Myeloid sarcoma: clinical and morphologic criteria useful for diagnosis.

Extramedullary accumulation of myeloblasts or immature myeloid cells form tumors called myeloid sarcoma in the WHO classification. Such tumors develop in lymphoid organs, bone (skull, orbit, etc.), skin, soft tissue, various mucosae and organs, and the CNS. They may precede or occur concurrently with acute myeloid leukemia, or reveal blastic transformation of chronic myeloproliferative disorders or myelodysplastic syndromes. They may also reveal relapses in treated patients. They are constituted by a diffuse infiltrate made up of medium-to-large cells. The cells are difficult to identify. Imprints are very useful. Immunohistochemistry can help diagnose and distinguish four variants: granulocytic myeloperoxidase (MPO+, CD 68+ [KP1+/-, PGM1-] lysozyme+, CD 34+/-), monoblastic (MPO-, CD 68+, [KP1+, PGM1+] lysozyme+, CD 34-), myelomonoblastic (MPO-, CD 68+, [KP1+, PGM1+] lysozyme+, CD 34-), or megakaryoblastic (positivity for factor VIII, CD 61, CD 31). Immunohistochemistry sometimes demonstrates expression of CD 43, CD 7, CD 79a, and CD 56 (particularly the monoblastic variant with t[8;21]). Recently the demonstration of CD 99 and CD 117, which can now be done on paraffin sections, may be useful to identify blasts of granulocytic origin. The diagnosis is missed in about 50% of cases when immunohistochemistry is not used. Patients with myeloid sarcomas should be treated in the same way as patients with acute myeloblastic leukemia. Disease progression and prognosis are similar for the two conditions.

Primary histiocytic sarcoma of the spleen associated with erythrophagocytic histiocytosis.

We report an exceptional case of a histiocytic sarcoma presenting as a primary isolated spleen tumor in a 71-year-old woman. The neoplastic cells in the cords and sinuses of the red pulp formed multiple lobulated tumors, which were detected in vivo by ultrasound scan. The medium cells, large cells and the giant cells expressed CD68, a histiocyte-associated marker, lysozyme and S100 protein. All these cells were negative for B- and T-cell markers, cytokeratins, melanosome markers (HMB45) and CD1a (Langerhans' cells). Many tumor cells displayed strong erythrophagocytosis and sometimes lymphocytophagocytosis. In addition, numerous histiocytes with morphology indistinguishable from reactive macrophages also exhibited a strong erythrophagocytosis, and were found in the tumor as well as in the normal splenic parenchyma. Despite multi-agent chemotherapy, the patient suffered from a relapse in the liver, with a rapid fatal outcome. A literature review showed that such a primary splenic presentation with multiple tumors is rare. In contrast, in systemic malignant histiocytosis, secondary spleen involvement occurs more frequently but with diffuse infiltration. The association with a reactive histiocytosis with erythrophagocytosis corresponds to "histiocytic medullary reticulosis", as previously described by Scott and Robb-Smith.

Expression and prognostic significance of survivin in de novo acute myeloid leukaemia.

Survivin is an inhibitor of apoptosis (programmed cell death) overexpressed in various human cancers, but undetectable in normal differentiated tissues. A potential distribution and prognostic significance of survivin in patients with de novo acute myeloid leukaemia (AML) was investigated. By immunofluorescence of bone marrow specimens and peripheral blood mononuclear cells, survivin was detected in 75 out of 125 interpretable AML cases (60%), with reactivity in 50-90% of AML cells. Survivin expression correlated with a lower white blood cell count (WBC) (P = 0.008 by the Mann-Whitney test) and was associated, in the 55 cases of FAB M0/M1/M2, with leukaemic granulocytic maturation (one out of five M/L0, 11 out of 22 M/L1 and 23 out of 28M/L2; P = 0.007 by the Fisher test). In 69 patients treated with the Acute Leukaemia French Association (ALFA) 9000 protocol, survivin expression was significantly associated with a lower WBC (P = 0.03 by the Mann-Whitney test) and favourable/intermediate cytogenetics (P= 0.03 by the Fisher test). There was no significant difference in complete remission rate or overall survival between survivin-positive and survivin-negative AML patients (P = 0.15 by the log-rank test). However, survivin expression became an independent negative prognostic factor for survival when adjusted with the Cox model for established prognostic factors in AML (cytogenetics, age and WBC) or for the ALFA 9000 treatment arm (RR = 2.8 and P = 0.026, by the likelihood-ratio test). These data suggest that survivin expression may be considered as a new unfavourable prognostic factor of de novo AML and suggest a role for apoptosis inhibition in influencing disease outcome.

