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Plasma and pleural fluid analysis of fibrinolytic system in malignant pleural mesothelioma
268
Ahsti-ucts
I Lung
staining of most adenocarcinoma, is probably a cell marker more closely related to the epithelial differentiation ofMM.
PLASMA ANB PLEURAL FLUID ANALYSIS OFFIBRLNOLYTIC SYSTEMlNMALlGNANTPLEURAL~OMA S. EmriI, O.&demir, Y. Karakoca, L. mpltl, S. Dtindar, I. Baris. Hacettepe University Medical School, Department ef Chest Diseases and Hematology, Ankara. Turkey. The interaction between neoplasms and hemostatic system is of paramount importance in the pathophysiology of not only hemorrhagic and thrombotic events but also in tumor invasion and metastasis. Umkinase type plasminogen activator (uPA) has been extensively studied in cancer since it converts plasminogen toplasmin which degrades fibrin and some extracellular matrix components. This study was planned to describe the alterations oftibrinolytic system in plasma and pleural elfusion in malignant pleural mesothelioma (MPM). The study group included 38 patients with pleural effusion (17 MPM, 8 lung cancer and 13 benign effusion) and 30 healthy volunteers. Immunologic and activity assays of tissue-type plasminogen activator (t-PA), u-PA and plasminogen activator inhibitor-l (PAI-1) were performed from plasma samples of all subjects and from pleural fluid samples of patients. While median plasma t-PA antigen levels were higher in MPM and in lung cancer groups than in the control group, median t-PA activity was higher only in lung cancer group compared to the control group. The median plasma PAI-I antigen level was higher in MPM group than in the control group. PAI- activity did not show any diKerence between groups. The median plasma uPA antigen was higher in both mahgnant groups than the control group. However, the median plasma uPA activity was higher only in benign et&ion group. In samples from pleural fluids, medii uPA antigen and uPA activity were higher in both malignant groups. The pleural fluid-plasma gradient (which was defined as ccpleural fluid level minus plasma level*) for uPA activity and also pleural fluid/plasma uPA activity ratio were signiticantly higher in mahgnant groups than the benign group. Increased plasma level of t-PA antigen in both cancer groups may bc regarded as an evidence for endothelial stimulation and/or damage. In MPM group, it seems that excess t-PA and PAI-I form complexes with each other in plasma, since t-PA antigen and PAI- antigen levels are increased but activities of t-PA and PAI-I are not different from control group. In lung cancer group, t-PA activity is increased, probably due to elevated t-PA antigen not opposed by PAI-I. PIeural fluid data of MPM and lung cancer groups are similar; pleural fluid u-PA activity are increased in both groups. It is concluded that plasma tibrinolytic system is partially balanced in MPM, but totally unbalanced in lung cancer. On the other hand, pleural fluid exhibits excess uPA activity in both malignant groups. Whether this accounts for tumor invasion will be further investigated.
