Predictive value of pre-clinical work for vaccine safety assessment.
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Animals↗
Biomedical subjects
F Verdier
Publications and source records attributed to F Verdier.
Recombinant viruses as a tool for therapeutic vaccination against human cancers.
Viral vectors can be used to express a variety of genes in vivo, that encode tumor associated antigens, cytokines, or accessory molecules. For vaccination purposes, the ideal viral vector should be safe and enable efficient presentation of expressed antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its industrialization. The characteristics of the most promising viral vectors, including retroviruses, poxviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and alphaviruses, will be reviewed in this communication. Such recombinant viruses have been successfully used in animal models as therapeutic cancer vaccines. Based on these encouraging results, a series of clinical studies, reviewed herein, have been undertaken. Human clinical trials, have as of today, allowed investigators to establish that recombinant viruses can be safely used in cancer patients, and that such recombinants can break immune tolerance against tumor-associated antigens. These promising results are now leading to improved immunization protocols associating recombinant viruses with alternate antigen-presentation platforms (prime-boost regimens), in order to elicit broad tumor-specific immune responses (humoral and cellular) against multiple target antigens.
Antigens, Neoplasm↗
Proteasomes regulate the duration of erythropoietin receptor activation by controlling down-regulation of cell surface receptors.
The binding of erythropoietin (Epo) to its receptor leads to the transient phosphorylation of the Epo receptor (EpoR) and the activation of intracellular signaling pathways. Inactivation mechanisms are simultaneously turned on, and Epo-induced signaling pathways return to nearly basal levels after 30-60 min of stimulation. We show that proteasomes control these inactivation mechanisms. In cells treated with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal (LLnL) or lactacystin, EpoR tyrosine phosphorylation and activation of intracellular signaling pathways (Jak2, STAT5, phosphatidylinositol 3-kinase) were sustained for at least 2 h. We show that this effect was due to the continuous replenishment of the cell surface pool of EpoRs in cells treated with proteasome inhibitors. Proteasome inhibitors did not modify the internalization and degradation of Epo.EpoR complexes, but they allowed the continuous replacement of the internalized receptors by newly synthesized receptors. Proteasome inhibitors did not modify the synthesis of EpoRs, but they allowed their transport to the cell surface. N-Ac-Leu-Leu-norleucinal, but not lactacystin, also inhibited the degradation of internalized Epo.EpoR complexes, most probably through cathepsin inhibition. The internalized EpoRs were not tyrosine-phosphorylated, and they did not activate intracellular signaling pathways. Our results show that the proteasome controls the down-regulation of EpoRs in Epo-stimulated cells by inhibiting the cell surface replacement of internalized EpoRs.
Cell Membrane↗
Erythropoietin induces the tyrosine phosphorylation of GAB1 and its association with SHC, SHP2, SHIP, and phosphatidylinositol 3-kinase.
Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.
Adaptor Proteins, Signal Transducing↗
Quinine disposition in globally malnourished children with cerebral malaria.
BACKGROUND: Both malnutrition and malaria affect drug disposition and are frequent among children in the tropics. We assessed their respective influence on quinine distribution. METHODS: Forty children were divided into 4 groups: children with normal nutritional status without (group 1) or with (group 2) cerebral malaria, and malnourished children without (group 3) or with (group 4) cerebral malaria. All children received an infusion of 8 mg/kg of a combination solution of cinchona alkaloids that contained 96.1% quinine, 2.5% quinidine, 0.68% cinchonine, and 0.67% cinchonidine (corresponding to 4.7 mg/kg quinine base). The children with malaria then received repeated infusions every 8 hours for 3 days. Pharmacokinetic profiles of plasma and erythrocyte quinine were determined during the first 8 hours, together with quinine protein binding. Additional measurements of plasma quinine concentrations were used to simulate quinine concentrations profiles in children with malaria with and without malnutrition. Clinical recovery and parasitemia clearance times were determined in the children with malaria. RESULTS: Compared with control children, malaria and malnutrition increased plasma concentrations of quinine and reduced both the volume of distribution and the total plasma clearance. Simultaneously, alglycoprotein plasma concentrations and protein-bound fraction of the drug were increased. Erythrocyte quinine concentrations correlated strongly with free plasma quinine but not with the extent of parasitemia. Similar effective and nontoxic quinine concentration profiles were obtained in malaria with and without malnutrition. CONCLUSIONS: Severe global malnutrition and cerebral malaria have a similar effect on quinine pharmacokinetics in children. Moderate malnutrition does not potentiate cerebral malaria-mediated modifications of quinine disposition. These results suggest that current parenteral quinine regimens can be used, unmodified, to treat children with both malaria and malnutrition.
Antimalarials↗
Preclinical safety evaluation of human gene therapy products.
