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In many domains regulating chemicals and chemical products, there is a legal requirement to determine skin sensitivity to allergens. While many in vitro assays to detect contact hypersensitivity have been developed as alternatives to animal testing over the past ten years and significant progress has been made in this area, there is still a need for continued investment in the creation of techniques and strategies that will allow accurate identification of potential contact allergens and their potency in vitro. In silico models are promising tools in this regard. However, none of the state-of-the-art systems seems to function well enough to serve as a stand-alone hazard identification tool, especially in evaluating the possible allergenicity effects in humans. The Universal Immune System Simulator, a mechanistic computational platform that simulates the human immune system response to a specific insult, provides a means of predicting the immunotoxicity induced by skin sensitisers, enriching the collection of computational models for the assessment of skin sensitization. Here, we present a specific disease layer implementation of the Universal Immune System Simulator for the prediction of allergic contact dermatitis induced by specific skin sensitizers.

We previously reported that the IL-2 Luc LTT can detect immunosuppressive effects of drugs that are attributed to their antimitotic activity. Here, we report an official validation study of the IL-2 Luc LTT. In the Phase I study that evaluated five coded chemicals, the within-laboratory reproducibility of three independent laboratories was 100.0%. In the combined results of the Phase I and II studies that evaluated 20 coded chemicals, the between-laboratory reproducibility was 92.0%. When compared with the reference data based on the previously-reported immunotoxicological information, the predictivity of the combined Phase I and II studies was 76.0% for Lab A and 72.0% for Labs B and C. In contrast, in the study in which the lead laboratory examined 37 non-pharmaceutical chemicals, the predictivity of the IL-2 Luc LTT and the IL-2 Luc assay was 48.6% and 64.9%, respectively, whereas that of the combined assays was 74.3%. It is clear that an integrated approach combining multiple assays is necessary for the development of in vitro immunosuppression testing. These data suggest that the IL-2 Luc LTT alone is not sufficient as a component of the integrated approach, but the combination of the IL-2 Luc assay and IL-2 Luc LTT is promising.

We have previously demonstrated that RACK1, which expression is under steroid hormone control, plays an important role in the activation of immune cells and its expression can be useful to evaluate the immunotoxic profile of endocrine disrupting chemicals (EDCs). Hence, we investigated the effects of three contaminating and persistent pesticides: the fungicide vinclozolin (VIN), the herbicide atrazine (ATR) and the insecticide cypermethrin (CYP) on RACK1 expression and on innate immune response. VIN resulted in modest alteration of RACK1 while ATR and CYP reduced in a dose dependent manner RACK1 expression, ultimately leading to the decrease in lipopolysaccharide-induced IL-8 and TNF-α release and CD86 and CD54 surface marker expression. Moreover, our data indicate that, after exposure to EDCs, alterations of RACK1 expression can also occur with mechanisms not directly mediated by an interaction with a nuclear or membrane steroid receptors. Therefore, RACK1 could represent a useful EDCs screening tool to evaluate their immunotoxic potential and to dissect their mechanisms of action.

We previously demonstrated that based on their potency, contact allergens differently modulate Blimp-1/NLRP12 expression in human keratinocytes, with the extreme allergen 2,4-dinitrochlorobenzene (DNCB) more rapidly upregulating Blimp-1, leading to downregulation of NLRP12, and to the production of interleukin-18 (IL-18). The purpose of this study was to further investigate the effects of DNCB and para-phenylenediamine (PPD) on the expression of the proteins of the inflammasome, namely NLRP3, ASC and caspase 1 by western blot analysis; to define the intracellular localization and co-localization of NLRP3 and NLPR12 by immunoprecipitation and immunohistochemistry; and to define the role of NF-κB in Blimp-1 induction by pharmacological inhibition. The human keratinocyte cell line NCTC2544 was used for all experiments. Dose and time course experiments were performed to evaluate the effect of the selected contact allergens on the parameters investigated. Results indicate, that consistent with previous finding, DNCB more rapidly (3 h) induces NLRP3, ASC protein expression and caspase-1 activation compared to PPD. Immunoprecipitation studies show the recruitment of ASC to the inflammasome following exposure to both allergens, while high level of NLRP12 and less ASC protein were found associated in control cells. By immunohistochemistry, we found increased NLRP3 expression following exposure to contact allergens, and observed a nuclear co-localization of the two proteins, indicating the NLRP12 likely acts preventing the cytosolic localization of NLRP3 and inflammasome assembly. Finally, contact allergen-induced Blimp-1 mRNA and protein expression can be completely blocked by inhibiting NF-κB activation, confirming the central role of NF-κB in contact allergen-induced keratinocyte activation.