THE ROLE OF MESOTHELIAL CELLS IN PLEURAL. FIBROSLS : EFFECT OF ASBESTOS FIBRES AND CYTOKINES ON PROCOLIAGENPRODUCl’IONINVTTRO *SE Mutsaers, *RJ McAnulh: #M Nemethova. #G Lubec, +M Versnel, *GJ Laurent. *Centre for Respiratory Research, University College London, lJK, #Deparhnents ofPoedioh?cs, Universi@ of Henna, Austr@, and +ImmunoIogy Erasmus Universiteit, Rotterdam. Netherlands. Following accumulation of asbestos fibres in the lung many individuals develop sign&ant interstitial and pleural fibrosis. The mechanisms involved are uncertain but asbestos may activate cells directly to synthesize and deposit collagen or may stimulate release of profibrotic cytoklnes by activated macrophages. The effect of asbestos tibres on net fibroblast collagen production remains unclear. There have been no studies in mesothelial cells. The aim of this study was therefore to determine the effect of asbestos on the rate of collagen production in mesothelial cells. Three potentially profibrotic cytokines were also
Cancer
I5 (1996)
259-279
examined to determine if collagen synthesis is cytokine dependent in thesecells. A primary mesothelial cell line (NM 2O/l)wasused todetennine the effect of fibres (crocidolite and chrysotile asbestos and a control mineral fibre) and optimal concentrations of the cvtokines transformina growth factor beta I (TGF-OlO.4 pM-O.4 nM), mmour necrosis factor alpha (TNPa, 5.7 PM-2.9 t&f) and platelet-derived growth factor AB (PDGF-AR, 3.7 pM-1.85 nM) on collagen production in vitro. Collagen production was analyzed using a HPLC method developed in this laboratory. Pmliminaty cytotoxicity studies demonstrated that the highest non toxic tibre dose was IO &ml. Mesothelial cells cultured in control medium synthesized 1.52iO.09 nmobs procoIlagen/well (mean l SEM, n = S-6). No stimulation of collagen production was shown following incubation of cells with I and IO pg/ml of all three tibre types. TGF-81 significantly stimulated collagen production at 4,40 and 400 pM with maximalstimuJationat400pM(5.05+~O.18mmoles&U pcO.01). PDGP-Al3 stimulated collagen production at 1.85 nM (3.708i 0.56 nmoles/well pcO.01). ‘INPudid not significantly stimulatecollagen production above control medium. There was no signiftcant change in the number of cells/ well of confluent cells (4x 105 cells/well) following incubation with any concentration ot cytokine examined. The data presented in this study confirms that mesothelial cells synthesize collagen and demonstrates that the rate of synthesis is increased following exposure to cytokines in vitro, thus supporting a role for these cells in fibrosis. It appears unlikely that pleural fibrosis results from the direct effect of tibres on the mesothelium but may be due to stimulation of these cells by cytokines present in the inflammatory milieu.
CD44,HYALURONAN,ANDHUMANhlESOTEIEUOMAS
M. B. Penno, F. B. Askin, Harly Ma, M. Carbone, M. l? Vargas, and H. 1. Pass. Malignant pleural mesotheliotna (MPM) is a rare malignancy with major environmental implications regarding passive asbestos exposure, We have conducted an immunohistochemical and functional study to determine whether CD44, a glycopmtein implicated in the progression of a number of cancers, is expressed on the surface of human MPM cells. Immunohistochemical staining with a monoclonal antibody (H4C4) recognizing a constant region of human CD44 demonstated that 34 (92%) of the MPMs examined expressed CD44 on 50- 100% of the cells. The extent of CD44 expression was appamndy related to the histologic subtype with the highest expression seen in the epithelioid mesotheliomas and the least in the sarcomatoid tumors. Tumor cell lines established from the primary tumors or elliisions of six of the MPM Patients showed high expression of the <~standardn from of CD44 (CD44s). Four of thesesell lines exhibited strong attachment to hyaluronan (HA) in an in vitro attachment assay indicating that their CD44 was functional with respect to HA anchorage. HA attaclunent was specific in that it could be abolished by meincubation with euitoue specific anti CD44 antibodies or soluble substrate or by hyalur&&se treatment of attachment surfaces. We conclude that CD44 is highly expressed in MPM and that it may contribute to disease progression through its association with HA, a major component of pleural fluid.
UNEXPECTEBEXFKESSIONOFCARBOII~DRATESTRUCIURE.S WITH TERMINAL a-CAL&TOSYL RESIDUES BY MESOTHELIoMAcFALLINEsmm Y. Pilatte, D. Pradel, A. Buard, A. GretBrd, L. Saint-Etiemte, J. Bignon and M.C. Jaurand. INSERM Unit4 139. Instituf Uondor de Medecine MoI&uIaire - Facult& de Medecine, 8 rue du G&&al Sorroil. 94010 CrPteil Cedex, France Aberrations in cell surface carbohydrates are probably among the more dramatical changes associated with malignant transformation. Several lines of evidence indicate that these changes in glycosylation
Ahsti-ucts
I Lung
staining of most adenocarcinoma, is probably a cell marker more closely related to the epithelial differentiation ofMM.
