Biomedical subjects

M Pallardy

Publications and source records attributed to M Pallardy.

At least 19 recordsLinked to original sources

PubMed2006

In vitro tests to evaluate immunotoxicity: a preliminary study.

The implementation of Registration, Evaluation and Authorisation of new and existing Chemicals (REACH) will increase the number of laboratory animals used, if alternative methods will not be available. In the meantime, REACH promotes the use of in vitro tests and, therefore, a set of appropriated alternative testing methods and assessment strategies are needed. The immune system can be a target for many chemicals including environmental contaminants and drugs with potential adverse effects on human health. The aim of this study was to evaluate the predictivity of a set of in vitro assays to detect immunosuppression. The tests have been performed on human, rat and murine cells. Different endpoints have been assessed: cytotoxicity, cytokine release, myelotoxicity and mitogen responsiveness. For each of these endpoints IC50s values have been calculated. Six chemical substances, representative of the full range of in vivo responses and for which good human and/or animal data are available either from databases or literature, have been selected: two chemicals classified as not immunotoxic (Urethane and Furosemide), and four (tributyltin chloride (TBTC), Verapamil, Cyclosporin A, Benzo(a)pyrene) with different effect on immune system. All the tests confirmed the strong immunotoxic effect of TBTC as well as they confirmed the negative controls. For one chemical (Verapamil) the IC50 is similar through the different tests. The IC50s obtained with the other chemicals depend on the endpoints and on the animal species. The clonogenic test (CFU-GM) and the mitogen responsiveness showed similar IC50s between human and rodent cells except for Cyclosporin A and TBTC. All different tests classified the compounds analyzed in the same way.

Animals↗

PubMed2005

Gene therapy bio-safety: scientific and regulatory issues.

We report here the topics discussed during the round table of the 2nd European Conference & Practical Course: Towards Clinical Gene Therapy: Preclinical Gene Transfer Assessment, held in Bellaterra (Spain), 1-14 February, 2004. First, how to predict the risk of pathologies generated by changes of the gene expression after proviral genome integration. In the light of the scientific information that emerged after the SAEs occurred in three X-SCID patients treated in France, (a) it is necessary to take into the account the dose of vector used in transduction protocols, in order to minimize the risk to target potentially pathogenic loci. Namely, low vector doses are recommended to minimize the number of vector genomes inserted per cell. (b) The potency of vector elements (ie promoter and transgene), in terms of activation of undesired cell function(s), should be elucidated to devise safe transduction protocols. (c) Target cells should be better characterized before and after transduction to avoid reinfusion into patients' cells, with proviral integration that may be pathogenic. (d) The possibility of replacing onco-retroviruses with other vector systems should be envisaged, for example, nonintegrative gene correction strategies. Second, adequate animal models are required in preclinical experimentation before going to clinics. Although animal models are not yet predictive for risk assessment of proviral insertion, they allow validation of the proof of principle of gene therapy strategies and pharmacological characterization of gene transfer products. Third, a dialogue between researchers and members of regulatory agencies is necessary to implement the regulatory frame where gene therapy products are to be used as new bio-pharmaceuticals. This will implement the whole gene therapy process development at both preclinic (research, development and clinical designs) and postclinic (follow-up of patients) stages. Hence, a European cooperation between professionals (researchers, physicians, industries, patients' associations, investors, etc) will allow implementation of gene therapy regulation in Eastern European countries.

Animals↗

PubMed2001

Th(1)/Th(2) responses to drugs.

T lymphocytes can be characterized by their pattern of cytokine secretion and be divided into type I (Th(l)/Tc(l)) and type 2 (Th(2)/Tc(2)) subsets. The involvement of type-1 or type 2-like responses in sensitization has been studied in the mouse, with reference contact and respiratory contact sensitizers. One interesting feature with certain drugs, such as beta-lactam antibiotics, is the diversity of clinical manifestations associated with immune-mediated hypersensitivity reactions in humans: immediate reactions such as urticaria, Quincke oedema and anaphylactic shock, and delayed hypersensitivity reactions, such as maculopapular rashes, allergic contact dermatitis and skin reactions of other types. In the mouse, Th(1) and Th(2) cytokines have been shown to regulate primary and secondary benzylpenicilloyl- (BPO-) specific antibody responses. Peripheral blood lymphocytes isolated from patients with a clear history of beta-lactam allergy were assessed for type-1 and type-2 phenotypes. Immediate reactions involved mixed Th(1), Tc(1), and Tc(2) responses, whereas allergic contact dermatitis involved Tc(1) and Th(1) cells. Other delayed hypersensitivity reactions to beta-lactams were restricted to Th(1) responses. It has been demonstrated that both CD4(+) and CD8(+)-lidocaine-specific T cell clones isolated from patients with allergic contact dermatitis produced IFN-gamma, even though CD8(+) clones only produce IFN-gamma, while IFN-gamma producing CD4(+) cells concomitantly produced IL-5 and IL-4. Together these data illustrate the heterogeneity of drug-specific T-cell responses.

