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Biomedical subjects

B Grouix

Publications and source records attributed to B Grouix.

12 recordsLinked to original sources
PubMed2000

In vitro and in vivo properties of bicyclic lactam inhibitors: a novel class of low molecular weight peptidomimetic thrombin inhibitors.

We have developed potent and selective thrombin inhibitors with a novel non-peptidic structure. A bicyclic lactam was used as the scaffold on which various P1 and P3 motifs were substituted. Herein, we report the in vitro and in vivo properties of four representatives of this novel class of inhibitors. Their Ki values were less than 10 nM, they inhibited equally both free and clot-bound thrombin, and they displayed high level of specificity for thrombin over other serine proteases (trypsin, factor Xa, activated Protein C, and plasmin). They prolonged the clotting time of human plasma to twice the control value in coagulation assays (TT, APTT, and PT) at a concentration below 3 microM. Their anticoagulant activities using rat plasma were similar to, although slightly weaker, than with human plasma. Furthermore, they inhibited thrombin-induced platelet aggregation (human and rat) at concentrations close to their Ki values for thrombin. These molecules demonstrated similar dose response antithrombotic efficacy in rat arterial and venous thrombosis models when given as i.v. bolus followed by infusion. Antithrombotic efficacy of 85% and greater was observed at a dose of 5-7 microM/kg/hour in each model. Bicyclic lactam inhibitor 3, at a dose which caused a complete inhibition of visible thrombus formation in the venous and arterial models of thrombosis, showed a 1.9-2.1 and a 4.0-4.8-fold shift in APTT and TT, respectively. Unfortunately, the bicyclic lactam inhibitors exhibited low oral bioavailability in rats. Therefore, this novel class of bicyclic lactam thrombin inhibitor has the potential to be promising intravenous antithrombotic agents for the treatment of arterial as well as venous thrombosis and warrants further investigation.

Animals↗
PubMed1997

Coordinate expression of group II phospholipase A2 and the acute-phase proteins haptoglobin (HP) and alpha1-anti-chymotrypsin (ACH) by HepG2 cells.

The early response to inflammation is characterized by the synthesis of a variety of proteins under cytokine and glucocorticoid control. During episodes of infection or inflammation, a secretory phospholipase A2 (sPLA2) appears in the circulation along with a variety of acute-phase proteins (APP), suggesting possible common regulatory elements amongst sPLA2 and APP. Using the human hepatoma line, HepG2, regulation of sPLA2 expression was examined in relation to synthesis of HP and ACH. The patterns of induction of sPLA2, HP and ACH were distinct for each of IL- 1, tumour necrosis factor (TNF) and IL-6, oncostatin M, IL-11 and leukaemia inhibitory factor. Dexamethasone had an enhancing effect on IL-6-induced expression of HP and ACH, but inhibited sPLA2 expression by 50%. Both 8-bromo-cAMP and dibutyryl cAMP increased sPLA2 expression (48.8-fold and 64.2-fold, respectively), whereas KT5720, an inhibitor of protein kinase A, down-regulated cytokine-induced sPLA2 synthesis by 51%. These data show that a panel of cytokines induced varying patterns of up-regulation of sPLA2, ACH and HP. Although dexamethasone potentiated IL-6-induced APP expression in HepG2 cells, it suppressed sPLA2 expression in a dose-dependent manner. In several respects, sPLA2 regulation is similar to that of HP and ACH, but a notable difference is the reciprocal effect of glucocorticoids on sPLA2 expression compared with that of ACH and HP.

Acute-Phase Proteins↗
PubMed1996

Secretory non-pancreatic phospholipase A2 and cyclooxygenase-2 expression by tracheobronchial smooth muscle cells.

Lipid mediators of inflammation, contribute to airway hyper-reactivity in asthma. Since production of lipid mediators is largely regulated by phospholipase A2 (PLA2), and since PLA2 expression in mesenchymal cells is induced by cytokines and other signals, we examined PLA2 expression by rat tracheobronchial smooth muscle cells (TBSMC). PLA2 expression in TBSMC cultures was markedly increased by tumour-necrosis factor (TNF) alpha (130-fold) and interleukin-1beta (IL-1beta) (7.4-fold). Lipopolysaccharide (LPS;100 ng/ml) resulted in a 51-fold increase in extracellular PLA2 activity. PLA2 expression by LPS-stimulated or cytokine-stimulated cells was downregulated by dexamethasone. Whereas forskolin or dibutyrl cAMP increased PLA2 activity, inhibition of protein kinase A but not tyrosine kinase reduced PLA2 expression. Northern blot analysis showed that TNF alpha and IL-1beta increased both PLA2 and inducible cyclooxygenase (Cox-2) mRNA transcription. Addition of dexamethasone substantially blunted the increase in PLA2 and Cox-2 mRNA. In contrast, the level of Cox-1 mRNA was very low and did not change with the various treatments. Since proinflammatory lipid mediators have been implicated in the pathogenesis of asthma and PLA2 activity regulates generation of these lipid mediators, cytokine-stimulated synthesis and release of PLA2 by airway smooth cells may contribute to the potentiation of airway inflammation in asthma.