Endogenous steroid hormones and endocrine disrupting compounds (EDC) can significantly affect the immune responses. While it is well established that hormonal homeostasis is important for keeping a competent and healthy immune system, the impact of EDC on immunity is less studied. Accumulating evidence indicates that environmental factors, in addition to genetic factors, may contribute to immune-mediated disorders, including hypersensitivity and cancer. Steroid hormones can modulate the function of immune cells, influencing the initiation of the immune response and the maintenance of peripheral tolerance to self-antigens. Hormones, including estrogens, androgens, progestogens, and glucocorticoids may profoundly affect dendritic cells differentiation, maturation and function leading to either a pro-inflammatory or an anti-inflammatory (or tolerogenic) phenotype. If not properly regulated, these processes can lead to immunotoxicity.

Particulate matter (PM) is acknowledged to have multiple detrimental effects on human health. In this review, we report literature results on the possible link between outdoor PM and health outcomes with a focus on pulmonary infections and the mechanisms responsible for observed negative effects. PM physical and chemical properties, such as size and chemical composition, as well as major emission sources are described for a more comprehensive view about the role played by atmospheric PM in the observed adverse health effects; to this aim, major processes leading to the deposition of PM in the respiratory tract and how this can pave the way to the onset of pathologies are also presented. From the literature works here reviewed, two ways in which PM can threaten human health promoting respiratory infectious diseases are mostly taken into account. The first pathway is related to an enhanced susceptibility and here we will also report on molecular mechanisms in the lung immune system responsible for the augmented susceptibility to pathogens, such as the damage of mechanical defensive barriers, the alteration of the innate immune response, and the generation of oxidative stress. The second one deals with the relationship between infectious agents and PM; here we recall that viruses and bacteria (BioPM) are themselves part of atmospheric PM and are collected during sampling together with particles of different origin; so, data should be analysed with caution in order to avoid any false cause-effect relation. To face these issues a multidisciplinary approach is mandatory as also evident from the ongoing research about the mechanisms hypothesized for the SARS-CoV-2 airborne spreading, which is still controversial and claims for further investigation. Therefore, we preferred not to include papers dealing with SARS-CoV-2.

Glyphosate (G) is the active ingredient of the most widely used herbicide products. It targets the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which lacks in humans, suggesting to confer a low mammalian toxicity to G-based herbicides (GBHs). Despite this, the use of G is currently under intense debate. Many studies indicating its hazard and toxicity on non-target organisms are emerging, and associations between GBHs and immune-endocrine disturbances have been described. This review aims to investigate, based on recent epidemiological studies and studies performed in vitro and in vivo in animals, the possible association between GBHs and immune-endocrine alterations. Published data suggest that GBHs have endocrine disrupting potentiality targeting sex and thyroid hormones, although its relevance for humans will require further investigations. Evidence of immunotoxicity are limited compared to those on endocrine effects, but overall highlight possible noxious effects, including lung inflammation and rhinitis. An attractive hypothesis could be the one that connects microbiota dysbiosis with possible immune-endocrine outcomes. Indeed, several intestinal microorganisms express the enzyme EPSPS and, studies are emerging that highlight a possible G-induced dysbiosis. Considering the wide use of GBHs in agriculture, further studies investigating their noxious effects at levels relevant for human exposure should be performed. A critical analysis of emerging evidence of G toxicity is required to better characterize its safety profile. In addition, attention should be paid to the differences between G alone and its formulations, which, containing substances able to increase G absorption, may present a different toxicity profile.

Potassium polyaspartate (A-5D K/SD) is proposed for use as a stabiliser in wine, with a maximum use level of 300 mg/L and typical levels in the range of 100–200 mg/L. Potassium polyaspartate (A-5D K/SD) tested negative in a bacterial reverse mutation assay performed in accordance with OECD TG 471 and in an in vitro mammalian cell micronucleus test performed in accordance with OECD TG 487. From a 90-day oral toxicity study in male and female Wistar rats performed in accordance with OECD TG 408, a no observed adverse effect level (NOAEL) was set at 1000 mg/kg bw per day, the highest dose tested. In its opinion adopted on 9 March 2016, the EFSA-ANS Panel (European Food Safety Authority - Panel on Food Additives and Nutrient Sources added to Food), considering these data, concluded that “there was no safety concern from the proposed use and use levels of potassium polyaspartate (A-5D K/SD)”.

