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480 Platelet adhesion induced by tissue-type plasminogen activator and its reversal by vitamin E
138
POSTER PRESENTATIONS P-XXII Cellular Fibrinolysis
478
479
PRODUCTION OF STAPHYLOKINASE BY S T A P H Y L O C O C C U S A U R E U S STRAINS ISOLATED FROM BACTEREMIC PATIENTS
EXPRESSION OF U R O K I N A S E - TYPE P L A S M I N O G E N A C T I V A T O R AND P L A S M I N O G E N A C T I V A T O R INHIBITOR TYPE 1 IN H U M A N BRAIN TUMORS PRIMARY CULTURES. lFarah S., 1,2Chinot O., IOuafik L'H., l M a r t i n P.M. ILaboratoire de Canctmlogic Exp&imcntale, CJF INSERM 93-I 1, Facult~ de M~.deeineHerd, IFR Jean Roche, Bd P. Dramard, 13916 Marseille Codex 20 and 2Service de Neurochirurgie, CHU Timone, Bd Jean Moulin, 13385 Marseille, Codex 5, France.
Mi~lk~aen T, Kuikka A and Kuusela P
Department of Bacteriology and Immunology, The Haartman Institute, University of Helsinki, Finland. Synthesis of staphylokinase (SAK) by 85 Staphylococcus aureus strains isolated from bacteremic patients was quantitated by using an enzymatic and an ELISA assay. Three fourths of the strains produced 568 ng/ml of SAK while one fourth turned out to be SAK non producers. Generally, SAK levels obtained by the two methods correlated~ ~1.l. There were, however, differences in ratios of values by ELISA andenzymatic assays indicating that the specific SAK activity may vary in individual strains. The role of SAK in staphylococcal invasion and penetration was favoured by the finding that 21% of the strains isolated from patients with a primary focus only were SAK non producers while only one out of 14 strains (7%) associated with a secondary focus (endocarditis, spondylitis, deep abscessus) did not produce SAK. Additionally, when the proportion of SAK non producers varied between 25% and 29% among strains associated with the focus in skin, bone and joints and in heart, the corresponding proportion was only 9% among strains isolated from patients with a primary or secondary pulmonary focus. Although the findings do not directly show the virulence characteristics of SAK some of the results strongly favour the idea that SAK plays a role in invasivion and penetration of S. aureus into tissues. Other factors such as the plasminogen receptor status, the variation of SAK activity in individual staphylococcal strains and the sensitivity of various SAKs to physiological inhibitors require further investigation.
The plasminogen activators are enzymatic proteins that play an important role in the degradation of the extracellular matrix in physiological conditions but also in the tumor invasion. Recently, an expression of urokinase-type plasminogen activator (u-PA) and its inhibitor (PAI-1) has been described in rive in gliomas and seems to be correlated with the histologicalgrade and the tumor agressiveness. The intratumoral expression of u-PA and PAI-I has been shown by in situ hybridization in the tumor cells and endothelial cells, which suggests their implication in tumoral in~'asion and angiogenesis phenomenons. In the present study, we have established an in vitro model to determine the cellular source and the role of u-PA and PAl- I . This study has been performed on 22 primary cultures obtained from fresh human tumors at neuro-surgical
operations. The 22 primary cultures include 13 gliomas (5 low-grade and g highgrade), 2 ependymomas, 2 medulloblastomas, 3 metastasis, I neurinoma and 1 meningioma, u-PA and PAI- 1 mRNAs have been detected by Northern blot and in situ hybridization. We observed an heterogeneity, among the studied gliomas, in the expression of u-PA and its inhibitor. None of the 5 benign gliomas exhibit expression of u-PA and PAI-I, which was restricted to malignant gliomas. In those aggressive tumor, mRNA expression of u-PA alone, PAI- 1 alone, and u-PA+PAI- 1 was observed respectively in 2/8, 5/7 and 2/7 gliomas, So, the expression of u-PA and/or PAl- 1 was observed in 5/8 malignant gliomas. The higher expressions have been shown, by Northern blot and in situ hybridization, in gliomas which the clinical evolution has been the most pejorative, in accordance to the in rive study. In another one, a medulloblastoma and a metastasis eoexpres~ u-PA and PAI-I. The epondymoma, the neurinoma and the mcninglomastudied express neither u-PA nor PAl- 1. Our results provide that tumoral glial cells are able to express u-PA and PAI-I individually. This expression, observed only in primary cultures of malignant gliomas, is in accordance with the heterogeneous profiles of u-PA and PAI-I we previously observed in vivo. This in vitro model will permitt to study the different 10ossibleregulations of the u-PA and PAl- I activity expression.