Animals↗
PubMed1995

Inhibition of human group II phospholipase A2 by C-reactive protein in vitro.

Endotoxin or cytokine-induced expression of a secretory non-pancreatic phospholipase A2 (sPLA2) has been implicated in the pathogenesis of multisystem organ dysfunction or failure in patients with septic shock. Circulating sPLA2 levels increase as much as 1000-fold during the course of septic shock. However, the mode of regulation of the activity of this lipolytic enzyme is unknown, since circulating inhibitors have not been identified. We investigated the potential inhibitory activity of the acute phase reactant, C-reactive protein (CRP), a phospholipid-binding protein whose expression increases as much as 1000-fold during severe infections. Serum CRP and sPLA2 profiles were highly concordant in patients with septic shock. In studies in vitro, human CRP inhibited hydrolysis of PC-lyso-PC (2:1) multilamellar liposomes by human recombinant sPLA2 in a dose-dependent manner, with an apparent IC50 of 50 micrograms/ml CRP and maximal inhibition at 100 micrograms/ml. Inhibition of sPLA2 activity by CRP was substrate concentration-dependent, and increasing substrate concentrations reversed the inhibitory effect of CRP using the PC-lyso-PC system. Preincubation of CRP with phosphorylcholine led to a concentration-dependent loss of CRP-induced inhibition of substrate hydrolysis. These observations are consistent with a substrate-depletion model of inhibition of sPLA2 activity by CRP.

C-Reactive Protein↗
PubMed1995

Induction of release of secretory nonpancreatic phospholipase A2 from human articular chondrocytes.

OBJECTIVE: Secretory nonpancreatic phospholipase A2 (sPLA2) is a known inducer/promoter of the inflammatory process in the joints. It correlates with disease activity in adult and juvenile rheumatoid arthritis. Synovial fluids contain high concentrations of sPLA2. We discovered that human articular cartilage contains large quantities of sPLA2 and that culture chondrocytes constitutively synthesize and release sPLA2. To test the mechanism controlling the release of sPLA2, we exposed cultured human articular chondrocytes to cytokines and other agents, known to induce sPLA2 in other cells. METHODS: Chondrocytes obtained from cartilage of normal appearance from rheumatoid and osteoarthritic joints, and from normal, neonatal joints were compared to rabbit articular chondrocytes. Radiolabeled Escherichia coli derived phospholipid assay and ELISA technique using monoclonal antibodies against recombinant human synovial type sPLA2 were employed. The cells were grown as monolayers as well as in alginate beads. RESULTS: Human articular chondrocytes from both arthritic and neonatal joints released sPLA2 constitutively but could not be further stimulated with interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), IL-6, oncostatin M, lipopolysaccharide (LPS), or forskolin. Marked stimulation was observed when the cells were exposed to 8-bromo cyclic adenosine monophosphate (cAMP). Growing the cells as monolayers or in alginate beads did not change the results. In contrast to human cells, rabbit chondrocytes responded to IL-1 beta and IL-1/TNF, but not to TNF-alpha alone, with a very marked increase in extracellular sPLA2 activity. CONCLUSION: Human articular chondrocytes synthesize and constitutively release sPLA2. Such continuous release is most probably responsible for the high concentration of sPLA2 in articular cartilage and may be the source of synovial fluid sPLA2. To our knowledge, human articular chondrocytes are the only sPLA2 producing cells tested to date that do not respond to cytokine stimulation with increased sPLA2 activity; yet enhancement was seen with 8-bromo cAMP. It seems therefore that, human articular chondrocytes possess signalling mechanisms for the release of sPLA2 unlike those from other mammalian cells. The significance of this observation remains to be elucidated.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Induction of TNF-alpha and proinflammatory secretory phospholipase A2 by intravenous administration of CAMPATH-1H in patients with rheumatoid arthritis.

OBJECTIVE: To test the effect of infusions of CAMPATH-1H on levels of proinflammatory secretory phospholipase A2 (sPLA2) and tumor necrosis factor alpha (TNF-alpha) in patients with rheumatoid arthritis (RA). METHODS: Two patients with RA were infused with CAMPATH-1H; extracellular nonpancreatic sPLA2 activity was tested using radiolabelled E. coli membrane phospholipid, and circulating TNF-alpha levels were tested by ultrasensitive immunoassay. RESULTS: Circulating TNF-alpha began to rise within the first 2 h after infusion, reaching > 1000-fold values compared to preinfusion levels. Circulating sPLA2 activity began to rise a few hours after the start of infusion and reached extremely high values in 12 h, concomitant with fever and hypotension. The activity of sPLA2 decreased to pretreatment values in 3-18 days after infusion. CONCLUSION: The mechanism leading to the increase of TNF-alpha and hyperphospholipasemia A2 has not been elucidated. It is possible that CAMPATH-1H activates cells that synthesize and release TNF-alpha and sPLA2, and/or that it induces interleukin 2 release, which in turn activates TNF-alpha, with subsequent release of sPLA2.