Over the last two decades, substantial progress has been made in the field of in vitro immunotoxicology. Unfortunately, only few in vitro methods have been adopted for routine testing for skin sensitization, while for immunosuppression little has changed in the current practice. In the latter case, current guidelines still rely on animal tests, which include general immune endpoints in repeated dose studies and trigger dedicated tests on a case-by-case basis. To date, only US-EPA requires functional tests for the registration of pesticides. Several in vitro approaches have been proposed for screening and prioritization of chemicals for in vivo testing, which may offer opportunities to replace animal testing in the near future. From the improved understanding of the pathways underlying immunotoxicity induction, new approaches are emerging which should be applied for assessment of the immunomodulatory properties of chemicals and included in integrated testing strategies. The purpose of this review is to give a general overview of in vitro immunotoxicology, dedicated contributions by experts are part of the Special Issue ‘Opportunities and challenges for in vitro assessment of immunotoxicity’.

The Receptor for Activated C Kinase 1 (RACK1) is a scaffold protein for different kinases and membrane receptors. RACK1 can shuttle proteins to their sites of action, facilitate cross-talk among distinct signaling pathways or recruit other signaling proteins into the complexes. Therefore, it is a key mediator of various pathways and is involved in various biological events including development, immune response, brain activity and cancer. Because of its importance, it is of extreme significance to understand the transcriptional mechanisms governing its expression. The identification of regulatory elements in the promoter of RACK1 shed some light on its transcriptional modulation in physiological and pathological context. Literature data support the existence of a complex hormonal balance, between glucocorticoids and androgens, in the control of RACK1 expression due to specific and complex interactions on the RACK1 promoter. These and other informations suggest that a better understanding of RACK1 transcriptional regulation is essential to unravel its role. Furthermore, the modulation of its expression in physiological or pathological conditions may be of interest in different context, such as aging and cancer.

We recently demonstrated the existence of a complex hormonal balance between steroid hormones in the control of RACK1 (Receptor for Activated C Kinase 1) expression and immune activation, suggesting that this scaffold protein may also be targeted by endocrine disrupting chemicals (EDCs). As a proof of concept, we investigated the effect of the doping agent nandrolone, an androgen receptor (AR) agonist, and of p,p'DDT (dichlorodiphenyltrichloroethane) and its main metabolite p,p'DDE (dichlorodiphenyldichloroethylene), a weak and strong AR antagonist, respectively, on RACK1 expression and innate immune response. In analogy to endogenous androgens, nandrolone induced a dose-related increase in RACK1 transcriptional activity and protein expression, resulting in increased LPS-induced IL-8 and TNF-α production and proliferation in THP-1 cells. Conversely, p,p'DDT and p,p'DDE significantly decrease RACK1 expression, LPS-induced cytokine production and CD86 expression; with p,p'DDE exerting a stronger repressor effect than p,p'DDT, consistent with its stronger AR antagonistic effect. These results indicate that RACK1 could be a relevant target of EDCs, responding in opposite ways to agonist or antagonist of AR, representing a bridge between the endocrine system and the innate immune system.

Hypersensitivity drug reactions (HDRs) are common among drugs, despite this, there are no validated in vitro or in vivo methods for screening the sensitizing potential of drugs in the preclinical phase. We previously developed the THP-1 activation assay, based on CD86 upregulation and IL-8 production, for the in vitro identification of drugs able to induce selective dendritic cell activation. In this paper, we investigated the predictive capacity of the method toward drugs associated with HDRs for which a correlation with specific human leukocyte antigens (HLA) have been demonstrated. For that purpose, abacavir, carbamazepine and clozapine were used. Metformin was used as negative control. Dose- and time-course experiments were conducted. The surface markers CD86, CD54 and HLA-DR were evaluated by flow cytometry analysis, whereas IL-8 release by ELISA. Abacavir, carbamazepine and clozapine gave positive results with CD86 upregulation and/or IL-8 release, with abacavir also inducing HLA-DR. The test reveals the ability of drugs to induce dendritic cell activation (signals 1/2), that preceded the adaptive immune response, which will be manifested only in a minority of patients carrying the specific HLA genotypes. The idea is to integrate this simple method during drug development to identify the potential of drugs to induce hypersensitivity reactions in the pre-clinical phase.

Keratinocytes have a key role in all phases of allergic contact dermatitis. We have recently identified the possibility to use IL-18 production for the in vitro identification of contact allergens. The purpose of this study was to characterize the molecular mechanisms underlying allergen-induced IL-18 production, in order to identify the cellular source of reactive oxygen species (ROS) and the danger signals involved. The NCTC2544 cell line was exposed to three contact allergens, namely p-phenylenediamine (PPD), 2,4-dinitrochlorobenzene (DNCB), and citral, in the presence or absence of diphenylene iodonium (DPI), allopurinol, and rotenone to identify the source of ROS, and to anti-Toll-like receptor 4 antibody and glycirrizic acid to characterize the danger-associated molecular pattern molecules. In the case of PPD, the induction of IL-18 can be modulated by rotenone, allopurinol, and DPI. In the case of DNCB, rotenone completely prevents the induction of IL-18, whereas for citral, DPI completely prevents the induction of IL-18. We demonstrated the ability of all allergens tested to induce the release of high-mobility group protein B1 (HMGB1). Its sequester by glycirrizic acid significantly modulates PPD-induced IL-18 production and completely prevents DNCB- and citral-induced IL-18. We found that different intracellular sources of ROS are triggered by contact allergens, and an important role for HMGB1 in chemical allergen-induced IL-18 production was demonstrated.