Allergens↗

PubMed2001

Apoptosis can be a confusing factor in in vitro clastogenic assays.

Among the tests used to determine the mutagenic potential of chemicals, the chromosomal aberrations and micronucleus assays play an important role. These tests score either chromosomal structural aberrations at metaphase or micronuclei at interphase. One of the hallmarks of apoptosis is DNA fragmentation into 50-300 kpb leading to oligonucleosomal fragmentation that can interfere with the results of clastogenic assays. In this case, apoptosis may be a confusing factor in the evaluation of the mutagenic potential of molecules and lead to false positive results. For these reasons we have developed a cell line able to demonstrate the interference of apoptosis in two mutagenicity tests: the in vitro micronucleus test and metaphase analysis in vitro. We used a murine cytotoxic T cell line, CTLL-2 Bcl2, in which a stably transfected bcl2 gene is known to protect these cells from apoptosis induced by various stimuli. A comparison between results obtained in parental CTLL-2 cells and in CTLL-2 Bcl2 cells treated with non-genotoxic apoptosis inducers, such as dexamethasone or gliotoxin, leads us to conclude that apoptosis could give false positive results due to DNA fragmentation. Moreover, with etoposide, a clastogen that also induces apoptosis, we observed that the percentages of aberrant cells and numbers of micronuclei were significantly increased in CTLL-2 cells compared with CTLL-2 Bcl2 cells. This observation suggests that apoptosis leads to an overestimation of the genotoxic potential of chemicals. Finally, with nocodazole, an aneugen, we confirm that this model can also detect agents that have only genotoxic potential and thus allows a better estimation of the genotoxic threshold in studies with aneugens, thus avoiding overestimation of the mutagenic risk of such a compound.

Animals↗

PubMed2001

Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1.

Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells.

Animals↗

PubMed2000

The glucocorticoid receptor and STAT6 physically and functionally interact in T-lymphocytes.

In lymphocytes, glucocorticoids (GC)- and interleukin-4-signaling pathways are known to interact, as evidenced by inhibition of IL-4-mediated proliferation by dexamethasone or suppression of GC-induced apoptosis by IL-4. In this study, we characterized the molecular basis for this reciprocal interference. We report that, in murine CTLL-2 cells, IL-4 inhibits GC-induced MMTV (mouse mammary tumor virus) promoter transactivation, and that GC suppress IL-4-induced transactivation of a STAT6 (signal transducers and activators of transcription 6)-responsive promoter without affecting IL-4-stimulated STAT6 DNA-binding. Moreover, we evidenced a physical association between GC receptor and STAT6, which proved to be functionally relevant, since STAT6 overexpression increased the IL-4 inhibitory effect on GC-induced MMTV transactivation.

Animals↗

PubMed2000

[Mode of action of glucocorticoids].

RECEPTORS: The effect of glucocorticoids is mediated by a receptor mainly found in the cytoplasm. The glucocorticoid receptor is a member of the nuclear receptor superfamily. RECEPTOR ACTIVATION: When unstimulated, the glucocorticoid receptor is inactivated by its integration within a multiple-protein complex associating heat shock proteins, immunophilins and cyclophilins. When the hormone binds to its receptor, the complex dissociates and the receptor migrates to the nucleus. In the nucleus, the activated receptor provokes an upregulation or downregulation of target gene expression. GENE REGULATION: Gene expression may be regulated via an interaction between specific nucleic acid sequences of the glucocorticoid receptor and nuclear DNA (direct mechanism) or by protein-protein interactions (indirect mechanism). The expression of target genes is either inhibited or stimulated. PHARMACOLOGICAL CONSEQUENCES: The main pharmacological applications of glucocorticoids can be explained by these different mechanisms. The antiinflammatory action of glucocorticoids results from an inhibition of the transcription of the collagenase gene via an interaction with the AP-1 transcription factor. The anticancer action of glucocorticoids results from the induction of apoptotic cell death via a mechanism which would require the transcriptional activity of the glucocorticoid receptor.