Prognostic significance of survivin expression in diffuse large B-cell lymphomas.

Survivin is an inhibitor of apoptosis overexpressed in various human cancers but undetectable in normal differentiated tissues. A potential expression and prognostic significance of survivin was studied in 222 patients with diffuse large B-cell lymphomas (centroblastic, 96%; immunoblastic, 4%). All patients were enrolled between 1987 and 1993 (median follow-up, 7 years) in the LNH87 protocol of the Groupe d'Etudes des Lymphomes de l'Adulte (GELA) and treated either with the reference ACVBP arm (doxorubicin, cyclophosphamide, vindesine, bleomycin, and prednisone)[AU3A] (n = 79) or other experimental anthracycline-containing regimens (n = 143). The characteristics of these patients were median age of 56 years; serum lactate dehydrogenase (LDH) greater than 1N, 60%; stage III-IV, 55%; performance status, according to the Eastern Cooperative Oncology Group (ECOG) scale, more than 1, 23%; extranodal sites more than 1, 29%; mass more than 10 cm, 44%; bone marrow involvement, 15%. Of the 222 patients studied, 134 (60%) revealed survivin expression in virtually all tumor cells by immunohistochemistry. The overall 5-year survival rate was significantly lower in patients with survivin expression than in those without (40% vs 54%, P =.02). Multivariate analysis incorporating prognostic factors from the International Prognostic Index (IPI) identified survivin expression as an independent predictive parameter on survival (P =.03, relative risk [RR] = 1.6) in addition to LDH (P =.02, RR = 1.6), stage (P =.03, RR = 1.7), and ECOG scale (P =.05, RR = 1.6). A second analysis incorporating IPI as a unique parameter demonstrated that survivin expression (P =.02, RR = 1.6) remained a prognostic factor for survival independently of IPI (P =.001, RR = 1.5). Survivin expression may be considered a new unfavorable prognostic factor of diffuse large B-cell lymphoma. (Blood. 2000;96:1921-1925)

Control of apoptosis during angiogenesis by survivin expression in endothelial cells.

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor alpha/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.

Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting.

Survivin is a new IAP apoptosis inhibitor expressed during development and in human cancer in vivo. The coding strand of the survivin gene was extensively complementary to that of effector cell protease receptor-1 (EPR-1), prompting the present investigation on the origin and functional relationship of these two transcripts. Southern blots of genomic DNA were consistent with the presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes. By pulsed field gel electrophoresis and single- and two-color fluorescence in situ hybridization, these were contained within a contiguous physical interval of 75-130 kilobases (kb) on chromosome 17q25. In Northern blots, a single strand-specific probe identified a 1.3-kb EPR-1 mRNA broadly distributed in normal adult and fetal tissues, structurally distinct from the 1.9-kb Survivin transcript expressed in transformed cell lines. Transient co-transfection of an EPR-1 cDNA potentially acting as a Survivin antisense with a lacZ reporter plasmid resulted in loss of viability of HeLa cells. In contrast, co-transfection of an antisense cDNA of intercellular adhesion molecule-1 or a sense-oriented Survivin cDNA was without effect. In stably transfected HeLa cells, ZnSO4 induction of an EPR-1 mRNA under the control of a metallothionein promoter suppressed the expression of endogenous survivin. This resulted in (i) increased apoptosis as detected by analysis of DNA content and in situ internucleosomal DNA fragmentation and (ii) inhibition of cell proliferation as compared with induced vector control transfectants. These findings suggest the existence of a potential EPR-1/survivin gene cluster and identify survivin as a new target for disrupting cell viability pathways in cancer.