We demonstrated that cortisol reduces the expression of RACK-1 (Receptor for Activated C Kinase-1), a protein required for immune cell activation. The aim of this study was to evaluate whether and to what extent other clinically relevant corticosteroids may modulate RACK-1 expression. We used the human promyelocytic cell line THP-1 to investigate the effects of cortisol, prednisone, prednisolone, budesonide, betamethasone and methylprednisolone on RACK-1 expression and cytokine production. As anticipated, all corticosteroids inhibited at non-cytotoxic concentrations in a dose and time related manner LPS-induced TNF-α and IL-8 release, with budesonide, betamethasone and methylprednisolone being the most active followed by prednisolone, cortisol and prednisone. To a similar extent, all corticosteroids also reduced RACK-1 mRNA expression and RACK-1 protein levels as assessed by Real Time PCR and Western blot, respectively. Prednisone was the least potent compound while betamethasone and methylprednisolone where the most active. A good correlation was observed between RACK-1 mRNA or protein levels and cytokine release (Pearson r=0.7376, p=0.0471 for RACK-1 mRNA and TNF-α release, and Pearson r=0.8108, p=0.0252 for RACK-1 protein and IL-8 release). Mifepristone, a potent glucocorticoid receptor (GR) antagonist, completely prevented the effect of cortisol, demonstrating that RACK-1 downregulation is via GR. Furthermore, to by-pass the defective PKC activation due to the decrease in RACK-1, we used a RACK-1 pseudosubstrate, that directly activates PKC-beta. RACK-1 pseudosubstrate was able to restore LPS-induced cytokine production affected by cortisol, supporting the role of RACK-1 in the anti-inflammatory effect of corticosteroids. These results confirm the involvement of RACK-1 in immune cell activation and identify this protein as a novel transcriptional target of corticosteroid-induced anti-inflammatory effects.

Purpose Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of keratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously shown that IL-18 production in the human keratinocyte cell line NCTC 2455 is a good model for the in vitro identification of contact sensitizers. The purpose of this manuscript is to summarize data obtained in the NCTC 2544 assay as an in vitro model to identify photoallergens and discriminate them from phototoxic chemicals. Methods The effect of UVA radiation (3.5 J/cm 2 ) over NCTC 2544 cells irradiated and non irradiated, and treated with increasing concentrations of various compounds including negative compounds (irritants and allergens), ibuprofen and acridine (photoirritants); ketoprofen, promethazine and chlorpromazine (photoirritants/photoallergens); benzophenone, 4-tert-butyl-4-methoxy-dibenzoylmethane, 2-ethylexyl-p-methoxycinnamate and 6-methylcumarin (photoallergens) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay, while a commercially available ELISA kit was used to assess the intracellular content of IL-18. Result At no cytotoxic concentrations, allergens and photoallergens induce a dose-related increase in the production of IL-18, whereas irritants and photoirritants failed, indicating the possibility to use the NCTC 2544 assay to identify in vitro photoallergens.

Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to chemical allergens. It describes the adverse effects that may results when exposure to a chemical elicits a T cell-mediated inflammatory skin disease. The ability of contact sensitizers to induce the oxidative stress pathway in keratinocytes and dendritic cells has been confirmed by several authors. Reactive oxygen species (ROS) can serve as essential second messengers mediating cellular responses resulting in immune cells activation. Oxidative stress may be the starter point, as it leads to the activation of transcription factors and signaling pathways, including NF-kB and p38 MAPK, which leads to the release of cytokines and chemokines. ROS are also involved in the activation of the NLRP3/NALP3 inflammasome, which is required to direct the proteolytic maturation of inflammatory cytokines such as IL-1β and IL-18, which are all integral to the process of dendritic cells mobilization, migration and functional maturation. Moreover, emerging evidence correlates ROS to changes in the constitution of the extracellular microenvironment found to facilitate ACD. The purpose of this review is to provide both conceptual and technical frameworks on the role of oxidative stress in chemical allergy.