Anti-Inflammatory Agents↗

PubMed2000

Il-4 and IFN-gamma mRNA induction in human peripheral lymphocytes specific for beta-lactam antibiotics in immediate or delayed hypersensitivity reactions.

Beta-lactam antibiotics elicit CD4+ and CD8+ T-cell-mediated immune responses that play a central role in allergic reactions. However, the involvement of a type 1- (Th1 or Tc1) or a type 2-like (Th2 or Tc2) differentiation in drug allergy remains unclear. We investigated the expression of interleukin 4 (IL-4) and interferon gamma (IFN-gamma) mRNA by quantitative reverse transcription and polymerase chain reaction (RT-PCR) in patient-derived peripheral blood lymphocytes following specific in vitro stimulation. Samples were collected from a total of 19 patients who had developed immediate or delayed clinical manifestations of hypersensitivity to beta-lactam and from 11 control subjects. Peripheral blood mononuclear cells (PBMCs) were stimulated with either free antibiotics or antibiotic-human serum albumin (HSA) conjugates. Specific induction of IFN-gamma mRNA expression was observed in 11 of 11 allergic patients with immediate reactions, in 6 of 8 patients with delayed reactions, and in 4 of 11 control subjects. IL-4 mRNA expression was induced in 5 of 11 allergic individuals with immediate reactions but in none of the 8 patients with delayed responses and none of the 11 control subjects. IL-4 mRNA expression was only induced following activation with free drugs, while IFN-gamma mRNA expression was predominantly induced in CD4+ T cells following stimulation with HSA-conjugated drugs. Immediate-type hypersensitivity to beta-lactams was not associated with a pure type 2-like response when PBMCs were specifically stimulated in vitro: Some patients with well-documented history of beta-lactam-induced immediate allergic reaction showed a high IFN-gamma response. Contact dermatitis involved Tc1 and Th1 cells and other delayed hypersensitivity reactions to beta-lactams were restricted to Th1 responses.

Adolescent↗

PubMed1999

Assessment of apoptosis in xenobiotic-induced immunotoxicity.

Generation of immunity is a highly complex process in which proliferation and differentiation of immune-competent cells regulated by cytokines and cell-cell interactions play a major role. Reducing the number of immune-competent cells or altering the function, selection, and differentiation of lymphocytes after xenobiotic treatment may lead to serious adverse effects. Programmed cell death, or apoptosis, is a highly regulated process by which an organism eliminates unwanted cells without eliciting an inflammatory response. However, xenobiotics are also able to trigger unwanted apoptosis or to alter the regulation of programmed cell death. Cytological characteristics of apoptosis are generally different from those seen in acute pathological cell death resulting from cell injury. The morphological characteristics of apoptosis are unique including cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, disruption of the nuclear lamina, nuclear fragmentation, and emergence of apoptotic bodies. It is now established that apoptosis plays a critical role in both development and homeostasis of the immune system: thymic selection, cytotoxicity, deletion of autoreactive cells, and regulation of the size of the lymphoid compartment. Assessment of apoptosis relies on the morphological and biochemical modifications of the dying cells. As a rule, and because an apoptotic cell rarely displays all of the characteristic apoptotic features, several criteria should be monitored in parallel including morphological examination. The techniques described in this paper have been divided into five categories: analysis of cell morphology by microscopy, identification of DNA fragmentation, determination of mitochondrial membrane potential, detection of plasma membrane changes, analysis of caspase activation.

Animals↗

PubMed1999

Quantitation of cytokine mRNA expression as an endpoint for prediction and diagnosis of xenobiotic-induced hypersensitivity reactions.

