Genome-wide expression screening in the cardiac embryonic stem cell test shows additional differentiation routes that are regulated by morpholines and piperidines.

The cardiac embryonic stem cell test (ESTc) is a well-studied non-animal alternative test method based on cardiac cell differentiation inhibition as a measure for developmental toxicity of tested chemicals. In the ESTc, a heterogenic cell population is generated besides cardiomyocytes. Using the full biological domain of ESTc may improve the sensitivity of the test system, possibly broadening the range of chemicals for which developmental effects can be detected in the test. In order to improve our knowledge of the biological and chemical applicability domains of the ESTc, we applied a hypothesis-generating data-driven approach on control samples as follows. A genome-wide expression screening was performed, using Next Generation Sequencing (NGS), to map the range of developmental pathways in the ESTc and to search for a predictive embryotoxicity biomarker profile, instead of the conventional read-out of beating cardiomyocytes. The detected developmental pathways included circulatory system development, skeletal system development, heart development, muscle and organ tissue development, and nervous system and cell development. Two pesticidal chemical classes, the morpholines and piperidines, were assessed for perturbation of differentiation in the ESTc using NGS. In addition to the anticipated impact on cardiomyocyte differentiation, the other developmental pathways were also regulated, in a concentration-response fashion. Despite the structural differences between the morpholine and piperidine pairs, their gene expression effect patterns were largely comparable. In addition, some chemical-specific gene regulation was also observed, which may help with future mechanistic understanding of specific effects with individual test compounds. These similar and unique regulations of gene expression profiles by the test compounds, adds to our knowledge of the chemical applicability domain, specificity and sensitivity of the ESTc. Knowledge of both the biological and chemical applicability domain contributes to the optimal placement of ESTc in test batteries and in Integrated Approaches to Testing and Assessment (IATA).

Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Molecular neural crest cell markers enable discrimination of organophosphates in the murine cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) originally used the differentiation of beating cardiomyocytes for embryotoxicity screenings of compounds. However, the ESTc consists of a heterogeneous cell population, including neural crest (NC) cells, which are important contributors to heart development in vivo. Molecular markers for NC cells were investigated to explore if this approach improved discrimination between structurally related chemicals, using the three organophosphates (OP): chlorpyrifos (CPF), malathion (MLT), and triphenyl phosphate (TPP). To decrease the test duration and to improve the objective quantification of the assay read-out, gene transcript biomarkers were measured on study day 4 instead of the traditional cardiomyocyte beating assessment at day 10. Gene expression profiling and immunocytochemistry were performed using markers for pluripotency, proliferation and cardiomyocyte and NC differentiation. Cell proliferation was also assessed by measurements of embryoid body (EB) size and total protein quantification (day 7). Exposure to the OPs resulted in similar patterns of inhibition of beating cardiomyocyte differentiation and of myosin protein expression on day 10. However, these three chemically related compounds induced distinctive effects on NC cell differentiation, indicated by changes in expression levels of the NC precursor (Msx2), NC marker (Ap2α), and epithelial to mesenchymal transition (EMT; Snai2) gene transcripts. This study shows that investigating NC markers can provide added value for ESTc outcome profiling and may enhance the applicability of this assay for the screening of structurally related test chemicals.

Immunotoxicity of poly (lactic-co-glycolic acid) nanoparticles: influence of surface properties on dendritic cell activation.

Modified nanoparticles (NPs) can interact with the immune system by causing its activation to fight tumors or for vaccination. During this activation, dendritic cells (DCs) are effective in generating robust immune response. However, the effect of nanomaterials on dendritic cell (DC) maturation, and the associated adjuvant effect, should be assessed as a novel biocompatibility criteria for biomaterials since immune consequences may constitute potential complications in nanomedicine. Among emerging biomaterials, poly(lactic-co-glycolic acid) NPs (PLGA NPs) are widely explored for various applications in which the degree of desired adjuvant effect may vary. As contradictory results are reported regarding their effects on DCs, we aimed at clarifying this point with particular emphasis on the relative impact of particle surface properties. To that end, NP uptake and effects on the viability, phenotype, and secretory activity of DC primary cultures. Intracellular signaling pathways were explored and evaluated. Immature human and murine DCs were exposed to cationic, neutral, or anionic PLGA NPs. Particle uptake was assessed by both confocal microscopy and flow cytometry. Cell viability was then evaluated prior to the study of maturation by examination of both surface marker expression and cytokine release. Our results demonstrate that PLGA NPs are rapidly engulfed by DCs and do not exert cytotoxic effects. However, upon exposure to PLGA NPs, DCs showed phenotypes and cytokine secretion profiles consistent with maturation which resulted, at least in part, from the transient intracellular activation of mitogen-activated protein kinases (MAPKs). Interestingly, NP-specific stimulation patterns were observed since NP surface properties had a sensible influence on the various parameters measured.