480
481
PLATELET ADHESION INDUCED BY TISSUE-TYPE PLASMINOGEN ACTIVATOR AND ITS REVERSAL BY VITAMIN E J e n C J, D o n g H P , L a i K C a n d C h e n H I D e p a r t m e n t o f Physiology, National C h e n g - K u n g University, Tainan, Taiwan, R.O.C.
F I B R I N O L Y T I C C O M P O N E N T S IN N A S A L M U C O S A AND N A S A L S E C R E T I O N Yasuda T, 2Madoiwa S, JKitamura K, 2Mimuro J, 2Matsuda M, an~'d-~-ata Y 'Department of Otolaryngology and 2Division of Thrombosis and Hemostasis, Jichi Medical School, Tochigi, Japan
T h r o m b o l y t i c therapy is k n o w n to induce platelet-related side effects. W e used a parallel-plate rectangular flow c h a m b e r to measure platelet adhesion under various conditions. For experiments e x r i v e , tissue-type p l a s m i n o g e n activator (tPA), vitamin E (vit E), and the combination o f these two were intravenously a d m i n i s t e r e d to the rat for 60 rain. Platelet adhesion m e a s u r e m e n t s were p e r f o r m e d via the femoral artery before drug infusion, and at 30, 60, and 120 min after the initiation o f drug infusion. Our results indicated that 1) tPA e n h a n c e d platelet a d h e s i o n at 60 and 120 min; 2) vit E tended to reduce platelet adhesion; 3) tPA/vit E r e d u c e d t P A - e n h a n c e d platelet adhesion; and 4) adherent platelets in the tPA-treated group d e m o n s t r a t e d severe m o r p h o l o g i c a l c h a n g e s w h i c h could be p r e v e n t e d b y vit E. In v i t r o experiments using citrated h u m a n w h o l e blood s h o w e d similar results, i.e., tPA, but not heatinactivated tPA, e n h a n c e d platelet a d h e s i o n and this p h e n o m e n o n could be b l o c k e d by various p l a s m i n inhibitors. Moreover, vit E prevented t P A - e n h a n c e d platelet adhesion in v i t r o . T h e s e data suggest that tPA e n h a n c e s platelet adhesion and that this adverse effect can be suppressed by vit E.
We evaluated a possible role for fibrinolytic components in nasal secretion by tissue localization with immunohistochemical techniques and by measuring their antigen concentrations in nasal discharge by means of enzyme-linked immunosorbent assays(ELISA) and by fibrinautography. Nasal mucosa was obtained surgically from the inferior turbinate of normal volunteers. Urokinase-type plasminogen activator (uPA) specific staining was observed in pseudostaratified ciliated epithelium and predominantly in mucous cells of seromucinous gland, while serous cells were almost devoid of stain. The pattem of staining of plasminogen activator inhibitor-2 was similar to that of uPA. In contrast, plasminogen activator inhibitor- I(PAI- 1) immunoreactive material was localized exclusively in serous cells of seromucinous gland. Positive staining for tissue-type PA(t-PA) was observed in endothelial cells and basal cells, which differentiate into either ciliated or goblet cells and are resting directly on the basement membrane. Then nasal secretion before and after stimulation by methacholine was examined by ELISA and fibrinantography. The nasal discharge contained single chain uPA, PAI- 1 and small amount of t-PA. After simulation, the level of t-PA tentatively increased but gradually decreased to its normal value. These differential staining of fibrinolytic components and the existence of PAs and PAI-1 in the nasal discharge suggest that fibrinolytic system may play a critical role in nasal secretion.