Xenobiotic-induced hypersensitivity reactions are immune-mediated effects that involve specific antibodies and/or effector and regulatory T lymphocytes. Cytokines are key mediators of such responses and must be considered as possible endpoints for predicting sensitizing potency of drugs and chemicals, as well as for helping diagnosis of allergy. Detecting cytokine production at the protein level has been shown to not be always sensitive enough. This paper describes three examples of the utilization of semiquantitative or competitive reverse transcription polymerase chain reaction analysis of interleukin-4, interferon gamma, and interleukin-1beta mRNAs as endpoints for assessing T-cell or dendritic cell responses to sensitizing drugs (beta-lactam antibiotics) or chemicals (dinitrochlorobenzene).

Animals↗

PubMed1999

Mechanisms of drug-induced allergic contact dermatitis.

Allergic contact dermatitis is induced by a wide variety of drugs that trigger specific immune responses following topical exposure. Identified chemical structures involved in such reactions include the mercuric and thiosalicylic acid groups of thimerosal, the diphenylketone group of the anti-inflammatory drug ketoprofen, the amide or ester structure of local anesthetics, and the side-chain and thiazolidine ring of beta-lactams. The T cell responses to such compounds involve CD4+ and CD8+ alphabeta+ T lymphocytes and also CD4 /CD8 gammadelta+ T cells. Although "T helper 2" cytokine production by drug-specific human T cells from patients with allergic contact dermatitis has been described, T helper 1-like and T cytotoxic 1-like responses clearly play key roles in this cutaneous reaction.

Allergens↗

PubMed1999

Synergism between interleukin-6 and interleukin-1beta in hypothalamic serotonin release: a reverse in vivo microdialysis study in F344 rats.

The effects of interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) perfused locally into the anterior hypothalamus (AHY) on serotonin (5-hydroxytryptamine, 5-HT) release were investigated in the same region using in vivo microdialysis in conscious, freely moving F344 rats. IL-1beta (1 ng/rat) or IL-6 (50 ng/rat) injected directly into the AHY elicited a rapid and transient statistically significant increase in extracellular 5-HT levels (to 161% and 145% of the respective AUC (area under the curve) basal value, 100%). Intra-hypothalamic infusion of IL-1-receptor antagonist IL-1Ra (2 mug/rat) prevented this effect of IL-1beta, but not that of IL-6, suggesting an IL-1beta-independent mechanism for hypothalamic 5-HT release by this latter cytokine. Furthermore, intra-hypothalamic co-perfusion of IL-6 with IL-1beta at sub-optimal doses (10 ng/rat and 0.5 ng/rat, respectively) synergized in releasing hypothalamic 5-HT, thus providing in vivo evidence that both cytokines, IL-6 and IL-1beta are able to modulate the neuronal 5-HT response in the rat AHY.

Animals↗

PubMed1999

[Principal cellular and molecular mechanisms of xenobiotic-induced hypersensitivity reactions].

Sensitization to xenobiotic first involves an interaction between the compound and antigen presenting cells such as Langerhans cells within the epidermis. This leads to the transdermal migration of this presenting cells towards draining lymph nodes. A second step consists in activation, proliferation and differenciation of CD4+ T cells, and CD8+ T lymphocytes involved in delayed hypersensitivity reactions such as contact dermatitis. T helper lymphocytes can differenciate into Th1 (producing interferon-gamma and interleukin (IL)-2) or Th2 (producing IL-4, IL-5, IL-10, IL-13) lymphocytes. Some experimental models allowed to demonstrate a link between Th1- or Th2-type responses and different hypersensitivity reactions. Specific antibody production also plays a key role in xenobiotic-induced allergy, especially IgE production involved in mastocytes degranulation and immediate hypersensitivity reactions.

Animals↗

PubMed1998

Testing strategies in immunotoxicology.

Developing a battery of immune function assays to screen potential immunotoxic compounds has been a major issue these past years. Improving this approach is possible using new probes and parameters that are now available from recent knowledge on how the immune system is working (apoptosis, RT-PCR for cytokine mRNA expression). Immunotoxic outcome generally results in serious adverse effects, thus it seems appropriate to evaluate this risk early in drug development. This is especially true in the context of the emerging combinatorial chemistry techniques and high throughput screening in pharmacology resulting in probably numerous molecules to test in toxicology. In this case, screening for adverse effects (genotoxicity, hepatotoxicity, immunotoxicity) that may compromise definitely the development of a molecule should be a help in the decision process since more than one molecule with equivalent pharmacological properties may be available.