Identification of T-cell epitopes from benzylpenicillin conjugated to human serum albumin and implication in penicillin allergy.

BACKGROUND: There is in vitro evidence that T cells from allergic patients react to benzylpenicillin-human serum albumin (BP-HSA) bioconjugates. Our group has recently shown the existence of naïve CD4+ T cells recognizing BP-HSA in healthy donors. However, BP-haptenated peptides from HSA participating in the immunization of allergic patients have never been identified. The purpose of the present study is to identify immunodominant BP-haptenated peptides from HSA involved in immunization of patients to BP and to refine the frequency calculation of naïve CD4+ T cells recognizing BP. METHODS: Co-cultures were established with CD4+ T cells from non-allergic donors and mature autologous dendritic cells (DCs) loaded with BP-HSA or BP-haptenated peptides from HSA. The CD4+ T-cell response specific for BP-HSA or for individual BP-haptenated peptides was measured using an interferon-γ (IFN-γ) ELISpot assay. The frequency of BP-specific CD4+ T cells was then calculated using the Poisson distribution. BP-HSA and BP-haptenated peptides recognition by allergic patients was evaluated on peripheral blood mononuclear cells (PBMCs) using a lymphocyte transformation test (LTT). RESULTS: Results showed that BP-HSA and BP-haptenated peptides were recognized by naïve T cells from 15/16 and 13/14 tested healthy donors, respectively. Most donors responded to 3 peptides with BP covalently bound on lysines 159, 212, and 525. Two of these benzylpenicilloylated peptides (lysines 159 and 525) were also found to induce PBMCs proliferation in patients with allergic reaction to penicillins. CONCLUSION: This study identifies and characterizes for the first time the BP-haptenated peptides from HSA involved in the immunization of patients to penicillins.

Alteration of Nrf2 and Glutamate Cysteine Ligase expression contribute to lesions growth and fibrogenesis in ectopic endometriosis.

The redox-sensitive nuclear factor erythroid-derived 2-like 2 (NRF2) controls endogenous antioxidant enzymes' transcription and protects against oxidative damage which is triggered by inflammation and known to favor progression of endometriosis. Glutamate Cysteine Ligase (GCL), a target gene of NRF2, is the first enzyme in the synthesis cascade of glutathione, an important endogenous antioxidant. Sixty-one patients, with thorough surgical examination of the abdominopelvic cavity, were recruited for the study: 31 with histologically-proven endometriosis and 30 disease-free women taken as controls. Expressions of NRF2 and GCL were investigated by quantitative RT-PCR and immunohistochemistry in eutopic and ectopic endometria from endometriosis-affected women and in endometrium of disease-free women. Ex vivo stromal and epithelial cells were extracted and purified from endometrial and endometriotic biopsies to explore expression of NRF2 and GCL in both stromal and epithelial compartments by western blot. Finally, in order to strengthen the role of NRF2 in endometriosis pathogenesis, we evaluated the drop of NRF2 expression in a mouse model of endometriosis using NRF2 knockout (NRF2-/-) mice. The mRNA levels of NRF2 and GCL were significantly lower in ectopic endometria of endometriosis-affected women compared to eutopic endometria of disease-free women. The immunohistochemical analysis confirmed the decreased expression of both NRF2 and GCL in ectopic endometriotic tissues compared to eutopic endometria of endometriosis-affected and disease-free women. Immunoblotting revealed a significant decreased of NRF2 and GCL expression in epithelial and stroma cells from ectopic lesions of endometriosis-affected women compared to eutopic endometria from controls. Using a murine model of endometriosis, NRF2-/- implants were more fibrotic compared to wild-type with an increased weight and volume. These findings indicate that expression of the transcription factor NRF2 and its effector GCL are both profoundly deregulated in endometriotic lesions towards increased growth and fibrogenetic processes.

Low dose evaluation of the antiandrogen flutamide following a Mode of Action approach.

The dose-response characterization of endocrine mediated toxicity is an on-going debate which is controversial when exploring the nature of the dose-response curve and the effect at the low-end of the curve. To contribute to this debate we have assessed the effects of a wide range of dose levels of the antiandrogen flutamide (FLU) on 7-week male Wistar rats. FLU was administered by oral gavage at doses of 0, 0.001, 0.01, 0.1, 1 and 10mg/kg/day for 28 days. To evaluate the reproducibility, the study was performed 3 times. The molecular initiating event (MIE; AR antagonism), the key events (LH increase, Leydig cell proliferation and hyperplasia increases) and associated events involved in the mode of action (MOA) of FLU induced testicular toxicity were characterized to address the dose response concordance. Results showed no effects at low doses (<0.1mg/kg/day) for the different key events studied. The histopathological changes (Leydig cell hyperplasia) observed at 1 and 10mg/kg/day were associated with an increase in steroidogenesis gene expression in the testis from 1mg/kg/day, as well as an increase in testosterone blood level at 10mg/kg/day. Each key event dose-response was in good concordance with the MOA of FLU on the testis. From the available results, only monotonic dose-response curves were observed for the MIE, the key events, associated events and in effects observed in other sex related tissues. All the results, so far, show that the reference endocrine disruptor FLU induces threshold effects in a standard 28-day toxicity study on adult male rats.

