases and several reagents. The addition of plasminogen up to 25 micrograms/ml to the gels significantly increased the extent of tube formation of BCE in a dose-dependent manner. Basic fibroblast growth factor (10 ng/ml) increased tube formation only in the presence of plasminogen. These enhancing effects on angiogenesis appeared to be related to the activation of fibrinolysis by u-PA derived from BCE, because they were suppressed by the addition of anti-u-PA IgG and anti-plasmin reagents such as aprotinin and alpha 2 anti-plasmin. Transforming growth factor beta also enhanced tube formation of BCE, but tumor necrosis factor alpha and interleukin-1 suppressed the tube formation. The quantitative assay of angiogenesis may be useful for clarifying the mechanism of neovascularization under pathological conditions.     

Fibrinolytic activity in rheumatoid arthritis and the effects of prednisolone therapy

The relationship between disease activity and fibrinolytic activity was investigated in 40 patients with rheumatoid arthritis (RA). There was no difference in global fibrinolytic activity between RA and controls, 220 (200–330) and 280 (167–346) min respectively although inhibition of plasminogen activator activity (PAI) was lower in RA 8.2 (5.1–9.7) vs 9.9 (8.0–15.7) IU/ml than controls, p=0.01. There were no significant differences between RA and controls in tPA:Ag 4.8ng/ml (3.1–7.8) vs 7.1(5.2–9.3) or PAI-1:Ag 8.Ong-ml (4.8–10.8) vs 9.1 (3.6–22.3). 9 patients with severe RA received prednisolone 40 mg daily for 7 days. This improved disease activity clinically and was associated with a fall in fibrinogen from 4.4. g/l (3.4–5.2) to 2.32 (2.31–2.63), p<0.0005 and plasminogen from 117.6% (107–121) to 101.5 (88.7–110.4), p<0.005. In addition PAI-1:Ag increased from 5.4ng/ml (2.77–10.38) to 11.0 (4.45–16.65), p<0.005 and PAI from 8.51U/ml (6.49–8.99) to 10.1 (8.5–16.5), p<0.005. In this study PAI was lower in patients with RA. Prednisolone improved disease activity, reduced Fibrinogen and plasminogen and increased PAI and PAI-1:Ag.     

高血糖通过AMPK/SIRT1调控CDK5通路介导阿尔兹海默症样分子病理改变

炎症性肠病 (Inflammatory Bowel Disease,IBD) 包括溃疡性结肠炎 (Ulcerative Colitis,UC) 和克罗恩病 (Crohn''s Disease,CD)。营养不良在IBD患者中很常见。与UC相比,CD中更易出现严重的营养不良。IBD患者肌肉减少症 (肌少症) 的患病率很高,肌少症对IBD患者的术后并发症和住院时间有负面影响,是IBD患者中需重视的问题。肌少症可通过多种机制发展,包括营养不良、慢性炎症、脂肪组织的炎症、维生素缺乏和肌-肠轴失衡。除了IBD患者的临床缓解和内镜下黏膜愈合外,对肌少症进行适当的治疗很重要。本综述旨在总结IBD和肌少症的相关研究成果,可能为IBD的诊治提供一种新的思路。     

Urokinase-type plasminogen activator (u-PA) antigen is a predictor of early relapse in breast cancer

Cancer tissue contains elevated levels of the urokinase-type plasminogen activator (u-PA). Experimental evidence supports the assumption that u-PA is related to invasion and metastasis. The aim of this study was to determine whether the u-PA content of human breast cancer tissue is a prognosis-associated factor. Tissue samples from breast cancer patients were assessed for u-PA antigen by ELISA and immunohistochemistry applying monoclonal antibodies. u-PA in breast cancer tissue was localised in the cytoplasm and plasma membrane of tumour cells.u-PA was quantitated in detergent-extracted tissue samples (1% Triton X-100 in TBS) obtained from patients with breast cancer (n =115) and benign lesions (n = 30). Median values of 2.62 ng versus 0.23 ng u-PA/mg protein were determined. Patients with high u-PA content (u-PA > 2.6 ng/mg protein) already showed a statistically significant higher incidence of relapse compared to patients with low u-PA content (26% versus 2%) after a median follow-up of 12.5 months (range 2–26 months). Multivariate regression analysis revealed that compared with established prognostic factors, u-PA had the strongest impact on relapse rate (relative risk RR=21.1), followed by hormone receptor status (RR = 5.8) and lymph node involvement (RR = 3.0).We conclude that the u-PA content of breast cancer tissue is an independent prognostic factor in predicting early relapse. High or low risk patients can be selected by u-PA determination within the stated risk groups defined by locoregional extension and hormone receptor status.     

Enzyme-Linked Immunosorbent Assay for Human Urokinase-Type Plasminogen Activator and its Proenzyme Using a Combination of Monoclonal and Polyclonal Antibodies

Abstract

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for human urokinase-type plasminogen activator (u-PA) and its inactive proenzyme (pro-u-PA) was developed. A monoclonal antibody was used as solid-phase antibody, while rabbit antibodies against human u-PA followed by peroxidase-conjugated third antibody were used for detection of bound u-PA. No reaction was observed with tissue-type plasminogen activator or with a variety of other human proteins. The assay was used for quantitation of u-PA in human urine and in culture fluid from human tumor cells. The recovery of added pro-u-PA was > 95%. A good agreement with the results obtained by enzymatic assays was found. The detection limit was less than 0.1 ng per ml, both for u-PA and pro-u-PA. The advantages of the use of ELISA compared with enzymatic assays and radioimmunoassays for quantitation of u-PA and pro-u-PA in biological samples are discussed.     