PLASMA ANB PLEURAL FLUID ANALYSIS OFFIBRLNOLYTIC SYSTEMlNMALlGNANTPLEURAL~OMA S. EmriI, O.&demir, Y. Karakoca, L. mpltl, S. Dtindar, I. Baris. Hacettepe University Medical School, Department ef Chest Diseases and Hematology, Ankara. Turkey. The interaction between neoplasms and hemostatic system is of paramount importance in the pathophysiology of not only hemorrhagic and thrombotic events but also in tumor invasion and metastasis. Umkinase type plasminogen activator (uPA) has been extensively studied in cancer since it converts plasminogen toplasmin which degrades fibrin and some extracellular matrix components. This study was planned to describe the alterations oftibrinolytic system in plasma and pleural elfusion in malignant pleural mesothelioma (MPM). The study group included 38 patients with pleural effusion (17 MPM, 8 lung cancer and 13 benign effusion) and 30 healthy volunteers. Immunologic and activity assays of tissue-type plasminogen activator (t-PA), u-PA and plasminogen activator inhibitor-l (PAI-1) were performed from plasma samples of all subjects and from pleural fluid samples of patients. While median plasma t-PA antigen levels were higher in MPM and in lung cancer groups than in the control group, median t-PA activity was higher only in lung cancer group compared to the control group. The median plasma PAI-I antigen level was higher in MPM group than in the control group. PAI- activity did not show any diKerence between groups. The median plasma uPA antigen was higher in both mahgnant groups than the control group. However, the median plasma uPA activity was higher only in benign et&ion group. In samples from pleural fluids, medii uPA antigen and uPA activity were higher in both malignant groups. The pleural fluid-plasma gradient (which was defined as ccpleural fluid level minus plasma level*) for uPA activity and also pleural fluid/plasma uPA activity ratio were signiticantly higher in mahgnant groups than the benign group. Increased plasma level of t-PA antigen in both cancer groups may bc regarded as an evidence for endothelial stimulation and/or damage. In MPM group, it seems that excess t-PA and PAI-I form complexes with each other in plasma, since t-PA antigen and PAI- antigen levels are increased but activities of t-PA and PAI-I are not different from control group. In lung cancer group, t-PA activity is increased, probably due to elevated t-PA antigen not opposed by PAI-I. PIeural fluid data of MPM and lung cancer groups are similar; pleural fluid u-PA activity are increased in both groups. It is concluded that plasma tibrinolytic system is partially balanced in MPM, but totally unbalanced in lung cancer. On the other hand, pleural fluid exhibits excess uPA activity in both malignant groups. Whether this accounts for tumor invasion will be further investigated.