The immune system can be the target of many chemicals, with potentially severe adverse effects on the host's health. In Western countries pesticides, together with new and modified patterns of exposure to chemicals, have been implicated in the increasing prevalence of diseases associated with alterations of the immune response, such as hypersensitivity reactions, certain autoimmune diseases and cancers. Xenobiotics may initiate, facilitate or exacerbate pathological immune processes, resulting in immunotoxicity by induction of mutations in genes coding for immunoregulatory factors, modifying immune tolerance and activation pathways. The purpose of this article is to update the evidence of pesticide immunotoxicity. Even if experimental data as well as sporadic human studies indicate that some pesticides can affect the immune system, overall, existing epidemiological studies are inadequate to raise conclusions on the immunotoxic risk associated to pesticide exposure. The available studies on the effects of pesticides on human immune system have several limitations including poor indication on exposure levels, multiple chemical exposures, heterogeneity of the approach, and difficulty in giving a prognostic significance to the slight changes often observed. Further studies are necessary, and they should be preferably carried out through comparison of pre and post-exposure findings in the same group of subjects with a matched control group. Attempt should be made to define the prognostic significance of slight changes often observed. Animal and in vitro studies are also important and necessary to scientifically support epidemiological evidences on pesticide-induced immunotoxicity.

Progress in understanding the mechanisms of skin sensitization, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitization testing. Keratinocytes play a key role in all phases of skin sensitization. We have recently identified interleukin-18 (IL-18) production in keratinocyte as a potentially useful endpoint for determination of contact sensitization potential of low molecular weight chemicals. The aim of the present article is to further exploit the performance of the NCTC 2544 assay. NCTC 2544 is a commercially available skin epithelial-like cell line originating from normal human skin, which posses a good expression of cytochrome P450-dependent enzymatic activities. Cells were exposed to contact allergens (2-bromo-2-bromomethyl glutaronitrile, cinnamaldehyde, citral, diethylmaleate, dinitrochlorobenzene, glyoxal, 2-mercaptobenzothiazole, nickel sulfate, 4-nitrobenzylbromide, oxazolone, penicillin G, resorcinol, tetramethylthiuram disulfide), to pre- pro-haptens (cinnamyl alcohol, eugenol, isoeugenol, p-phenylediamine), to respiratory allergens (ammonium hexachloroplatinate, diphenylmethane diisocyanate, glutaraldehyde, hexamethylenediisocyanate, maleic anhydride, trimellitic anhydride) and to irritants (benzaldehyde, cholorobenzene, diethylphtalate, hydrobenzoic acid, lactic acid, octanoic acid, phenol, salicylic acid, sodium lauryl sulphate, sulfamic acid). Cell associated IL-18 was evaluated 24 later by ELISA. At not-cytotoxic concentrations (cell viability higher of 80%, as assessed by MTT reduction assay), all contact sensitizers, including pre-pro-haptens, induced a dose-related increase in IL-18, whereas both irritants, with the exception of sulfamic acid, and respiratory allergens failed. A total of 33 chemicals were tested, with an overall accuracy of 97%. Overall, results obtained indicated that cell-associated IL-18 might provide an in vitro tool for identification and discrimination of contact vs. respiratory allergens and/or irritants.

Several European Union legislations request the use of in vitro methods for toxicological evaluations, including sensitization, in order to increase consumer safety but also to reduce the use of animals. The EU project SENS-IT-IV addresses the need of developing predictive in vitro tests to assess contact and respiratory hypersensitivity reactions. In this context, we have recently reported the possibility to use IL-18 production in the human keratinocyte cell line NCTC 2544 to discriminate contact sensitizer from irritants and low molecular weight respiratory allergens. The aims of the present study were to further develop this assay in order to optimize experimental conditions; to develop a 96-well plate format to establish a high throughput assay; to test the performance of other available keratinocyte cell lines, and to understand the signal transduction pathway involved in p-phenylenediamine (PPD)-induced IL-18 production. If cells reach confluence at the moment of treatment, the ability to identify contact allergens is lost; therefore a careful check for the optimal cell density using PPD as reference contact allergen is critical. In our hands, a cell density of 1-2.5 × 10(5)cells/ml gave optimal stimulation. In order to develop a high throughput test, cells seeded in 96-well plate were exposed to contact allergens (2,4-dinitrochlorobenzene, p-phenylenediamine, isoeugenol, cinnamaldehyde, tetramethylthiuram disulfite, resorcinol, cinnamic alcohol and eugenol), irritants (phenol, sodium laurel sulphate, lactic acid and salicylic acid) and respiratory allergens (hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride). A selective increase in total (intracellular plus released) IL-18 was observed 24h later in cells treated with contact allergens, whereas no changes were observed following treatment with respiratory allergens and irritants, confirming previous results obtained in a 24-well format assay. A selective induction of IL-18 was also obtained testing with PPD other keratinocyte cell lines, namely HPKII and HaCaT, with the HPKII showing the highest stimulation index. Regarding the signal transduction pathway, we could demonstrate using selective inhibitors a role for oxidative stress, NF-κB and p38 MAPK activation in PPD-induced IL-18 production. In conclusion, results obtained suggest that the production of IL-18 represents a promising endpoint for the screening of potential contact allergens. The assay can be performed in a 96-well plate format, different keratinocyte cell lines can be used, and a role for oxidative stress in contact allergen-induced IL-18 was demonstrated.