Standardizing terms, definitions and concepts for describing and interpreting unwanted immunogenicity of biopharmaceuticals: recommendations of the Innovative Medicines Initiative ABIRISK consortium.

Biopharmaceuticals (BPs) represent a rapidly growing class of approved and investigational drug therapies that is contributing significantly to advancing treatment in multiple disease areas, including inflammatory and autoimmune diseases, genetic deficiencies and cancer. Unfortunately, unwanted immunogenic responses to BPs, in particular those affecting clinical safety or efficacy, remain among the most common negative effects associated with this important class of drugs. To manage and reduce risk of unwanted immunogenicity, diverse communities of clinicians, pharmaceutical industry and academic scientists are involved in: interpretation and management of clinical and biological outcomes of BP immunogenicity, improvement of methods for describing, predicting and mitigating immunogenicity risk and elucidation of underlying causes. Collaboration and alignment of efforts across these communities is made difficult due to lack of agreement on concepts, practices and standardized terms and definitions related to immunogenicity. The Innovative Medicines Initiative (IMI; www.imi-europe.org), ABIRISK consortium [Anti-Biopharmaceutical (BP) Immunization Prediction and Clinical Relevance to Reduce the Risk; www.abirisk.eu] was formed by leading clinicians, academic scientists and EFPIA (European Federation of Pharmaceutical Industries and Associations) members to elucidate underlying causes, improve methods for immunogenicity prediction and mitigation and establish common definitions around terms and concepts related to immunogenicity. These efforts are expected to facilitate broader collaborations and lead to new guidelines for managing immunogenicity. To support alignment, an overview of concepts behind the set of key terms and definitions adopted to date by ABIRISK is provided herein along with a link to access and download the ABIRISK terms and definitions and provide comments (http://www.abirisk.eu/index_t_and_d.asp).

Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients.

BACKGROUND: Sublingual immunotherapy (SLIT) applied to type I respiratory allergies is commonly performed with natural allergen extracts. Herein, we developed a sublingual tablet made of pharmaceutical-grade recombinant Bet v 1.0101 (rBet v 1) and investigated its clinical safety and efficacy in birch pollen (BP)-allergic patients. METHODS: Following expression in Escherichia coli and purification, rBet v 1 was characterized using chromatography, capillary electrophoresis, circular dichroism, mass spectrometry and crystallography. Safety and efficacy of rBet v 1 formulated as a sublingual tablet were assessed in a multicentre, double-blind, placebo-controlled study conducted in 483 patients with BP-induced rhinoconjunctivitis. RESULTS: In-depth characterization confirmed the intact product structure and high purity of GMP-grade rBet v 1. The crystal structure resolved at 1.2 Å documented the natural conformation of the molecule. Native or oxidized forms of rBet v 1 did not induce the production of any proinflammatory cytokine by blood dendritic cells or mononuclear cells. Bet v 1 tablets were well tolerated by patients, consistent with the known safety profile of SLIT. The average adjusted symptom scores were significantly decreased relative to placebo in patients receiving once daily for 5 months rBet v 1 tablets, with a mean difference of 17.0-17.7% relative to the group treated with placebo (P < 0.025), without any influence of the dose in the range (12.5-50 µg) tested. CONCLUSION: Recombinant Bet v 1 has been produced as a well-characterized pharmaceutical-grade biological drug. Sublingual administration of rBet v 1 tablets is safe and efficacious in patients with BP allergic rhinoconjunctivitis.

Enhanced glucocorticoid-induced leucine zipper in dendritic cells induces allergen-specific regulatory CD4(+) T-cells in respiratory allergies.