Human metalloprotease-disintegrin Kuzbanian regulates sympathoadrenal cell fate in development and neoplasia.

The development of the sympathetic nervous system involves cell-cell interactions that regulate the fate and migration of progenitor neural cells. Recent evidence shows that focal membrane-bound protease activity is critical for such interactions. The Drosophila kuzbanian (kuz) gene is required in neurogenesis and encodes a highly conserved, membrane-bound metalloprotease- disintegrin closley related to theTNF-alphaconvertingenzyme (TACE). We have characterized the human and mouse kuz homologs and mapped human kuz to chromosome 15q22. During mouse embryonic development Kuz is expressed mainly in the sympathoadrenal and olfactory neural precursors. Once sympathoadrenal cells differentiate into chromaffin cells in the adult adrenal medulla, they no longer express Kuz. However, we found that tumors of sympathoadrenal origin, such as pheochromocytomas and neuroblastomas, overexpress Kuz. Further, transfection of a kuz construct lacking the protease domain, but not the full-length construct, induces neurite formation in PC12 chromaffin tumor cells. Taken together our results suggest a critical role for Kuz in regulation of sympathoadrenal cell fate.

Developmentally regulated expression of the novel cancer anti-apoptosis gene survivin in human and mouse differentiation.

Inhibitors of programmed cell death (apoptosis) may regulate tissue differentiation and aberrantly promote cell survival in neoplasia. A novel apoptosis inhibitor of the IAP gene family, designated survivin, was recently found in all of the most common human cancers but not in normal, terminally differentiated adult tissues. The expression of survivin in embryonic and fetal development was investigated. Immunohistochemistry and in situ hybridization studies demonstrated strong expression of survivin in several apoptosis-regulated fetal tissues, including the stem cell layer of stratified epithelia, endocrine pancreas, and thymic medulla, with a pattern that did not overlap with that of another apoptosis inhibitor, bcl-2. A sequence-specific antibody to survivin immunoblotted a single approximately 16.5-kd survivin band in human fetal lung, liver, heart, kidney, and gastrointestinal tract. In mouse embryo, prominent and nearly ubiquitous distribution of survivin was found at embryonic day (E)11.5, whereas at E15 to -21, survivin expression was restricted to the distal bronchiolar epithelium of the lung and neural-crest-derived cells, including dorsal root ganglion neurons, hypophysis, and the choroid plexus. These data suggest that expression of survivin in embryonic and fetal development may contribute to tissue homeostasis and differentiation independently of bcl-2. Aberrations of this developmental pathway may result in prominent re-expression of survivin in neoplasia and abnormally prolonged cell viability.

A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.

Inhibitors of programmed cell death (apoptosis) aberrantly prolonging cell viability may contribute to cancer by facilitating the insurgence of mutations and by promoting resistance to therapy. Despite the identification of several new apoptosis inhibitors related to bcl-2 or to the baculovirus IAP gene, it is not clear whether apoptosis inhibition plays a general role in neoplasia. Here, we describe a new human gene encoding a structurally unique IAP apoptosis inhibitor, designated survivin. Survivin contains a single baculovirus IAP repeat and lacks a carboxyl-terminal RING finger. Present during fetal development, survivin is undetectable in terminally differentiated adult tissues. However, survivin becomes prominently expressed in transformed cell lines and in all the most common human cancers of lung, colon, pancreas, prostate and breast, in vivo. Survivin is also found in approximately 50% of high-grade non-Hodgkin's lymphomas (centroblastic, immunoblastic), but not in low-grade lymphomas (lymphocytic). Recombinant expression of survivin counteracts apoptosis of B lymphocyte precursors deprived of interleukin 3 (IL-3). These findings suggest that apoptosis inhibition may be a general feature of neoplasia and identify survivin as a potential new target for apoptosis-based therapy in cancer and lymphoma.

Effector cell protease receptor-1, a platelet activation-dependent membrane protein, regulates prothrombinase-catalyzed thrombin generation.

