Biomedical subjects
P Prigent
Publications and source records attributed to P Prigent.
Preclinical profile of the monodisperse iodinated macromolecular blood pool agent P743.
RATIONALE AND OBJECTIVES: To summarize the chemical synthesis, physicochemical characterization, pharmacokinetic behavior, and biological evaluation of P743, a new macromolecular iodinated contrast medium. METHODS: The synthesis and molecular modeling of the iodinated macromolecule P743 are described. The pharmacokinetic profile was established in rabbits and rats. Acute toxicity in mice, renal tolerance in normal rabbits, and renal tolerance in uninephrectomized, dehydrated rats undergoing selective intrarenal injection was evaluated. In vitro permeability effects on isolated mastocytes and on the coagulation pathways were carried out. Computed tomography vascular imaging was performed after intravenous injection of P743 (300 mg I/kg) in rabbits and compared with the nonspecific nonionic agent iobitridol. RESULTS: P743 is a monodisperse, macromolecular iodinated contrast medium. In both rabbits and rats, P743 showed a pharmacokinetic profile consistent with that of a rapid-clearance blood-pool agent. Its diffusion through the endothelium was found to be low in vitro, thus confirming early confinement of this macromolecule, unlike nonspecific contrast media. In both species, P743 was excreted by glomerular filtration. Acute toxicity disclosed no mortality at the highest volume that could be injected into mice, leading to a median lethal dose greater than 8.9 g I/kg. Renal tolerance was found to be good in both euvolemic rabbits and uninephrectomized, dehydrated rats. No histamine or leukotriene B4 release was found on RBL-2H3 isolated mastocytes. P743 did not interfere with the coagulation pathways. Imaging experiments confirmed that P743 remains in the vascular compartment for a longer time than does iobitridol, thus allowing vascular enhancement that is twice as high as that of iobitridol in the recirculation phase. CONCLUSIONS: The pharmacokinetic and imaging profiles of P743, a new, monodisperse, macromolecular blood-pool iodinated contrast medium, were consistent with those of a rapid-clearance blood-pool agent. Its initial safety profile is satisfactory. Further experimental imaging studies are required to define the clinical interest in such molecules.
Lymphocyte activation gene-3 induces tumor regression and antitumor immune responses.
The lymphocyte activation gene-3 (LAG-3) product is an MHC class II ligand related to CD4. We investigated whether LAG-3 could be used in vivo to stimulate MHC class II(+) antigen-presenting cells (APC), such as resident macrophages or dendritic cells known to play a crucial role in processing and presenting of antigens to the immune system. We first introduced human (h) LAG-3 or mouse LAG-3 into three types of tumor cells (MCA 205, TS / A and RENCA) to evaluate its capacity to stimulate a tumor-specific immune response in vivo. In contrast to the progressive growth of wild-type cells in syngeneic mice, LAG-3-transfected tumors completely regressed or their growth was markedly reduced. Mice were significantly to completely protected against a rechallenge with parental tumor cells. Protection induced by hLAG-3(+) tumor cells involved recruitment of a CD8(+) T cell response since nu / nu mice and CD8-depleted mice did not reject tumors, and a systemic tumor-specific CTL activity was induced. Co-administration of soluble LAG-3 with wild-type tumor cells also markedly reduced primary tumor growth. Interestingly, immunization with LAG-3(+) tumor cells or co-administration of soluble LAG-3 with irradiated wild-type tumor cells reduced the growth of pre-established tumors. We therefore suggest that LAG-3 could be used as a vaccine adjuvant for its ability to trigger APC via MHC class II molecules.
Characterization of the major histocompatibility complex class II binding site on LAG-3 protein.