POSTER PRESENTATIONS P-XXII Cellular Fibrinolysis
478
479
PRODUCTION OF STAPHYLOKINASE BY S T A P H Y L O C O C C U S A U R E U S STRAINS ISOLATED FROM BACTEREMIC PATIENTS
EXPRESSION OF U R O K I N A S E - TYPE P L A S M I N O G E N A C T I V A T O R AND P L A S M I N O G E N A C T I V A T O R INHIBITOR TYPE 1 IN H U M A N BRAIN TUMORS PRIMARY CULTURES. lFarah S., 1,2Chinot O., IOuafik L'H., l M a r t i n P.M. ILaboratoire de Canctmlogic Exp&imcntale, CJF INSERM 93-I 1, Facult~ de M~.deeineHerd, IFR Jean Roche, Bd P. Dramard, 13916 Marseille Codex 20 and 2Service de Neurochirurgie, CHU Timone, Bd Jean Moulin, 13385 Marseille, Codex 5, France.
Mi~lk~aen T, Kuikka A and Kuusela P
Department of Bacteriology and Immunology, The Haartman Institute, University of Helsinki, Finland. Synthesis of staphylokinase (SAK) by 85 Staphylococcus aureus strains isolated from bacteremic patients was quantitated by using an enzymatic and an ELISA assay. Three fourths of the strains produced 568 ng/ml of SAK while one fourth turned out to be SAK non producers. Generally, SAK levels obtained by the two methods correlated~ ~1.l. There were, however, differences in ratios of values by ELISA andenzymatic assays indicating that the specific SAK activity may vary in individual strains. The role of SAK in staphylococcal invasion and penetration was favoured by the finding that 21% of the strains isolated from patients with a primary focus only were SAK non producers while only one out of 14 strains (7%) associated with a secondary focus (endocarditis, spondylitis, deep abscessus) did not produce SAK. Additionally, when the proportion of SAK non producers varied between 25% and 29% among strains associated with the focus in skin, bone and joints and in heart, the corresponding proportion was only 9% among strains isolated from patients with a primary or secondary pulmonary focus. Although the findings do not directly show the virulence characteristics of SAK some of the results strongly favour the idea that SAK plays a role in invasivion and penetration of S. aureus into tissues. Other factors such as the plasminogen receptor status, the variation of SAK activity in individual staphylococcal strains and the sensitivity of various SAKs to physiological inhibitors require further investigation.
The plasminogen activators are enzymatic proteins that play an important role in the degradation of the extracellular matrix in physiological conditions but also in the tumor invasion. Recently, an expression of urokinase-type plasminogen activator (u-PA) and its inhibitor (PAI-1) has been described in rive in gliomas and seems to be correlated with the histologicalgrade and the tumor agressiveness. The intratumoral expression of u-PA and PAI-I has been shown by in situ hybridization in the tumor cells and endothelial cells, which suggests their implication in tumoral in~'asion and angiogenesis phenomenons. In the present study, we have established an in vitro model to determine the cellular source and the role of u-PA and PAl- I . This study has been performed on 22 primary cultures obtained from fresh human tumors at neuro-surgical
operations. The 22 primary cultures include 13 gliomas (5 low-grade and g highgrade), 2 ependymomas, 2 medulloblastomas, 3 metastasis, I neurinoma and 1 meningioma, u-PA and PAI- 1 mRNAs have been detected by Northern blot and in situ hybridization. We observed an heterogeneity, among the studied gliomas, in the expression of u-PA and its inhibitor. None of the 5 benign gliomas exhibit expression of u-PA and PAI-I, which was restricted to malignant gliomas. In those aggressive tumor, mRNA expression of u-PA alone, PAI- 1 alone, and u-PA+PAI- 1 was observed respectively in 2/8, 5/7 and 2/7 gliomas, So, the expression of u-PA and/or PAl- 1 was observed in 5/8 malignant gliomas. The higher expressions have been shown, by Northern blot and in situ hybridization, in gliomas which the clinical evolution has been the most pejorative, in accordance to the in rive study. In another one, a medulloblastoma and a metastasis eoexpres~ u-PA and PAI-I. The epondymoma, the neurinoma and the mcninglomastudied express neither u-PA nor PAl- 1. Our results provide that tumoral glial cells are able to express u-PA and PAI-I individually. This expression, observed only in primary cultures of malignant gliomas, is in accordance with the heterogeneous profiles of u-PA and PAI-I we previously observed in vivo. This in vitro model will permitt to study the different 10ossibleregulations of the u-PA and PAl- I activity expression.