Animals↗

PubMed1998

Apoptosis: identification of dying cells.

Cell death is usually classified into two broad categories: apoptosis and necrosis. Necrosis is a passive, catabolic process, always pathological, that represents a cell's response to extreme accidental or toxic insults. Apoptosis, in contrast, occurs under normal physiological conditions and is an active process requiring energy. However, apoptosis can also be elicited in a pathological way by toxic injury or during disease processes. In these nonphysiological conditions, both types of cell death can be encountered following the same initial insult and the balance between death by apoptosis and by necrosis appears to depend upon the intensity of the injury and the level of available intracellular ATP. It is important, however, to discriminate between apoptosis and necrosis in pathological conditions, as therapeutic intervention could be considered in apoptotic cell death with putative new pharmacological agents aimed at interfering with the key molecular events involved. In most cases, none of the current laboratory techniques used alone allows for unambiguous identification of apoptotic cells. Some of the most common methods based on morphology, biochemistry, and plasma membrane changes are discussed in terms of specificity and possible sources of error in data interpretation. As a rule, classification of cell death in a given model should always include morphological examination coupled with at least one of the other assays.

Adenosine Triphosphate↗

PubMed1998

[Induction of apoptosis in lymphocytes by glucocorticoids: between physiology and pharmacology].

Glucocorticoids are physiological molecules that are also extensively used in clinics as anti-inflammatory, immunosuppressive or anti-tumoral agents. Glucocorticoids can induce apoptosis on normal lymphoid cells and play a key role in the physiology of thymic selection. In clinics these molecules are also used for their potencies in inducing apoptosis of malignant lymphoid cells. Glucocorticoids are mediating their effects after binding to an intracellular receptor belonging to the steroid receptor superfamily: the glucocorticoid receptor (GR). Once activated, the GR, can mediate his effects through direct binding on the DNA or via protein/protein interactions with transcription factors. Depending on the type of lymphocytes, the mechanism of apoptosis induced by glucocorticoids fall roughly in two categories: induction of "death genes" by the activated GR (I kappa B, c-jun) or repression of survival factors (AP-1, c-Myc). In the case of thymic selection the mechanism is more subtle depending on the mutual repression of Nur77 and GR.

Animals↗

PubMed1997

Cyclosporin-A inhibits human endothelial cells proliferation through interleukin-6-dependent mechanisms.

Cyclosporine A (CsA) is a widely used immunosuppressant which possesses significant side effects including nephrotoxicity and systemic arterial hypertension. In this work we evaluated the effects of CsA on human umbilical endothelial cell proliferation (HUVEC). Treatment of endothelial cells with CsA inhibited cell proliferation, and was correlated with an increase of interleukin-6 (IL-6) production and IL-6 mRNA expression. Addition of exogenous IL-6 also significantly altered cell proliferation. Furthermore, neutralization of endogenous IL-6 with a specific monoclonal antibody concomitantly with CsA treatment completely restored HUVEC proliferation. These results suggest that CsA-induced inhibition of HUVEC proliferation is mediated through an augmentation of IL-6 synthesis.

Antibodies↗

PubMed1996

Position 16 of the steroid nucleus modulates glucocorticoid-induced apoptosis at the transcriptional level in murine T-lymphocytes.

Synthetic glucocorticoids (GCs), which possess a different radical substituted in position 16 of the steroid nucleus structure, display various antiproliferative activities on activated lymphoid cells. We analysed this structure-function relationship between dexamethasone (DEX; methyl group in position 16 alpha) and beta-methasone (BM; methyl group in position 16 beta) with regard to two important aspects of GC activity, namely the activation of transcription and induction of apoptosis in IL-2-dependent murine lymphoid cells. DEX induced a higher percentage of apoptotic viable cells compared to BM. This structure-activity relationship was not related to differences in cytosolic glucocorticoid receptor (GR) affinity or kinetics of apoptosis. However, DEX was more efficient than BM in inducing transcriptional activation of an MMTV-CAT plasmid in transiently transfected CTLL-2 cells. In addition, DEX was more potent in inhibiting AP-1 DNA-binding activity compared to BM. These results suggest that the configuration in position 16 may influence the potency of GCs to induce apoptosis in lymphoid cells, mainly by modulating GR-induced transcription.

Animals↗