The objective of this study was to evaluate the potential of a high-relaxivity macromolecular gadolinium (Gd) chelate to target folate receptors (FRs). P866 is a dimeric high-relaxivity Gd chelate coupled to a folate moiety. Binding affinity, in vivo biodistribution studies in KB tumor-bearing mice at 1, 4, and 24 In, and dynamic contrast-enhanced (DCE)-MRI (2.35 T) over 4 h were assessed. Binding and internalization of P866 through the FR was demonstrated. Due to the high molecular volume of P866, the binding affinity compared to free FA was decreased (K-D = 59.3 +/- 1.8 nM and 5.9 +/- 0.2 nM, respectively). Tumor/muscle (T/M) uptake was 5.4 +/- 1.0, 4 In after injection of 15 mu mol/kg. Competition with free FA was less effective when the dose was increased due to a saturation of FR. At a dose of 5 mu mol/kg, a 70% difference in signal enhancement was observed between P866 and the nonspecific reference compound, thus demonstrating the specificity of FR targeting. While this high-relaxivity folate-Gd chelate has demonstrated its potential capacity to target in vivo FR on tumors, the sensitivity is probably limited to a certain extent by the saturation of the FR and by the decrease in the apparent relaxivity of the internalized part of P866 in the tumor cells. Magn Reson Med 60: 1337-1346, 2008. (C) 2008 Wiley-Liss, Inc.

The theory describing the magnetic interaction of superparamagnetic compounds with water protons has been described by different theoretical models derived from the classical outer-sphere paramagnetic relaxation model. Consequently, a full set of analytical methods should be used to characterize the efficacy, purity of nanoparticles, and reproducibility of the synthesis process. The distribution and elimination data obtained with C-ferumoxtran-10 are investigated, in comparison with Fe data, to understand the metabolism of nanoparticles. The introduction of superparamagnetic nanoparticle contrast agents in magnetic resonance imaging has constituted a major improvement of the range of tools available to clinicians. Bulk ferromagnetic materials are composed of fully magnetized domains of micron size. In the absence of an external magnetic field, a ferromagnetic material is not magnetized, as the magnetization of these ferromagnetic domains is oriented in directions. The synthesis of superparamagnetic nanoparticles is a complex process because of their colloidal nature.

Contrast-induced nephropathy (CIN) is a worrying concern in at-risk patients. Its pathophysiological mechanism remains speculative and is possibly modulated according to the risk factor(s) and clinical presentation of the patients. Overall, iodinated contrast media (CM) have been shown, in animal models, to induce medullary hypoxia. Furthermore, numerous studies have demonstrated that they have a direct cytotoxic potential on proximal (LLC-PK1) as well as distal (MDCK) tubular cell lines and mesangial cells. A pro-apoptotic potential of such molecules has been found on various cell types including renal tubular and mesangial cells, both in vitro and in vivo. This pro-apoptotic effect on tubular cells has been found to be concentration- and time-dependent. In addition to periprocedural hydration, which is the cornerstone of CIN, several drugs have been investigated for the pharmacological prophylaxis of CIN, either on pre-clinical models or in clinical studies. Some of them (theophylline, Nacetylcysteine, the prostacyclin analogue beraprost or taurine) are known to interfere with the apoptosis pathways. This article reviews and critically discusses the available data concerning the role of apoptosis in the mechanism of CIN and its pharmacological prophylaxis. It also reviews the putative interaction of gadolinium chelates, used as CM for magnetic resonance imaging, with apoptosis pathways.