At sites of vascular injury thrombin is generated via prothrombinase, a stoichiometric (1:1), Ca2+-dependent, and membrane-bound complex consisting of the nonenzymatic cofactor factor Va and the serine protease factor Xa. While the importance of anionic platelet membrane phospholipids in regulating thrombin generation is well recognized, the identification of regulatory protein receptors has eluded investigators. This study reports the first description of a human platelet membrane protein that regulates prothrombinase complex assembly and function. Direct platelet-protein binding studies indicated that, although required, platelet-bound factor Va alone is insufficient to mediate factor Xa binding, and that factor Va and factor Xa bind to discrete sites on activated platelets for which expression is independently regulated as a function of the agonist concentration. When specific monoclonal antibodies against effector cell protease receptor-1 (EPR-1, a 65-kDa membrane receptor for factor Xa) were used in Western blotting, immunohistochemical staining, and/or flow cytometric analyses, activated platelets and their precursors, megakaryocytes, were shown to express EPR-1. These results were confirmed by reverse transcription-polymerase chain reaction of mRNA extracted from megakaryocyte-like cell lines. Additional flow cytometric studies demonstrated that a platelet-bound factor Va/factor Xa complex precluded binding of the anti-EPR-1 antibody, B6, to activated platelets by approximately 50%. Likewise, the anti-EPR-1 antibody was shown to inhibit prothrombinase-catalyzed thrombin generation on activated platelets in a dose- and platelet donor-dependent manner, indicating that platelet-expressed EPR-1 mediates factor Xa assembly into the prothrombinase complex. These collective data indicate that both EPR-1 and membrane-bound factor Va are required to mediate factor Xa binding to the activated platelet to form a functional prothrombinase complex.

Protease receptors in Hodgkin's disease: expression of the factor Xa receptor, effector cell protease receptor-1, in Reed-Sternberg cells.

The expression of a cellular receptor for the blood-clotting protease factor Xa, designated effector cell protease receptor-1 (EPR-1), was investigated in lymphoma. Immunohistochemical analysis demonstrated prominent reactivity of monoclonal antibodies to EPR-1 with Reed-Sternberg cells in 30 of 35 cases of nodular-sclerosis, lymphocyte-depletion, and mixed-cellularity Hodgkin's disease (HD). In contrast, several non-Hodgkin's lymphomas, or the nonneoplastic cellular components of HD, did not react with anti-EPR-1 monoclonal antibodies. A single molecular species of approximately 62 kD, consistent with the size and structural organization of EPR-1, was immunoblotted by an anti-EPR-1 monoclonal antibody from tissue samples of HD, but not from normal lymph nodes. Expression of EPR-1 transcripts in Reed-Sternberg cells was demonstrated by in situ hybridization with an antisense EPR-1 riboprobe, and by amplification of reverse-transcribed HD RNA with EPR-1-specific primers. These findings identify the factor Xa receptor, EPR-1, as a novel marker of Reed-Sternberg cells, and suggest its potential role in the histopathogenesis of HD.

Induction of MHC class II molecules HLA-DR, -DP and -DQ and ICAM 1 in human podocytes by gamma-interferon.

MHC class II-encoded molecules HLA-DR, -DP and -DQ play a pivotal role in the human immune response. Their constitutive expression is restricted to a number of immunocompetent cells referred to as antigen-presenting cells. However, gamma-interferon (gamma-IFN) has been shown to induce MHC class II molecule expression in several epithelia. Using flow cytometric analysis, we show here that normal and SV40-transformed human podocytes in culture constitutively expressed gamma-IFN receptors. We also show that MHC class I molecules are constitutively expressed in these cells and that HLA-DR, -DP and -DQ expression, which is not found in unstimulated cells, can be induced by gamma-IFN stimulation. This induction was a time-dependent event, a lag phase of 24-48 h being necessary for MHC class II molecules to become detectable at the cell surface by flow cytometric analysis. Induction of MHC class II molecules in human podocytes also showed a concentration dependence, a plateau being reached at a concentration of 500 IU of gamma-IFN/ml of culture medium. This effect was blunted by coincubation of the cells with an antihuman gamma-IFN receptor monoclonal antibody. HLA-DR expression was associated with specific mRNA accumulation, as detected by Northern blot analysis. By indirect immunofluorescence, the intercellular adhesion molecule 1 was also induced by gamma-IFN stimulation. Induction of DR, DP and DQ in human podocytes may be involved in the pathogenesis of immune glomerulonephritis in man.

