Claire Barro

Human B lymphocytes synthesize the 92-kDa gelatinase, matrix metalloproteinase-9

Matrix metalloproteinases (MMPs) are involved in the remodeling of connective tissue as well as in disease states associated with acute and chronic inflammation or tumoral metastatic processes. Despite detailed and extensive studies of the mechanisms of lymphocyte extravasation, remarkably little is known about the expression and regulation of metalloproteinases involved in the migratory process. By using zymography and reverse transcription-polymerase chain reaction experiments, we have demonstrated that Epstein-Barr virus-immortalized B lymphocytes are able to secrete a 92-kDa metalloproteinase with gelatinolytic activity which has been purified and identified as being MMP-9. Moreover, the tissue inhibitor of metalloproteinase was shown to be constitutively expressed by the B cells. The expression of 92-kDa gelatinase is mediated by cytokines, growth factors, lipopolysaccharide, concanavalin A, and the tumor promotor phorbol 12-myristate 13-acetate. Time dependence activity increased rapidly up to 24 h of incubation with lipopolysaccharide or concanavalin A stimulation while it requires a delay and more time to have an optimum effect when cytokines were the stimulating agents; transforming growth factor-β abolished 92-kDa gelatinase production. Both staurosporine and wortmannin are inductive stimuli, and the level of MMP-9 secreted into the media is greater than that observed with other agents except concanavalin A. Elicitation of the chemotactic migration of B cells through a model basement membrane by lipopolysaccharide was shown to be correlated with gelatinase expression and inhibited by 7 mm captopril. Our study indicates that Epstein-Barr virus-B lymphocytes express 92-kDa gelatinase, the production of which can be modified by a variety of physiological and pharmacological signals which have been shown to differ according to the cell type.

Quantitative high D-dimer value is predictive of pulmonary embolism occurrence independently of clinical score in a well-defined low risk factor population

Summary.  We performed a prospective study to assess whether positive quantitative D‐dimer (DD) levels could be integrated for a selected population in a defined strategy to accurately diagnose pulmonary embolism (PE). For this purpose, 1528 in‐ or outpatients with clinically suspected PE were investigated according to our prescription rules. Clinical probability was defined as low, intermediate or high. Patients in whom DD levels were measured met criteria defined by our previously described decision‐making algorithm: in‐ and outpatients, < 80 years, without surgery in the previous 30 days or active cancer. Nine hundred and twenty‐three patients (60.4%) had quantitative DD measurement using automated latex DD assay (STA‐Liatest D‐Di®). According to our decision‐making algorithm, DD measurement was applied to 70.5% of out‐, and 55.7% of inpatients, and PE diagnosis was ruled out in 49.5% of the 923 patients. This allowed us to confirm prospectively that our specific rules greatly improve the DD testing efficiency. PE was diagnosed in 115 (12.5%) patients. For a 0.5 mg L−1 cut‐off, the test sensitivity was 97.4%, but its specificity was only 56.7%. However, PE prevalence increased gradually with DD levels. The true observed PE prevalence, according to the quantitative assessment of DD levels, differed from that predicted with pretest clinical probability only. Moreover, in this well‐defined patient group, a quantitative DD level > 2 mg L−1 was predictive of PE occurrence independently of the clinical score (odds ratio 6.9, 95% confidence interval 3.7, 12.8). As part of a defined strategy, knowledge of positive DD quantitative value, together with the clinical probability score, improves the PE predictive model. A clinical validation of these results in a follow‐up study would now be necessary before considering the implementation of this strategy into clinical practice.