Human gene therapy products include naked DNA and viral as well as non-viral vectors containing nucleic acids. There is limited experience on the preclinical toxicity studies necessary for the safety evaluation of these products, which have been outlined in several recently released guidelines. Requirements for the preclinical safety evaluation of human gene therapy products are both specific and non-specific. All key preclinical studies should be performed in compliance with Good Laboratory Practices. Non-specific requirements are in fact common to all pharmaceutical products. Critical specific issues to be addressed are: the safety evaluation of the vector and the toxicity of the expressed protein(s), which are the two components of gene therapy products, the quality of the test article, the selection of animal species, and the verification that the administration method successfully transports the gene of interest, with the vector, to the target site(s). The treatment schedule should mimic the intended human therapeutic design. The host's immune response against the gene therapy product has to be evaluated to detect possible adverse effects and immune neutralization by antibodies. The biodistribution of the gene of interest is also essential and can be evaluated by molecular biology techniques, such as PCR. Specific confinement is required for the safe manipulation of viral vectors.
Animals↗
Proteasomes regulate erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) activation. Possible involvement of the ubiquitinated Cis protein.
Cis is an Src homology 2 domain-containing protein, which binds to the erythropoietin receptor and decreases erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the erythropoietin receptor (Tyr401). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitors N-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In erythropoietin-stimulated UT-7 cells, the activation of the erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30-60 min of erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the erythropoietin receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-erythropoietin receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the erythropoietin receptor.
Acetylcysteine↗
A sequence of the CIS gene promoter interacts preferentially with two associated STAT5A dimers: a distinct biochemical difference between STAT5A and STAT5B.
Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.
Animals↗
The popliteal lymph node assay: results of a preliminary interlaboratory validation study.
The popliteal lymph node (PLN) assay was proposed to predict possible autoimmune effects of xenobiotics. A preliminary interlaboratory validation study of the PLN assay was conducted in Wistar rats. Three laboratories tested in blind fashion four compounds, namely chlorpromazine, zimeldine, hydrazine and streptozotocin, which were reported to cause autoimmune-like reactions in humans, and one compound, i.e. barbital, which was not, using strictly the same experimental procedure. All tested substances were injected into the hind footpad of rats on day 1, and PLN weight and cellularity were measured on day 8. Comparison of the controlateral PLN was used to calculate weight and cellularity indices. The results were independently analyzed in a fourth laboratory. All four positive compounds were detected by the three laboratories using both weight and cellularity indices, and the negative compound consistently proved negative. Despite variations in absolute values between laboratories, although not significant, these results provide further evidence of the potential predictive value of the PLN assay.
Animals↗
Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation.
In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine following treatment of UT-7 cells with erythropoietin. We have investigated the expression of IRS-1 and IRS-2 in several cell lines with erythroid and/or megakaryocytic features, and we observed that IRS-2 was expressed in all cell lines tested. In contrast, we did not detect the expression of IRS-1 in these cells. In response to erythropoietin, IRS-2 was immediately phosphorylated on tyrosine, with maximal phosphorylation between 1 and 5 min. Tyrosine-phosphorylated IRS-2 was associated with phosphatidylinositol 3-kinase and with a 140-kDa protein that comigrated with the phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase, SHIP. Moreover, IRS-2 was constitutively associated with the erythropoietin receptor. We did not observe the association of IRS-2 with JAK2, Grb2, or PTP1D. Using BaF3 cells transfected with mutated erythropoietin receptors, we demonstrate that neither the tyrosine residues of the intracellular domain nor the last 109 amino acids of the erythropoietin receptor are required for erythropoietin-induced IRS-2 tyrosine phosphorylation. Altogether, our results indicate that erythropoietin-induced IRS-2 tyrosine phosphorylation could account for the previously reported activation of phosphatidylinositol 3-kinase mediated by erythropoietin receptors mutated in the phosphatidylinositol 3-kinase-binding site (Damen, J., Cutler, R. L., Jiao, H., Yi, T., and Krystal, G. (1995) J. Biol. Chem. 270, 23402-23406; Gobert, S., Porteu, F., Pallu, S., Muller, O., Sabbah, M., Dusanter-Fourt, I., Courtois, G., Lacombe, C., Gisselbrecht, S., and Mayeux, P. (1995) Blood 86, 598-606).
Enzyme Activation↗
The popliteal lymph node assay in 1996.