Stromelysin 3 belongs to a subgroup of proteinases expressed in breast carcinoma fibroblastic cells and possibly implicated in tumor progression.

The expression of the stromelysin 3 (ST3) gene, which encodes a putative matrix metalloproteinase, was studied during breast cancer progression. The ST3 gene is expressed in all invasive breast carcinomas, in a number of their metastases, and in some in situ carcinomas where the probability of detecting ST3 transcripts correlates with the known risk of these carcinomas to become invasive. ST3 RNA and protein were specifically detected in fibroblastic cells immediately surrounding the neoplastic cells in both primary and metastatic tumors. This expression pattern distinguishes the ST3 gene from other matrix metalloproteinase genes, most notably from the 72-kDa type IV collagenase gene, which can be expressed in fibroblastic cells distributed throughout the stroma of primary breast carcinomas. Furthermore, high levels of 72-kDa type IV collagenase, but not of ST3 transcripts, are detected in benign breast fibroadenomas. Interestingly, the urokinase and ST3 genes exhibit very similar patterns of expression in breast carcinomas, which suggests that their products may cooperate during cancer progression.

Species-specific properties of the glomerular mesangium.

Mesangial cells play a central role in the physiology and pathophysiology of the glomerulus. To date, most of the in vitro studies have been performed in cultured rat mesangial cells, with only 10% of them performed in human mesangial cells. In this article, the major differences between results obtained with these two types of cultured cells will be reviewed. In rats and in humans, most of the mesangial cells appear to be of smooth muscle origin. In the rat, some of the cultured cells also express a phenotype suggesting a monocyte/macrophage origin. Phagocytosis and synthesis of cytokines or proinflammatory proteins that have been described in cultured rat cells seem mostly linked to this monocyte/macrophage subtype of resident mesangial cells. In humans, macrophages are only detected in pathologic conditions, suggesting that they are not resident but rather infiltrating cells. Mesangial receptors, most notably angiotensin II receptors, are clearly present on mesangial cell membranes and are linked to prostaglandin E2 synthesis and to cell contraction. In humans, spontaneous prostanoid synthesis is low and is increased by the induction of cyclooxygenase by sodium butyrate in the medium. Even so, the amount of prostaglandin E2 synthesized by human mesangial cells is quantitatively low comparatively with that in rats. In rats, accordingly, mesangial cells play a role in the regulation of single-nephron GFR. In humans, angiotensin II also exerts a control on GFR but it is more difficult to demonstrate its contractile effect on human than on rat mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor alpha increases antifibrinolytic activity of cultured human mesangial cells.

Tumor necrosis factor alpha (TNF alpha) is likely to exert a major influence in the pathogenesis of glomerulopathies. Besides its proinflammatory properties. TNF alpha interacts with cell growth and synthesis of components of the fibrinolytic system. In this study, we report the effects of recombinant human TNF alpha on the synthesis of tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) by human mesangial cells in culture. We first demonstrate that TNF alpha binds specifically to a single class of high affinity receptors (Kd 5.10(-11) M; 1500 receptors/cell). TNF alpha has an antimitogenic effect on human mesangial cells since it decreased DNA synthesis, measured by 3H-thymidine incorporation, in a dose-dependent manner. Release of cytosolic LDH and incorporated 51Cr was not increased by 100 ng/ml TNF alpha as compared with control, indicating that this monokine is not cytotoxic for cultured human mesangial cells. Zymographic analysis and reverse fibrin autography disclosed a 120 kD t-PA-PAI-1 complex and a 50 kD free form of PAI-1 in the supernatants of both unstimulated and TNF-stimulated cells; PAI-1 was released in excess and free t-PA was not observed. TNF alpha (0 to 100 ng/ml) had no effect on t-PA synthesis, but enhanced PAI-1 release in a time- and dose-dependent manner (97% increase of PAI-1 synthesis after a 24 hour incubation). This effect was abolished by cycloheximide, suggesting that protein synthesis was required. Northern blot analysis showed that TNF alpha increased the steady-state PAI-1 mRNA levels in a time-dependent manner, with a maximal effect at two hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional activation of the urokinase receptor gene by endothelin-1.