Plasma d-Dimer Testing Improves the Management of Thromboembolic Disease in Hospitalized Patients

in charge of thromboembolic disease (DVTE) diagnosis in hospitalized patients. Therefore we de

Differential expression and secretion of gelatinases and tissue inhibitor of metalloproteinase-1 during neutrophil adhesion

Transmigrating neutrophils secrete a 92 kDa gelatinase (MMP-9) in order to degrade type IV endothelial basement membrane collagen. A model system for neutrophil adhesion combining a short pre-adhesion time (30 min) in plastic or endothelium-coated wells, medium removal and addition of soluble stimuli (fMLP, TNF alpha), enabled us to induce the release of a basal level of gelatinase activity (> 12% total cell content) from tertiary granules, while the release of vitamin B12 binding protein from specific granules was limited to 4% total cell content. Neutrophil gelatinase activity in unfractionated supernatants from endothelium-coated wells was significantly reduced (P < 0.01) compared to levels obtained on plastic supports, even after TNF alpha treatment or when cell populations were physically separated by trans-well inserts. In contrast, gelatin zymograms of supernatants from plastic and endothelium-coated wells remained similar. These findings suggest that MMP-9 is equally secreted but differentially inhibited by the tissue inhibitor of metalloproteinase-1 originating from the neutrophils. MMP-9 RT-PCR from neutrophils, assessed after up to one hour adhesion on plastic, yielded a single 270 bp fragment which was almost undetectable in the endothelial RT-PCR counterpart, whereas the TIMP-1 PCR product was apparent in both cell types. Furthermore, neutrophil adhesion on endothelial cells and TNF alpha activation for one hour induced the disappearance of MMP-9 cDNA without changes in TIMP-1 and beta-actin PCR products. These results suggest the existence of a dual down-regulation during neutrophil-endothelial interaction, both at the level of secreted MMP-9 activity and of MMP-9 gene transcription.

Matrix Metalloproteinase Expression in Rat Pancreatic Islets

Matrix metalloproteinases (MMPs) are involved in the regulation of extracellular matrix turnover and tissue remodeling, through which they can influence the infiltration of a graft by immune-competent cells. Little is known about their role in islet allograft rejection. Therefore we investigated the expression of several MMPs and of two of their tissue inhibitors (TIMPs) in rat pancreatic islets. MMP and TIMP expression in isolated rat pancreatic islets was assessed by reverse transcriptase - polymerase chain reaction (RT-PCR) from total RNA. Several MMPs of different substrate specificities were found to be expressed in rat pancreatic islets, either shortly after islet isolation and in all conditions tested (MMP-9, TIMP-1) or after a lag time (MMP-2, MMP-3, MMP-14, TIMP-2). Fetal calf serum induced MMP-7 expression. The inflammatory cytokine interleukin-1P (IL-1p) did not induce MMP or TIMP expression. We showed that rat pancreatic islets are well equipped with MMPs and TIMPs, but the functional meaning of this expression remains to be elucidated. On the basis of the known effects on tissue remodeling and cytokine processing, we anticipate that they can influence islet engraftment and viability and participate to islet graft rejection.

INTER-REGULATED BALANCE BETWEEN GELATINASES AND TISSUE INHIBITOR (TIMP-1) IN ISOLATED HUMAN GLOMERULI

Leukocyte infiltration inside glomeruli necessitates basement membrane collagen i.v. breakdown and leads to mesangiolysis, cell proliferation and extracellular matrix synthesis during the repair process as observed in the course of acute glomerulonephritis, vasculitis and acute graft rejection. Two matrix metalloproteinases, MMP-2 and MMP-9 gelatinases, are expressed and co-secreted in balance with the tissue inhibitor of metalloproteinases-1 (TIMP-1) by activated neutrophils as well as by glomerular cells and are aimed to control basement membrane collage i.v. deposition. Using a conventional double mesh sieving method, pure populations of glomeruli were isolated from fresh human cortex specimen and maintained in short-term cultures. ELISA, zymography and immunoblotting of conditioned serum-free media revealed glomerular MMP-2, MMP-9 and TIMP-1 secretion and activity while reverse transcription-polymerase chain reaction amplification of cellular RNA demonstrated glomerular transcripts coding for these enzymes and their inhibitor. When purified neutrophils were allowed to adhere onto Transwell apparatus in contact with glomerular suspensions, neutrophil 92 kDa gelatinase seemed apparently inhibited mainly because the production of TIMP-1 was enhanced on both sides of the insert. Glomerular 72 kDa and 92 kDa gelatinases were activated shortly (1 to 6 h) after neutrophils had interacted with glomeruli and furthermore upon activation by inflammatory or vasoactive mediators such as phorbol. Decreased neutrophil MMP-9 activity together with reduced MMP-9 mRNA levels and protracted TIMP-1 transcription and secretion during cell-cell interaction could participate to cell detachment from degraded basement membranes and to increased collagen i.v. deposition leading to glomerulosclerosis after initial glomerular injury by inflammatory cells.