The popliteal lymph node (PLN) assay is based on the assumption that a mechanism similar to a graft-versus-host (GvH) reaction is involved in 'GvH-like' drug-induced side-effects, including generalized lymphadenopathy, serum sickness-like disease, scleroderma-like reaction and the lupus syndrome. An increased PLN weight 7-10 days after injection of the test article into the footpad is generally held as a positive response. Most, if not all compounds reported to induce pseudo-GvH side-effects in man (namely positive model compounds) have been shown to induce positive PLN responses in mice and/or rats. Reproducible results have been obtained in several laboratories, in some instances blindly. However, positive responses have also been obtained with the negative model compounds acetone and imipramine. Flow cytometry analysis and conventional histology failed to help differentiate between a true GvH response and a primary irritative effect. In order to confirm the potential value of the PLN assay to predict the risk for drug-induced GvH-like reactions, mechanistic studies are urgently needed.
Animals↗
Autoantibodies in conventional toxicity testing.
A number of drugs and chemicals can induce autoimmune disorders. The use of autoantibody assays in toxicology has been evaluated as a tool to predict and explain autoimmune reactions. Autoantibodies are divided between organ-specific and ubiquitous autoantibodies and several mechanisms have been proposed for their pathogenesis. Assay methods depend on the autoantibody investigated and the specificity of the assay required. There is only a partial correlation between the presence of autoantibodies in humans or animals and the risk of developing an autoimmune disease. Responses in animals vary according to genetic influence and extrapolation to humans is difficult. Therefore, autoantibody assays were not considered absolutely predictive of the potential of a new drug to trigger autoimmune diseases. However assays such as the Coombs test for hemolytic anemia or anti-thyroid antibody assay for thyroiditis, can be useful for explaining the possible mechanisms of autoimmune reactions which occur during toxicology studies.
Animals↗
Erythropoietin-induced erythroid differentiation of the human erythroleukemia cell line TF-1 correlates with impaired STAT5 activation.
The TF-1 cell line has been established from a patient with erythroleukemia. While various cytokines induce TF-1 cell proliferation, erythropoietin (Epo) only sustains the short-term growth of these cells and induces their differentiation along the erythroid lineage. A truncated Epo receptor (EpoR) is overexpressed in these cells. The truncation removed the 96 C-terminal amino acids, including seven tyrosine residues. An additional single mutation at position +3 of Tyr344 led to the replacement of leucine 347 by proline. Stimulation by Epo induced an impaired activation of the STAT5 transcription factor in these cells. The same defect in STAT5 activation was found in the murine FDCP-1 cell line transfected with a chimeric EpoR containing the abnormal TF-1 EpoR cytoplasmic domain. Infection of TF-1 cells with a retrovirus containing a normal murine EpoR was able to restore both Epo-induced STAT5 activity and cellular proliferation. In contrast, Epo-induced differentiation was reduced strongly in infected TF-1ER cells. These results suggest that Epo-induced differentiation correlates with impaired Epo-induced STAT5 activation.
Amino Acid Sequence↗
p53 gene mutation: software and database.
A large number of different mutations in the tumor suppressor gene p53 gene have been identified in all types of cancer. As of September 1995, this database contains over 4200 mutations. This substantial increase since our previous report can enable epidemiological analyses which were not previously possible. In order to capture all these new data, the software permitting analysis has been improved. This report describes the various improvements since first release of the database.
Animals↗
Efficacy and pharmacokinetics of a new intrarectal quinine formulation in children with Plasmodium falciparum malaria.
1. Three groups of seven children aged 2-14 years with acute uncomplicated Plasmodium falciparum malaria received 12.8 mg kg-1 quinine gluconate by the intrarectal route (new cream formulation) or 8 mg kg-1 Quinimax (a Cinchona alkaloid alkaloid combination) by the intramuscular or intravenous (4 h infusion) route every 8 h for 3 days. Clinical and parasitological status was similar in the three groups at enrolment. 2. At 36 h, body temperature of all children of the three groups was returned to normal and remained so until day 7. 3. The decrease in parasitaemia did not differ between the three groups and the time required for a 50% fall in parasitaemia relative to baseline was 12.3 +/- 5.4, 18.2 +/- 6.1 and 14.5 +/- 4.2 h in the intrarectal, intramuscular and intravenous treatment groups, respectively. Parasitaemia expressed as a percentage of initial values was not significantly different in the three groups after 48 h of treatment (7.4 +/- 16.0, 4.1 +/- 4.2 and 2.2 +/- 3.8% in the intrarectal, intramuscular and intravenous treatment groups, respectively). All the patients were aparasitaemic by day 7. 4. The tolerability of the three treatments was good; in particular, no rectal irritation was reported with the cream formulation. 5. The tmax occurred later after intrarectal (4.1 +/- 2.4 h) and intravenous infusion (3.8 +/- 0.5 h) than after intramuscular injection (1.6 +/- 1.3 h) (P = 0.02). Cmax was lower with the intrarectal (3.0 +/- 1.0 mg 1(-1)) and intramuscular routes (3.2 +/- 0.7 mg 1(-1)) than with the intravenous route (5.1 +/- 1.4 mg 1(-1)) (P = 0.003). Areas under the curve (AUC(0, 8 h)) were smaller with intrarectal (17.0 +/- 7 mg 1(-1) h) and intramuscular routes (19.4 +/- 4.8 mg 1(-1)) than with the intravenous route (27.8 +/- 8.2 mg 1(-1) h) (P = 0.02). The approximate bioavailability of intrarectal quinine from 0 to 8 h was 36% vs intravenous quinine and 51% vs intramuscular quinine. 6. The good tolerability and efficacy of this new intrarectal quinine formulation outweigh its low approximate bioavailability. This new approach might thus be a safe and effective alternative to intramuscular quinine injection for the treatment of children with acute uncomplicated Plasmodium falciparum malaria in the field.