Using an immortalized human glomerular epithelial cell line (E71 A1), we studied the effect of endothelin-1 (ET-1), a potent vasoconstrictor peptide, on the synthesis of urokinase type plasminogen activator (u-PA) and its receptor (u-PAR). The results show that ET-1 had no effect on u-PA synthesis but induced an increase in u-PAR number (2.8 +/- 0.6 x 10(4) vs 1.2 +/- 0.5 x 10(4) sites per cell, p less than 0.001) without change in receptor affinity (280 +/- 80 pM vs 250 +/- 50 pM, NS), maximal effect being observed at 10(-7) M. Time course shows that a plateau was reached after a 24 hour incubation. ET-1 induced-increase in binding capacity was abolished by cycloheximide. ET-1 also induced an increase in u-PAR mRNA level, which was completely blocked by alpha-amanitin (5 micrograms/ml). Cycloheximide (1 microgram/ml) alone induced an increase in u-PAR mRNA level and this effect was enhanced when cycloheximide was combined with ET-1. Our data show that ET-1 can induce an increase in membrane expression of u-PAR through activation of the transcription of the u-PAR gene and de novo protein synthesis.

Plasminogen activator inhibitor-1 deposition in the extracellular matrix of cultured human mesangial cells.

Human mesangial cells secrete tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), the latter being secreted in large excess in vitro. We demonstrate that PAI-1 is a major component of the extracellular matrix of cultured human mesangial cells, where its deposition is dependent on cell density. By immunogold silver staining, epipolarization microscopy and dispersive X-ray spectrometry, we have shown that matrix-associated PAI-1 is synthesized by spreading human mesangial cells, as indicated by the time-dependent accumulation of PAI-1 and the inhibitory effect of cycloheximide. Furthermore, by in situ hybridization, PAI-1 mRNA was detected in cultured mesangial cells. t-PA is present inside the cells, or at the cell surface, but is never associated with the extracellular matrix. Exogenous t-PA can remove matrix-associated PAI-1 without affecting cell adhesion. A similar effect was obtained by addition of urokinase-type plasminogen activator (u-PA) but not with fibrinolysis unrelated enzymes. In conclusion, PAI-1 is synthesized by human cultured mesangial cells and is deposited in the extracellular matrix by nonconfluent cells, whereas less PAI-1 is seen between confluent cells. This can explain the absence of detectable PAI-1 in normal human kidney biopsies. t-PA released by mesangial cells can bind and detach matrix PAI-1.

Thrombin signal transduction mechanisms in human glomerular epithelial cells.

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.

[Role of hemostasis in the formation of crescents in extracapillary glomerulonephritis]. / Rôle de l'hémostase dans la formation de croissants au cours des glomérulonéphrites extracapillaires.

Extracapillary glomerulonephritis are associated with fibrin deposition in the urinary space of the glomerulus. Such deposits were correlated with the severity of the disease and with a poor renal outcome. Fibrin formation involves an activation of the coagulation cascade either through the intrinsic pathway, Hageman factor being activated by the altered glomerular basement membrane, either by the extrinsic pathway, infiltrating monocytes and glomerular cells exhibiting a procoagulant activity i.e. thromboplastin or tissue factor. Treatments with heparin or warfarin were shown to decrease the severity of experimental glomerular diseases. A similar beneficial effect was obtained with a monocyte-depleting serum and more recently with a treatment by a tissue type plasminogen activator. Glomerular cells also produce a fibrinolytic activity which could be too low or uneffective on extracapillary fibrin deposits if they contain high amounts of plasminogen activator inhibitors. Thrombin has procoagulant activity, antifibrinolytic activity and has cellular chemotactic and proliferative effects. It could play a major role in the pathogenesis of crescent formation.