Adult↗
PubMed1987

Affinity purification of a high molecular weight human breast cancer-associated antigen identified by the BCD-B4 monoclonal antibody.

This study reports the purification and characterization of a high molecular weight human breast cancer-associated antigen identified by a previously described (1,2) murine monoclonal antibody, BCD-B4. Immunohistochemical analysis indicated that BCD-B4 recognizes an antigen expressed in an altered form on the human breast carcinoma cell line, BT-20, compared to the non-malignant human mammary epithelial cell line, HBL-100. Chemical treatments and enzymatic digestions suggested that the recognized moiety was a protein. The antigenic determinant was resistant to neuraminidase and periodate treatments but was sensitive to trypsin and proteinase K. The antigen was purified by affinity chromatography and its molecular weight, determined by SDS-PAGE analysis under non-reducing conditions, was proven to be 250 Kd. Under reducing conditions, the molecule dissociated into two polypeptides of 125 and 45 Kd, respectively. Both subunits could be isolated from normal HBL-100 and neoplastic BT-20 cellular protein extracts by affinity chromatography. The higher molecular weight subunit showed; however, qualitative and quantitative differences between the two cell lines: it was expressed in greater quantity on BT-20 cells and its molecular weight was 15 Kd higher. Both subunits could also be identified by immunoblots of BT-20 cells.

Antibodies, Monoclonal↗
PubMed1983

Role of self carriers in the immune response and tolerance. VIII. Suppression of the MOPC-104E idiotypic response by idiotype-modified self, but not by dextran-modified self.

We have investigated the suppression of the anti-dextran B1355S immune response using our model of modified self. The anti-dextran response is idiotypically well defined in BALB/c mice. This system enables us to examine the contribution of various predominant idiotypes to the antibody response under conditions of suppression by antigen or by idiotype-specific suppressor cells. Our results demonstrate that the total anti-dextran response can be inhibited by pretreatment of animals with dextran-coupled syngeneic spleen cells; however, the representation of major idiotypes constituting this response are not reduced in percentage. In contrast, pretreatment of mice with MOPC-104E-coupled spleen cells leads to a specific suppression of the private IdI-104E idiotype. The total anti-dextran response remains unchanged, as well as proportions of other major idiotypes known (IdI-588 and IdX). This suppression is mediated by Thy-1.2+, Lyt-2.2+ T cells, as demonstrated by adoptive transfer assays. This system will allow the molecular dissection of the regulation of an idiotypically well-defined system for the suppression by either antigen- or idiotype-specific suppressor T cells.

Animals↗
PubMedDate not supplied

Clinical value of immunolymphscintigraphy in patients with breast cancer.

Thirty four patients suspected of suffering from breast cancer were investigated by immunolymphscintigraphy (ILS) a few days before a planned operation. Patients received 500 micrograms of the BCD-F9 monoclonal antibody or its F(ab')2 fragments containing 1 mCi of Iodin-123. Each preparation was given by a subcutaneous injection into the fingerwebs between the 2nd and 3rd fingers of both hands. Three static scintigrams were taken 4, 8 and 24 hours following injection of the radiolabelled antibodies. ILS results were always compared to histopathological findings and gave for the intact antibody a sensitivity of 83%, a specificity of 93% and an accuracy of 89%, the positive predictive value was 91% and the negative predictive value was 80%. On the other hand, when the F(ab')2 fragments were used the sensitivity of the ILS technique was 75%, the specificity was 100% while the accuracy was 86%, the positive predictive value was 100% and the negative predictive value was 75%.

Antibodies, Monoclonal↗
PubMedDate not supplied

Immunolymphscintigraphy of axillary lymph node metastases in breast cancer patients using monoclonal antibodies: first clinical findings.

The results of preliminary investigations into the immunolymphscintigraphic (ILS) detection of axillary lymph node metastases in nine breast cancer patients by means of the 3C6F9 monoclonal antibody (MAs) are presented. The IgG2a monoclonal antibody detects a 37 KD antigen, consistently found on the surface of primary and metastatic breast tumors. Each patient received 1 mCi of I-123 (specific activity, 2 mCi per mg of antibody) as a subcutaneous injection between the 2nd and the 3rd finger of both hands, i.e., the healthy side serving as a control for the affected side. Clear images of lymph node metastases were visible 4 to 8 hours after injection of the antibody. Seven of the nine patients studied were positive by scanning and six showed positive lymph node involvement by histopathology (6/7; true positive = 86%). Two patients did not show any iodine uptake in the axilla and were subsequently found to be free of metastases (2/2; true negative = 100%). These data give an overall accuracy of ILS of 89% and demonstrate that 3C6F9 localizes preferentially in affected axillary lymph nodes compared to normal lymph nodes.

Antibodies, Monoclonal↗