THE ROLE OF MESOTHELIAL CELLS IN PLEURAL. FIBROSLS : EFFECT OF ASBESTOS FIBRES AND CYTOKINES ON PROCOLIAGENPRODUCl’IONINVTTRO *SE Mutsaers, *RJ McAnulh: #M Nemethova. #G Lubec, +M Versnel, *GJ Laurent. *Centre for Respiratory Research, University College London, lJK, #Deparhnents ofPoedioh?cs, Universi@ of Henna, Austr@, and +ImmunoIogy Erasmus Universiteit, Rotterdam. Netherlands. Following accumulation of asbestos fibres in the lung many individuals develop sign&ant interstitial and pleural fibrosis. The mechanisms involved are uncertain but asbestos may activate cells directly to synthesize and deposit collagen or may stimulate release of profibrotic cytoklnes by activated macrophages. The effect of asbestos tibres on net fibroblast collagen production remains unclear. There have been no studies in mesothelial cells. The aim of this study was therefore to determine the effect of asbestos on the rate of collagen production in mesothelial cells. Three potentially profibrotic cytokines were also
Cancer
I5 (1996)
259-279
examined to determine if collagen synthesis is cytokine dependent in thesecells. A primary mesothelial cell line (NM 2O/l)wasused todetennine the effect of fibres (crocidolite and chrysotile asbestos and a control mineral fibre) and optimal concentrations of the cvtokines transformina growth factor beta I (TGF-OlO.4 pM-O.4 nM), mmour necrosis factor alpha (TNPa, 5.7 PM-2.9 t&f) and platelet-derived growth factor AB (PDGF-AR, 3.7 pM-1.85 nM) on collagen production in vitro. Collagen production was analyzed using a HPLC method developed in this laboratory. Pmliminaty cytotoxicity studies demonstrated that the highest non toxic tibre dose was IO &ml. Mesothelial cells cultured in control medium synthesized 1.52iO.09 nmobs procoIlagen/well (mean l SEM, n = S-6). No stimulation of collagen production was shown following incubation of cells with I and IO pg/ml of all three tibre types. TGF-81 significantly stimulated collagen production at 4,40 and 400 pM with maximalstimuJationat400pM(5.05+~O.18mmoles&U pcO.01). PDGP-Al3 stimulated collagen production at 1.85 nM (3.708i 0.56 nmoles/well pcO.01). ‘INPudid not significantly stimulatecollagen production above control medium. There was no signiftcant change in the number of cells/ well of confluent cells (4x 105 cells/well) following incubation with any concentration ot cytokine examined. The data presented in this study confirms that mesothelial cells synthesize collagen and demonstrates that the rate of synthesis is increased following exposure to cytokines in vitro, thus supporting a role for these cells in fibrosis. It appears unlikely that pleural fibrosis results from the direct effect of tibres on the mesothelium but may be due to stimulation of these cells by cytokines present in the inflammatory milieu.
CD44,HYALURONAN,ANDHUMANhlESOTEIEUOMAS
M. B. Penno, F. B. Askin, Harly Ma, M. Carbone, M. l? Vargas, and H. 1. Pass. Malignant pleural mesotheliotna (MPM) is a rare malignancy with major environmental implications regarding passive asbestos exposure, We have conducted an immunohistochemical and functional study to determine whether CD44, a glycopmtein implicated in the progression of a number of cancers, is expressed on the surface of human MPM cells. Immunohistochemical staining with a monoclonal antibody (H4C4) recognizing a constant region of human CD44 demonstated that 34 (92%) of the MPMs examined expressed CD44 on 50- 100% of the cells. The extent of CD44 expression was appamndy related to the histologic subtype with the highest expression seen in the epithelioid mesotheliomas and the least in the sarcomatoid tumors. Tumor cell lines established from the primary tumors or elliisions of six of the MPM Patients showed high expression of the <~standardn from of CD44 (CD44s). Four of thesesell lines exhibited strong attachment to hyaluronan (HA) in an in vitro attachment assay indicating that their CD44 was functional with respect to HA anchorage. HA attaclunent was specific in that it could be abolished by meincubation with euitoue specific anti CD44 antibodies or soluble substrate or by hyalur&&se treatment of attachment surfaces. We conclude that CD44 is highly expressed in MPM and that it may contribute to disease progression through its association with HA, a major component of pleural fluid.
UNEXPECTEBEXFKESSIONOFCARBOII~DRATESTRUCIURE.S WITH TERMINAL a-CAL&TOSYL RESIDUES BY MESOTHELIoMAcFALLINEsmm Y. Pilatte, D. Pradel, A. Buard, A. GretBrd, L. Saint-Etiemte, J. Bignon and M.C. Jaurand. INSERM Unit4 139. Instituf Uondor de Medecine MoI&uIaire - Facult& de Medecine, 8 rue du G&&al Sorroil. 94010 CrPteil Cedex, France Aberrations in cell surface carbohydrates are probably among the more dramatical changes associated with malignant transformation. Several lines of evidence indicate that these changes in glycosylation