Discordant D‐dimer results of two rapid quantitative automated assays are related to age

We read with interest the recently published paper by Carrier et al. [1] on the use of D-dimer (DD) in the elderly. This study demonstrates that the combination of a low Wells pre-test probability with a negative DD value safely excludes deep vein thrombosis (DVT) in elderly patients (aged over 60 years). However, the results of this study, based on pooled results of three technically different DD assays, are not totally in agreement with three other studies [2–4], and the authors suggest that this discrepancy might be explained by differences in the specificity of the DD assays. DD assays have been extensively evaluated, and meta-analyses were recently performed to determine the best tests [5,6]. One limitation of these approaches is that the findings are mainly based on an indirect comparison of the test performance from various studies and hence, mostly from heterogeneous patient populations. In the study, we compared performances in two rapid quantitative automatedDDassays on the same population and demonstrated that age was the main clinical characteristic related to discordant results between the two tests. We previously published a management study of 1134 consecutive non-selected patients clinically suspected of pulmonary embolism (PE) [7]. In this study, DD (cut-off 0.5 mg L, quantitative ELISA test, Vidas D-Dimer; BioMerieux, Lyon, France): (V) was determined straight away for 656 patients who fulfilled the criteria of our decision-making. For 608 of these patients, citrated plasma was immediately stored at )80 C, and a second DD measurement performed retrospectively using a rapid immunoturbidimetric assay (cutoff 0.5 mg L, STA-Liatest D-DI; Stago, Paris, France): (L). Data were complete for 603 patients (five patients were lost at follow-up) who achieved a 3-month follow-up. DDwere found negative (<0.5 mg L) by both methods (L)/V)) in 259 patients (43%; mean age 50.4 years, SD 16.6). The mean DD values for the two tests were not statistically different. False negatives accounted for 1.92% (CI 95% 0.26–3.58%) and this was identical for the two methods. DD were found to be positive (‡0.5 mg L) by both methods (L+/V+) in 277 patients (mean age 64.2 years, SD 14). Results were not concordant for 67 (11%) patients, of these, 63 (94%) patients neither had PE nor develop PE during the 3-month follow-up. Among these 67 discordant results, 54 were L)/V+ and 13 L+/V). Three patients (4.5%, 95% CI 0–9%) had PE in the L)/V+ group, comparable with one (1.5%, 95% CI 0– 4.3%) (P = 0.6) in the L+/V) group. In fact, all these patients were at high clinical probability. Conversely, 51 of the 67 with discordant results (76.1%, 95%CI 65.9–86.3) were L)/ V+ and did not have any thromboembolic events, compared with 12 (17.9%, 95% CI 8–27.1%) in the L+/V) group (P < 0.0001). To explain this discrepancy in the positive rate between the two tests, we analysed the performances in each test related to the patients clinical characteristics. Both gender and ward origin of the 67 patients with discrepant results did not differ significantly from those of the 536 patients with concordant results. Interestingly, when V DD values only were positive, the patients mean age was 64.2 years (SD 14) but significantly lower, 54.3 years (SD 18), when LDD values were only positive (P = 0.03). Therefore, we tested the difference between DD using both methods according to age. For this purpose, DD obtained using the two tests were recorded for each patient, and DDD (i.e. DD Vidas minus DD Liatest) calculated and expressed for the three age groups. This analysis was performed for 420 patients out of 603, as values above 1 mg L (not given quantitatively by the ELISA method at the time of the study) were excluded. As shown in Fig. 1, there was a significant increase in the difference between the Vidas Correspondence: Gilles Pernod, Vascular Medicine Unit, PPM, CHU Grenoble, BP 217X, 38043 Grenoble cedex 09, France. Tel.: 33 476765717; fax: 33 476765524. E-mail: GPernod@chu-grenoble.fr