480
481
PLATELET ADHESION INDUCED BY TISSUE-TYPE PLASMINOGEN ACTIVATOR AND ITS REVERSAL BY VITAMIN E J e n C J, D o n g H P , L a i K C a n d C h e n H I D e p a r t m e n t o f Physiology, National C h e n g - K u n g University, Tainan, Taiwan, R.O.C.
F I B R I N O L Y T I C C O M P O N E N T S IN N A S A L M U C O S A AND N A S A L S E C R E T I O N Yasuda T, 2Madoiwa S, JKitamura K, 2Mimuro J, 2Matsuda M, an~'d-~-ata Y 'Department of Otolaryngology and 2Division of Thrombosis and Hemostasis, Jichi Medical School, Tochigi, Japan
T h r o m b o l y t i c therapy is k n o w n to induce platelet-related side effects. W e used a parallel-plate rectangular flow c h a m b e r to measure platelet adhesion under various conditions. For experiments e x r i v e , tissue-type p l a s m i n o g e n activator (tPA), vitamin E (vit E), and the combination o f these two were intravenously a d m i n i s t e r e d to the rat for 60 rain. Platelet adhesion m e a s u r e m e n t s were p e r f o r m e d via the femoral artery before drug infusion, and at 30, 60, and 120 min after the initiation o f drug infusion. Our results indicated that 1) tPA e n h a n c e d platelet a d h e s i o n at 60 and 120 min; 2) vit E tended to reduce platelet adhesion; 3) tPA/vit E r e d u c e d t P A - e n h a n c e d platelet adhesion; and 4) adherent platelets in the tPA-treated group d e m o n s t r a t e d severe m o r p h o l o g i c a l c h a n g e s w h i c h could be p r e v e n t e d b y vit E. In v i t r o experiments using citrated h u m a n w h o l e blood s h o w e d similar results, i.e., tPA, but not heatinactivated tPA, e n h a n c e d platelet a d h e s i o n and this p h e n o m e n o n could be b l o c k e d by various p l a s m i n inhibitors. Moreover, vit E prevented t P A - e n h a n c e d platelet adhesion in v i t r o . T h e s e data suggest that tPA e n h a n c e s platelet adhesion and that this adverse effect can be suppressed by vit E.
We evaluated a possible role for fibrinolytic components in nasal secretion by tissue localization with immunohistochemical techniques and by measuring their antigen concentrations in nasal discharge by means of enzyme-linked immunosorbent assays(ELISA) and by fibrinautography. Nasal mucosa was obtained surgically from the inferior turbinate of normal volunteers. Urokinase-type plasminogen activator (uPA) specific staining was observed in pseudostaratified ciliated epithelium and predominantly in mucous cells of seromucinous gland, while serous cells were almost devoid of stain. The pattem of staining of plasminogen activator inhibitor-2 was similar to that of uPA. In contrast, plasminogen activator inhibitor- I(PAI- 1) immunoreactive material was localized exclusively in serous cells of seromucinous gland. Positive staining for tissue-type PA(t-PA) was observed in endothelial cells and basal cells, which differentiate into either ciliated or goblet cells and are resting directly on the basement membrane. Then nasal secretion before and after stimulation by methacholine was examined by ELISA and fibrinantography. The nasal discharge contained single chain uPA, PAI- 1 and small amount of t-PA. After simulation, the level of t-PA tentatively increased but gradually decreased to its normal value. These differential staining of fibrinolytic components and the existence of PAs and PAI-1 in the nasal discharge suggest that fibrinolytic system may play a critical role in nasal secretion.