RACK1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. Previously, we characterized an age-dependent decline of RACK1 protein expression which could be counteracted with DHEA (dehydroepiandrosterone) [Corsini, E., et al. 2002. In vivo dehydroepiandrosterone restores age-associated defects in the protein kinase C signal transduction pathway and related functional responses. J. Immunol. 168, 1753-1758. and Corsini, E., et al. 2005. Age-related decline in RACK-1 expression in human leukocytes is correlated to plasma levels of dehydroepiandrosterone. J. Leukoc. Biol. 77, 247-256.]. Hypothesizing a direct control of RACK1 expression by DHEA we studied the not yet characterized human promoter region of its coding gene GNB2L1. The FLOE (Fluorescently Labeled Oligonucleotide Extension) was used to map the transcription start site and a novel Gateway luciferase vector (GW luc basic; Del Vecchio, I., Zuccotti, A., Canneva, F., Lenzken, S.C., Racchi, M., 2007. Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions. Plasmid 58, 269-274.) to obtain promoter region mutants. Human SH-SY5Y, THP1 and lymphoblastoid cells were used for transient transfections and treatments with lipopolysaccharide (LPS), phorbol myristate acetate (PMA), DHEA and cortisol (the first two molecules to differently activate NF-kB, a transcription complex able to regulate the murine Gnb2l1 gene expression, whereas DHEA and cortisol since they are known to be imbalanced during the aging and possess counteracting actions on the immune function). The primer extension demonstrated the existence of two alternative start sites of transcription respectively located at about 230 and 300 nt 5' of the Genbank mRNA entry for GNB2L1. Moreover, as a result of the luciferase study we were able to demonstrate that a little region of approximately 300 nt conserved sufficient elements for reporter expression. We also reported that the DHEA modulation of GNB2L1 endogenous expression could not be recapitulated with the luciferase assays. Indeed, the promoter was significantly modulated by means of LPS and PMA treatments but not using DHEA. Differently the use of cortisol led us to demonstrate a biologically significant decrease of luciferase activity only in the presence of a binding site for nuclear receptors of glucocorticoids. Interestingly, other binding sites for transcriptional factors were identified in silico: different c-Rel (NF-kB) and some cardiomyocitic specific cis-acting elements. All this data suggest that the DHEA mediated GNB2L1 regulation is modulated by distant elements (enhancers/silencers), whereas LPS, PMA and cortisol effect can act directly on the mapped GNB2L1 promoter. In conclusion we hypothesize that the imbalance between DHEA and cortisol during aging could be important in the previously demonstrated recovery of the RACK1 expression.