BACKGROUND: Respiratory allergies rely on a defect of IL-10-secreting regulatory CD4(+) T-cells (IL-10-Tregs ) leading to excessive Th2-biased immune responses to allergens. According to clinical data, the restoration of allergen-specific IL-10-Tregs is required to control respiratory allergies and cure patients. The discovery of mechanisms involved in the generation of IL-10-Tregs will thus help to provide effective treatments. We previously demonstrated that dendritic cells (DCs) expressing high levels of the glucocorticoid-induced leucine zipper protein (GILZ) generate antigen-specific IL-10-Tregs . OBJECTIVE: We suspect a defective expression of GILZ in the DCs of respiratory allergic patients and speculate that increasing its expression might restore immune tolerance against allergens through the induction of IL-10-Tregs . METHODS: We assessed GILZ expression in blood DCs of patients and healthy nonallergic donors by qPCR. We compared the ability of patients' DCs to induce allergen-specific IL-10-Tregs before and after an in vivo up-regulation of GILZ expression by steroid administration, steroids being inducers of GILZ. RESULTS: We report lower levels of GILZ in DCs of respiratory allergic patients that return to normal levels after steroid administration. We show that patients' DCs with increased levels of GILZ generate allergen-specific IL-10-Tregs again. We further confirm unequivocally that GILZ is required in patients' DCs to activate these IL-10-Tregs . CONCLUSION: This proof of concept study shows that the re-establishment of GILZ expression in patients' DCs to normal levels restores their capacity to activate allergen-specific IL-10-Tregs . We thus highlight the up-regulation of GILZ in DCs as a new interventional approach to restore the immune tolerance to allergens.

Identification and frequency of circulating CD4(+) T lymphocytes specific to Benzylpenicillin in healthy donors.

BACKGROUND: Drug hypersensitivity is known to rely on a drug-specific T-cell response. Amplitude of antigen-specific T-cell response is partly controlled by the size of the antigen-specific naïve CD4(+) T-cell repertoire, but estimate of this repertoire has never been investigated for allergenic drugs. The purpose of this study was to evaluate the frequency of benzylpenicillin-specific CD4(+) T lymphocytes in healthy donors. METHODS: Co-cultures were established with CD4(+) T lymphocytes from healthy donors and mature autologous dendritic cells loaded with benzylpenicillin coupled to human serum albumin. CD4(+) T lymphocytes were stimulated once a week for 4 weeks with benzylpenicillin coupled to human serum albumin. The CD4(+) T-cell response was measured using an interferon-γ ELISPOT assay. Frequency of benzylpenicillin-specific naive CD4(+) T lymphocytes was then calculated using the Poisson distribution law. RESULTS: Results showed the presence of benzylpenicillin-specific CD4(+) T lymphocytes in 9 of 10 tested healthy donors irrespective of their HLA typing, with a mean frequency of 0.29 cells per million of CD4(+) T cells. Experiments performed on naive (CD45RA(+) ) and on memory (CD45RO(+) ) CD4(+) T lymphocytes showed that these benzylpenicillin-specific CD4(+) T lymphocytes belonged to the naive T-cell subpopulation. CONCLUSION: This study showed for the first time the existence of naive CD4(+) T lymphocytes specific to benzylpenicillin in healthy donors.

Primate testing of TGN1412: right target, wrong cell.

The failure of toxicity studies in non-human primates to predict the cytokine release syndrome during a first-in-man study of the CD28-specific monoclonal antibody TGN1412 has remained unexplained so far. In this issue of the BJP, work from the NIBSC first identifies the effector memory subset of human T-lymphocytes as the most likely source of the pro-inflammatory cytokines released during the study, and goes on to show that in cynomolgus monkeys, this subset lacks CD28, the target molecule of TGN1412. We discuss the implications for the TGN1412 catastrophe and for preclinical evaluation of biologicals in animal models in general.

Monitoring the levels of deoxynivalenol (DON) in cereals in Lebanon and validation of an HPLC/UV detection for the determination of DON in crushed wheat (bulgur).

This paper reports for the first time a method for determining deoxynivalenol (DON) in crushed wheat (bulgur) using an high-performance liquid chromatography (HPLC) method with ultraviolet light detection. Linearity (r (2) > 0.999), selectivity and recovery (70-110%) were acceptable. Results show that the limit of detection (LOD) was 50 µg kg(-1) and the limit of quantification (LOQ) was 80 µg kg(-1). The level of DON was determined in 165 samples of cereals - wheat, forkha (wheat flour special for cake) and bulgur (crushed wheat) - marketed in Lebanon. The results showed that the contamination with DON was 8.75%, 7.5% and 5.0% in bulgur, wheat and forkha, respectively. The LOD and LOQ for wheat (or forkha) were, respectively, 40 and 50 µg kg(-1). The level of DON in all samples was below 1250 µg kg(-1) as recommended by European Union Directives (Commission Regulation (EC) No. 1126/2007), except for one sample of wheat which contained 2307 µg kg(-1). These data suggest that the Lebanese population is exposed to DON through food ingestion at concentrations lower than the tolerable daily intake (TDI) and suggest that measures must be performed routinely to avoid high levels of DON contamination to be found on Lebanese market.

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.

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.

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.

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.

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.

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.

[Mode of action of glucocorticoids]. / Mode d'action des glucocorticoïdes.

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.