Allergy-like reactions may occur following administration of iodinated contrast media (CM), mostly in at-risk patients (patients with history of previous reaction, history of allergy, co-treated with interleukin-2 or beta-blockers, etc.) but remain generally unpredictable. Severe and fatal reactions are very rare events. All categories of CM may induce such reactions, although first generation (high osmolar CM) have been found to induce a higher rate of adverse events than low osmolar CM. However, no differences were found between the two categories of CM with respect to mortality. Delayed reactions can also occur. There are no differences between the various categories of CM except for non-ionic dimers, which are more likely to induce such effect. Numerous clinical studies have evaluated the prophylactic value of drugs (mostly antihistamines and corticosteroids). Results are unclear and highly variable. Any prevention depends upon the mechanism involved. However, the mechanism of CM-induced allergy-like reaction remains disputed. Relatively recent data revived the hypothesis of a type-I hypersensitivity mechanism. Positive skin tests to CM have been reported. However, the affinity of IgE towards CM has been found to be very low in the only study which actually evaluated it. Other pathophysiological mechanisms (involving direct secretory effects on mast cells or basophils, or activation of the complement system associated or not with the plasma contact system) are also much debated. Anaphylaxis and anaphylactoid reactions are, in the end, clinically undistinguishable.

Rationale and objectivesSuperparamagnetic iron oxides (SPIO) used as magnetic resonance (MR) contrast agents undergo specific uptake by macrophages. The purpose of this study was first to determine the mechanism of macrophage uptake for Ferumoxides by using competition experiments with specific ligands of scavenger receptors SR-A (I/II) and second, to evaluate and compare the internalization of 2 different contrast agents, Ferumoxides (SPIO) and Ferumoxtran-10 (USPIO: ultrasmall superparamagnetic iron oxide) using macrophages obtained by chemical activation of human monocytic cells. MethodsFerumoxides and Ferumoxtran-10 are 2 MR contrast agents, composed of dextran-coated iron oxide nanoparticles. The endocytosis pathway of Ferumoxides was studied using competition experiments on mouse peritoneal macrophages in the presence of specific ligands of scavenger receptors SR-A (types I and II): polyinosinic acid and fucoidan. In vitro assays using THP-1 (human promonocyte) cells activated into macrophages were performed in the presence of the 2 superparamagnetic nanoparticles. The cellular uptake was determined by measuring the iron content using ICP-AES (inductively coupled plasma–atomic emission spectrometry) and by Prussian blue staining. ResultsIn the presence of polyinosinic acid or fucoidan, the endocytosis of Ferumoxides by mouse peritoneal macrophages was inhibited. This inhibition was obtained using 10 &mgr;g/mL of scavenger receptor ligands at a concentration of 62.5 &mgr;g Fe/mL of SPIO, and a dose-dependent relationship was observed. Without competitors, the percentage of uptake of Ferumoxides by mouse peritoneal macrophages ranged between 3 and 8%. On the human activated monocyte THP-1 cell assay, Ferumoxides underwent a higher macrophage uptake (between 1.1 and 3%) compared with Ferumoxtran-10 (between 0.03 and 0.12%). This difference is attributed to the larger size of Ferumoxides nanoparticles. ConclusionsCompetition experiments indicate that the cellular uptake of Ferumoxides involves scavenger receptor SR-A-mediated endocytosis. The comparison between Ferumoxides and Ferumoxtran-10 confirms that macrophage uptake of iron oxide nanoparticles depends mainly on the size of these contrast agents.

The sections in this article are Introduction Physicochemical Characteristics Physicochemical Properties of the Crystal Hydrodynamic Particle Size and Charge Pharmacology and Metabolism Role of Physicochemical Parameters Mechanism and Consequences of Interaction with Macrophages Pharmacokinetics Nanoparticle Vectorization Current Clinical Uses and Future Developments Gastrointestinal Tract Imaging Liver and Spleen Diseases Lymph Node Metastases Blood Pool Characteristics Characterization of the Atheromatous Plaque Other Potential Uses Stroke Cerebral Tumor Characterization Multiple Sclerosis Arthritis Infection Kidney Imaging Acute Cardiac Transplant Rejection In Vivo Monitoring of Cell Therapy T-staging of Uterine Neoplasms MRI-detectable Embolotherapy Conclusion

Idée JM, Port M, Robert P, et al. Preclinical profile of the monodisperse iodinated macromolecular blood pool agent P743. Invest Radiol 2001;36:41–49. 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 iodin ated 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.

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.

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 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.

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 (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. (C) 1995 Academic Press, Inc.

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.

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.