Neurological complications as well as movement disorders are relevant symptoms in animals and humans chronically exposed to dithiocarbamates. Using rat pheochromocytoma cells differentiated by NGF (PC12), we investigated whether propineb affects acetylcholine (Ach) release and the molecular mechanisms involved. Propineb(0.001-100 nM)dose-dependently increased Ach release from PC12.Thus, 0.001-1 nM propineb-induced Ach release, reaching a maximal effect (similar to 50%) at 0.1-1 nIV1. Higher concentrations of propineb(10-100 nM) caused a progressive disappearance of the effect. Chelation of extra- and intracellula Ca2+ did not affect Ach release by propineb, which was prevented by the actin stabilizer jasplakinolide (500nM). Accordingly, actin depolymerization was observed after exposure of differentiated PC12 to 0.1-1 nM propineb, a loss of effect was evident at higher concentrations (100nM), and the effect was Ca2+ -independent. Disulfiram, a related dithiocarbamate not coordinated with Zn2+, also depolymerized actin, suggesting the involvement of the organic structure of dithiocarbamates Father than the leakage of Zn2+. Nevertheless, propineb did not depolymerized actin in a cell-free system.These data suggest that dithiocarbamates, through the activation of intracellular cascade(s), impair cytoskeletal actin. This effect may contribute to affect synaptic vesicles processing resulting in an impaired cholinergic transmission, (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Interleukin-1beta is a proinflammatory cytokine implicated under pathological conditions involving NMDA receptor activation, including the AIDS dementia complex (HAD). No information is available on the molecular mechanisms recruited by native interleukin-1beta produced under this type of condition. Using a sandwich co-culture of primary hippocampal neurons and glia, we investigated whether native interleukin-1beta released by HIV-gp120-activated glia (i) affects NMDAR functions and (ii) the relevance on neuronal spine density and survival, two specific traits of HAD. Increased phosphorylation of NR2B Tyr-1472 was observed after 24 h of exposure of neurons to 600 pm gp120. This effect occurred only when neurons were treated in the presence of glial cells and was abolished by the interleukin-1 receptor antagonist (IL-1ra). Gp120-induced phosphorylation of NR2B resulted in a sustained elevation of intracellular Ca(2+) in neurons and in a significant increase of NR2B binding to PSD95. Increased intracellular Ca(2+) was prevented by 10 mum ifenprodil, that selectively inhibits receptors containing the NR2B, by interleukin-1ra and by Ca-pYEEIE, a Src family SH2 inhibitor peptide. These last two inhibitors, prevented also NR2B binding to PSD95. Finally, gp120 reduced by 35% of the total PSD95 positive spine density after 48 h of treatment and induced by 30% of the neuronal death. Again, both of these effects were blocked by Ca-pYEEIE. Altogether, our data show that gp120 releasing interleukin-1beta from glia increases tyrosine phosphorylation of NMDAR. Thus, tyrosine phosphorylation may contribute to the sensitization of the receptor increasing its function and synaptic localization. Both of these effects are relevant for neurodegeneration.

Mancozeb, a polymeric complex of manganese ethylenebisdithiocarbamate with zinc salt, is widely used in agriculture as fungicide. Literature data indicate that ethylenebisdithiocarbamates (EBDTCs) may have immunomodulatory effects in humans. We have recently found in agricultural workers occupationally exposed to the fungicide mancozeb a statistically significant decrease in lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF) production in leukocytes. TNF is an essential proinflammatory cytokine whose production is normally stimulated during an infection. The purpose of this work was to establish an in vitro model reflecting in vivo data and to characterize the molecular mechanism of action of mancozeb.The human promyelocytic cell line THP-1 was used as in vitro model to study the effects of mancozeb and its main metabolite ethylenthiourea (ETU) on LPS-induced TNF release. Mancozeb, but not ETU, at non-cytotoxic concentrations (1-100 mu g/ml), induced a dose- and time-dependent inhibition of LPS-induced TNF release. reflecting, in vivo data. The modulatory effect observed was not limited to mancozeb but also other EBDTCs, namely zineb and ziram, showed similar inhibitory effects. Mancozeb must be added before or simultaneously to LPS in order to observe the effect, indicating that it acts on early events triggered by LPS. It is known that nuclear factor-kappa B (NF-kappa B) tightly regulates TNF transcription. We could demonstrate that mancozeb, modulating LPS-induced reactive oxygen species generation, prevented I kappa B degradation and NF-kappa B nuclear translocation, which in turn resulted in decreased TNF production. To further understand the mechanism of the effect of mancozeb on TNF transcription, THP-1 cells were transfected with NF-kappa B promoter-luciferase construct, and the effect of mancozeb on luciferase activity was measured. Cells transfected with promoter constructs containing kappa B site showed decreased LPS-induced luciferase activity relative to control after mancozeb treatment, confirming NF-kappa B binding as an intracellular target of mancozeb.Overall, this study contributes to our understanding of the mechanism underlying mancozeb-induced immunotoxicity. (c) 2005 Elsevier Inc. All rights reserved.

Erythropoietin (Epo), the principal regulator of erythroids progenitor cells, greatly improves neuronal survival. A wide variety of experimental studies have shown that both Epo and the EpoR are functionally expressed in the nervous system and that this cytokine exerts a remarkable neuroprotection both in vitro against different neurotoxicants and in animal models of nervous system disorders. Aim of this review is to summarize the neuroprotective properties of Epo and to outline the molecular mechanisms involved in order to point out the signal transduction pathway which may represent the potential basis for future strategies against neuronal injury.

Industrialized countries are facing an increase of diseases attributable to an alteration of the immune system function, and concern is growing that this trend could be at least partially attributable to new and modified patterns of exposure to chemicals. Among chemicals matter of concern, pesticides can be included. The Authors have reviewed the existing evidence of pesticide immunotoxicity in humans, showing that existing data are inadequate to raise conclusions on the immunotoxic risk related to these compounds. The limits of existing studies are: poor knowledge on exposure levels, heterogeneity of the approach, and difficulty in giving a prognostic significance to the slight changes often observed. To overcome these limits, the Authors have proposed a tier approach, based on three steps: the first, addressed at pointing out a possible immunomodulation; the second, at refining the results and the third one, when needed, to finalize the study and to point out concordance with previous results. Studies should preferably be carried out through comparison of pre- and post-exposure findings in the same groups of subjects to be examined immediately after the end of the exposure. A simplification of the first step approach can be used by the occupational health physician and the occupational toxicologist. Conclusions on the prognostic significance of the slight changes often observed will be reached only by validating the hypothesis generated by field studies with an epidemiological approach. In this field, the most useful option is represented by longitudinal perspective studies.