Association of celiac disease genes with inflammatory bowel disease in Finnish and Swedish patients

Some genetic loci may affect susceptibility to multiple immune system-related diseases. In the current study, we investigated whether the known susceptibility loci for celiac disease (CelD) also associate with Crohn's disease (CD) and/or ulcerative colitis (UC), the two main forms of inflammatory bowel disease (IBD), in Finnish patients. A total of 45 genetic markers were genotyped in a Finnish data set comprising 699 IBD patients and 2482 controls. Single-marker association with IBD and its subphenotypes was tested. A meta-analysis with a Swedish UC data set was also performed. A total of 12 single-nucleotide polymorphisms associated with CD and/or UC (P<0.05). In the subphenotype analysis, rs6974491-ELMO1 (P=0.0002, odds ratio (OR): 2.20) and rs2298428-UBE2L3 (P=5.44 × 10(-5), OR: 2.59) associated with pediatric UC and CD, respectively. In the meta-analysis, rs4819388-ICOSLG (P=0.00042, OR: 0.79) associated with UC. In the subphenotype meta-analysis, rs1738074-TAGAP (P=7.40 × 10(-5), OR: 0.61), rs6974491-ELMO1 (P=0.00052, OR: 1.73) and rs4819388-ICOSLG (P=0.00019, OR: 0.75) associated with familial UC, pediatric UC and sporadic UC, respectively. Multiple CelD risk loci also confer susceptibility for CD and/or UC in the Finnish and Swedish populations. Certain genetic risk variants may furthermore predispose an individual for developing a particular disease phenotype.     

Serum laminin and collagen IV in inflammatory bowel disease

BACKGROUND:/Aims: Laminin and collagen IV have been proposed as extracellular matrix serum markers. Because fibrosis is a major complication of inflammatory bowel disease, serum concentrations of laminin and collagen IV were measured in patients with ulcerative colitis (UC) and Crohn's disease (CD) and compared with inflammatory and healthy controls. METHODS: Laminin and collagen IV serum concentrations were measured in 170 patients with inflammatory bowel disease (86 UC and 84 CD), in 23 patients with other causes of intestinal inflammation, and in 80 matched healthy controls using commercially available enzyme linked immunosorbent assays. Laminin and collagen IV concentrations were correlated with disease activity, type, localisation, and treatment. RESULTS: Mean (SD) serum laminin concentrations were 281.0 (110.1) ng/ml in patients with UC, 275.6 (106.7) ng/ml in patients with CD, 192.0 (17.8) ng/ml in healthy controls, and 198.5 (32.5) ng/ml in inflammatory controls. Mean (SD) serum collagen IV concentrations were 72.8 (22.9) ng/ml in patients with UC, 71.0 (18.2) in patients with CD, 79.8 (12.2) ng/ml in healthy controls, and 88.9 (24.6) ng/ml in inflammatory controls. There was a significant difference among the four groups (p < 0.0001) for both markers. There was a strong correlation between serum laminin, but not collagen IV, and disease activity in both diseases. No significant association was found between these markers and disease localisation or disease type. CONCLUSIONS: Serum concentrations of laminin are increased, whereas serum concentrations of collagen IV are decreased, in patients with inflammatory bowel disease. They may be useful surrogate markers for sustained inflammation and tissue remodelling.     

Cell invasion is affected by differential expression of the urokinase plasminogen activator/urokinase plasminogen activator receptor system in muscle satellite cells from normal and dystrophic patients

The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti-u-PA and anti-u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA-induced invasion, as well as in u-PA-induced proliferation.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases plasma fibrinolytic activity in vivo

Increased phagocyte adhesion to vascular endothelium in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) administration may affect endothelial integrity and functions such as the expression of fibrinolytic properties. In patients with lymphoma receiving GM-CSF (30 μg/m² over 2 h), over-all plasma fibrinolytic activity, as measured by fibrin plate assays, increased from 78±15% to 118±35% (p<0.05, n=4), while tissue plasminogen activator (t-PA) levels increased from 0.35±0.10 IU/mL to 1.14±0.2 IU/mL (245±139% over preinfusion levels, p<0.05) by the end of the infusion. This was accompanied by a rise in t-PA antigen levels to 50±32% above baseline (p<0.05), and a corresponding fall in plasminogen activator inhibitor levels from 14.5±3.2 IU/mL to 6.8±2.9 IU/mL (52±25% of baseline, p<0.05). In contrast, macrophage colony-stimulating factor (M-CSF), which activates and primes mononuclear phagocytic cells, but does not increase cell adherence to endothelium, has no effect on plasma fibrinolytic parameters at these times. Increased plasma t-PA activity following GM-CSF administration suggests an indirect effect on endothelial function in vivo, and may be relevant to the reported side effects of GM-CSF such as the capillary leak syndrome, as well as to the chronic bleeding seen in GM-CSF transgenic mice.     

Enzyme-linked immunosorbent assay for human urokinase-type plasminogen activator and its proenzyme using a combination of monoclonal and polyclonal antibodies

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for human urokinase-type plasminogen activator (u-PA) and its inactive proenzyme (pro-u-PA) was developed. A monoclonal antibody was used as solid-phase antibody, while rabbit antibodies against human u-PA followed by peroxidase-conjugated third antibody were used for detection of bound u-PA. No reaction was observed with tissue-type plasminogen activator or with a variety of other human proteins. The assay was used for quantitation of u-PA in human urine and in culture fluid from human tumor cells. The recovery of added pro-u-PA was greater than 95%. A good agreement with the results obtained by enzymatic assays was found. The detection limit was less than 0.1 ng per ml, both for u-PA and pro-u-PA. The advantages of the use of ELISA compared with enzymatic assays and radioimmunoassays for quantitation of u-PA and pro-u-PA in biological samples are discussed.