The lymphocyte activation gene-3 (LAG-3), selectively transcribed in human activated T and NK cells, encodes a ligand for major histocompatibility complex (MHC) class II molecules. Like CD4, LAG-3 ectodomain is composed of four Ig-like domains (D1-D4). Nothing is known about the LAG-3 regions or residues required to form a stable MHC class II binding site. In contrast to CD4, soluble LAG-3 molecules stably interact with MHC class II molecules expressed on the cell surface. In addition, the first two N-terminal domains of soluble LAG-3 (D1 and D2) molecules, alone, are capable of binding MHC class II. From a LAG-3 model structure, we designed mutants and tested their ability to bind MHC class II molecules in an intercellular adhesion assay. We found residues on the membrane-distal, CDR1-2-containing top face of D1 that are essential for either binding or repulsing MHC class II proteins. Most of these residues are clustered at the base of a large extra-loop structure that is a hallmark of the LAG-3 D1 Ig-like domain. In addition, as for CD4, oligomerization of LAG-3 on the cell surface may be required to form a stable MHC binding site because mutation of three residues in the ABED beta-strands containing side of D1 results in a dominant negative effect (i.e., binding inhibition of coexpressed wild-type LAG-3).
T cell major histocompatibility complex class II molecules down-regulate CD4+ T cell clone responses following LAG-3 binding.
T cell response to its antigen requires recognition by the T cell receptor together with a co-receptor molecule, either CD4 or CD8. Additional molecules have been identified that are capable of delivering the co-stimulatory signals provided by APC. Following T cell priming, a number of T cell activation antigens are expressed that may play a role in the inactivation phase of the T cell response. The lymphocyte activation gene (LAG)-3 protein and its counter-receptors, the major histocompatibility complex (MHC) class II molecules, are such activation antigens whose interaction may result in the down-regulation of the ongoing immune response. To investigate the role of LAG-3/class II molecule interaction, we produced a soluble form of LAG-3 by fusing the extracellular Ig domains of this membrane protein to the constant region of human IgG1 (LAG-3Ig). Here, we show a direct and specific binding of LAG-3Ig to class II molecules on the cell surface. In addition, we show that LAG-3/class II molecule interaction leads to the down-regulation of CD4+ Ag-specific T cell clone proliferation and cytokine secretion. This inhibitory effect is observed at the level of the effector cells and not the APC and is also found with anti-CD3 mAb, PHA + PMA or low-dose IL-2 driven stimulation in the absence of APC. These functional studies indicate that T cell MHC class II molecules down-regulate T cell proliferation following LAG-3 binding and suggest a role for LAG-3 in the control of the CD4+ T cell response.
[Dural fistulae of the cavernous sinus and interventional neuroradiology. Apropos of 4 cases].
PURPOSE: Our purpose was to show how difficult it is to diagnose a dural fistula of the cavernous sinus, which is an anomalous arteriovenous shunt within the dura mater extending from meningeal arteries to the cavernous sinus. CASE REPORT: A dural fistula was suspected in four female patients aged between 61 and 80, presenting with a red eye, dilated episcleral veins, exophthalmos and elevated intraocular pressure. A cerebral hyperselective angiography was performed in all cases. RESULTS: The cerebral angiography confirmed the diagnosis of a dural fistula in all cases, showing the early filling of the cavernous sinus followed by the draining vessel (posterior in case n. 4, anterior in cases n degrees 1, 2, 3). Case n degrees 2 was unilateral and cases n. 1, 3, 3 were bilateral. The blood flow was low in all cases. A successful embolization was performed in all patients with resolution of all symptoms. CONCLUSION: The diagnosis of dural fistulas is often difficult because of misleading clinical signs. It is documented by a cerebral angiography showing the feeding vessels and helping to choose either venous or arterial embolization which is the most suitable treatment.
CD4/major histocompatibility complex class II interaction analyzed with CD4- and lymphocyte activation gene-3 (LAG-3)-Ig fusion proteins.
We analyzed CD4 major histocompatibility complex (MHC) class II interactions with CD4 and lymphocyte activation gene (LAG)-3 recombinant fusion proteins termed CD4Ig and LAG-3Ig. CD4Ig bound MHC class II molecules expressed on the cell surface only when used in the micromolar range. This weak CD4Ig binding was specific, since it was inhibited by anti-CD4 and anti-MHC class II mAb. LAG-3Ig bound MHC class II molecules with intermediate avidity (Kd = 60 nM at 37 degrees C). Using LAG-3Ig as a competitor in a CD4/MHC class II-dependent cellular adhesion assay, we showed that this recombinant molecule was able to block CD4/MHC class II interaction. In contrast, no inhibition was observed in a CD4/MHC class II-dependent T cell cytotoxicity assay. Together, these results suggest that co-engagement of the TcR with CD4 alters the CD4/MHC class II molecular interaction to become insensitive to LAG-3Ig competition.