Administration, Rectal↗
[Effects of chloroquine on the replication of a murine retrovirus].
The wide use of chloroquine (Cq) for prophylaxis and chemotherapy of malaria in Africa, and the increased spread of AIDS in areas of this continent where malaria is endemic, raised the question of a possible interaction between chloroquine intake and HIV infection. Indeed, hydroxychloroquine and chloroquine itself have been shown to inhibit HIV-1 replication in vitro, hydroxychloroquine being proposed as a potential useful adjunctive therapy in the treatment of HIV-1 infection. On the other hand, chloroquine has been reported to enhance the replication of Semliki forest and encephalomyocarditis viruses in a mouse model. In an attempt to elucidate Cq effect on retroviral replication, we have studied the effect of various concentrations of chloroquine in vitro (0.1 nmol/l to 25 mumol/l) on Friend retrovirus (FV)-infected fibroblasts of mice and in vivo (2 to 30 mg/kg) on FV-infected mice. No reduction in the number of virus foci was found in chloroquine-treated fibroblasts cultures. In chloroquine treated-infected mice, no differences were observed in the spleen weights, except an increase at 10 mg/kg. A decrease in splenocyte virus titer was only observed at 10 and 30 mg/kg. No differences in the median survival time was observed up to 30 mg/kg. The authors concluded that chloroquine seemed to have variable effects on viral replication in vivo depending on the dosage, but has no influence on the course of FV-induced disease.
Animals↗
Determination of lymphocyte subsets and cytokine levels in cynomolgus monkeys.
Lymphocyte subset counts and cytokine assays are useful to investigate the interactions of pharmaceuticals, particularly new biotechnology products, with the immune system. As no specific reagents are available to label monkey lymphocytes or to assay monkey cytokines by ELISA, cross reactivities of a panel of monoclonal antibodies specific for human lymphocytes or cytokines were studied in the Cynomolgus monkey. The proportions of B, T, CD4+ and CD8+ cells were determined by flow cytometry using a whole blood technique with at least one monoclonal antibody for each subset. Background data were obtained for more than 300 samples. Monkey and human cultured white blood cells were stimulated with standard mitogens. PHA + LPS in humans and Con A + PWM in monkeys triggered the greatest proliferation. IL-1 beta IL-2, IL-6, IL-8, TNF-alpha, TNF-beta and IFN-gamma, but not IL-1 alpha, were detected in the monkey using human reagents. In addition, the cytokine profile and the kinetics of cytokine production compared well in humans and Cynomolgus monkeys.
Animals↗
Competitive ELISA to evaluate the immunogenicity of double-virus-inactivated factor VIII in rabbits.
Viral inactivation is a critical step during the manufacture of factor VIII formulations for use in humans. Indeed, viral inactivation procedures may alter the three-dimensional structure of factor VIII resulting in the formation of new epitopes which, in turn, may lead to the synthesis of inhibiting antibodies, and hence to a decreased therapeutic activity. A rabbit model was used to compare the immunogenicity of a solvent/detergent (SD)-inactivated formulation with a double-inactivated (SD plus heating in solution, SDP) formulation of factor VIII. Two groups of five rabbits were immunized by six s.c. injections of each formulation. The serum of each animal was incubated with various amounts of the competing antigens, e.g. factor VIII SD or factor VIII SDP. The remaining free polyclonal antibodies were assayed by ELISA. Curves obtained with both antigens were compared for each serum. Both factor VIII SD and factor VIII SDP decreased the amount of antibodies raised to either formulation in a dose-dependent manner without observable differences. These results suggest that no new epitopes were present on factor VIII SDP as compared with factor VIII SDP as compared with factor VIII SD and that no epitope deletions occurred, supporting the view that the double-inactivation procedure does not change the immunogenicity of factor VIII SD. This model is proposed as a tool to detect changes in the immunogenicity of proteins which may be modified.
Animals↗