Mercuric chloride-induced programmed cell death of a murine T cell hybridoma. I. Effect of the proto-oncogene Bcl-2.
Mercuric chloride (HgCl2) as well as several drugs can induce T cell activation leading to systemic immune-mediated diseases in genetically susceptible individuals or rodents. T cell hybridomas represent a well-characterized model system for in vivo mechanisms of various stimuli-induced cell death. The cellular response to HgCl2 was examined using various T cell lines and particularly the murine T cell hybridoma 2B4.11. Exposure to HgCl2 induced both necrosis and apoptosis in a dose- and time-dependent way as demonstrated by DNA fragmentation analysis, flow cytometry of the whole cells and of isolated nuclei, and morphological examination. HgCl2-induced cell death was partly inhibited by cycloheximide. The expression of human Bcl-2 in 2B4.11 cells after transfection significantly prevented HgCl2-induced cell death but did not affect the susceptibility to apoptosis induced by an anti-CD3 epsilon mAb. Subcytotoxic doses of HgCl2 enhanced metabolic activity of Bcl-2 transfectants in contrast with mock-transfected cell line. Thus, we conclude that apoptosis is part of the cell death process induced by HgCl2 and that the ability of Bcl-2 to prevent the death of one particular cell line is stimulus-dependent suggesting the existence of different pathways leading to cell death.
Mercuric chloride-induced programmed cell death of a murine T cell hybridoma. II. Opposite effect of interleukin-2 and interleukin-4.
In susceptible animals evidence is accumulating for a primary role for Th2 cells in the course of HgCl2-induced autoimmunity, and for a contribution of Th1 cells in the self-regulated phase of this disease. We have reported that incubation of 2B4.11 T cell hybridoma with HgCl2 induced programmed cell death. This paper shows that recombinant IL-2 significantly diminished HgCl2-induced 2B4.11 cell death. Although no effect was observed upon incubation with exogenous IL-4, we observed a significant protection by adding an anti-IL-4 monoclonal antibody to the culture. Accordingly, by RT-PCR we found the presence of IL-2 receptor-encoding mRNA, and by cytofluorometry, the expression of the protein was detected only after exposure to HgCl2. Moreover, upon HgCl2 treatment, 2B4.11 cells were induced to produce IL-4. Altogether these findings showed that cytokine environment, IL-2, IL-4 otherwise defining the Th1/Th2 dichotomy, in conjunction with a chemical may differentially influence the fate of cell populations, death or survival.
Mercuric chloride, a chemical responsible for T helper cell (Th)2-mediated autoimmunity in brown Norway rats, directly triggers T cells to produce interleukin-4.
Mercurials may induce immune manifestations in susceptible individuals. Mercuric chloride (HgCl2) induced autoimmunity in the Brown Norway (BN) strain but an immuno-suppression in the Lewis strain with, however, autoreactive anti-class II T cells present in both strains. In the present study we looked at modifications of cytokine production by PCR and cytofluorometric analyses in normal BN and Lewis rat splenocytes, cultured with or without HgCl2. Unfractionated BN rat splenocytes and purified T cells exposed to HgCl2 expressed high levels of IL-4 mRNA. Increase in class II and CD23 molecule expression on B cells was partly inhibited by anti-IL-4 mAb showing that IL-4 was produced. By contrast, no overexpression of IL-4 mRNA could be seen in Lewis rats. Although an increase in class II molecule expression was observed suggesting that other T helper cell 2 cytokines were produced, there was also a concomitant decrease in CD23 molecule expression that was abrogated after addition of an anti-IFN-gamma mAb to the culture. IFN-gamma mRNA production was induced in unfractionated spleen cells and T cells from both strains after HgCl2 exposure. Altogether these findings demonstrate that HgCl2 has very early direct effects on cytokine production and that these effects differ depending on the strain. The early effect on IL-4 production observed on BN rat spleen cells and T cells may explain that the autoreactive anti-class II T cells that are found in HgCl2-injected BN rats have a Th2 phenotype.
A safe and rapid method for analyzing apoptosis-induced fragmentation of DNA extracted from tissues or cultured cells.
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