In searching for pharmacologic agents able to reduce xenobiotic-induced skin irritation, we found that cyclosporine A exacerbates the skin irritation induced by tributyltin. We previously demonstrated the involvement of interleukin-1 alpha and tumor necrosis factor alpha in tributyltin-induced skin irritation. Here, we show that cyclosporine A (28 mg per kg), at a dose that results in systemic immunosuppression, potentiates tributyltin-induced skin irritation through increased tumor necrosis factor alpha production, associated with increased tributyltin-induced activation of transcription factor nuclear factor kappa B in cyclosporine-A-treated mice. On the other hand, under the same experimental conditions, cyclosporine A prevented the elicitation phase of oxazolone-induced contact allergy, but was ineffective in preventing benzalkonium-chloride-induced skin irritation. Using a murine keratinocyte cell line (HEL30) we demonstrated, also in vitro, that the cyclosporine A potentiates tributyltin-induced nuclear factor kappa B activation and cytokine production, this being preceded by an increase in cellular oxidative activity, essential for nuclear factor kappa B activation, that is time and dose (0.1-10 microM) dependent. This effect was not exclusive to tributyltin but could be extended to other mitochondrial poisons such as sodium arsenate. It has been reported that cyclosporine A binds to cyclophilins. An 18-mer antisense phosphorothioate oligodeoxynucleotide was used to target mitochondrial cyclophilin D mRNA. After 24 h exposure to the oligonucleotide, the amount of cyclophilin D in the cells was decreased by 54% as judged by Western blot analysis. Cyclophilin D suppression prevented cyclosporine A potentiation of tributyltin-induced cellular oxidative activity, indicating the key role of the binding of cyclosporine A to mitochondrial cyclophilin D in mediating this effect.

A progressive decline of cognitive and memory functions, compared to the average young-life performance, characterizes brain aging. The changes in performance may depend upon altered activity of neurotransmitters acting on attention and memory trace formation (acetylcholine, catecholamines, glutamate, for example) or the failure of the transduction mechanisms linked to receptor activation. One of the fundamental cellular changes associated with brain aging is the alteration of mechanisms involving the activity of the calcium-phospholipid-dependent protein kinase C (PKC). A crucial event for the activation of protein kinase C is its translocation from the cytosol to different intracellular sites and recent studies have demonstrated the key role played by several anchoring proteins in this mechanism. The defective activation of PKC-dependent pathways during aging is due to a defective mechanism of translocation of the kinase because of reduced levels of the major anchoring protein RACK-1 (receptor for activated C kinase). Pharmacological strategies aimed at the correction of age-associated memory deficits have been mostly focused on neurotransmitters using direct or indirect agonists. More recently, attention has been paid to the memory enhancing properties of some steroid hormones, namely 'neurosteroids'. Among these the activities of dehydroepiandrosterone (DHEA), pregnenolone (PREG) and their sulfates, have been extensively studied. These neuroactive steroids, can regulate neuronal function through their concurrent influence on transmitter-gated ion channels and gene expression. We addressed the possibility that DHEA, among other neurosteroids, could modulate directly the age-associated impairment of PKC signal transduction and provide experimental evidence that DHEA can revert the alteration of RACK-1 anchoring protein expression.

Exposure to pesticides can cause a number of effects on the immune system, varying from a slight modulation of immune functions to the development of clinical immune diseases. The aim of this study has been reviewing published data on immune effects of pesticides in humans, with particular attention for effects observed in absence of any other change, and to the possibility of identifying a dose effect relationship. Some evidence of immunotoxic effects in man involve organophosphorus compounds, some organochlorine insecticides (OC), some carbamates, some phenoxy herbicides, dithiocarbamates, and pentachlorophenol (PCP). The alterations are usually observed in absence of any other change; in some cases, data suggest the presence of a dose effect relationship. The prognostic significance of the observed changes is still unclear. The Authors propose a tier approach to assess immune effects in humans.

Tributyltin (TBT) salts are potent skin irritants both in humans and rodents. Data in the literature indicate mitochondria as target of TBT effects. The purpose of this study was to investigate the early intracellular molecular events that follow TBT treatment and mitochondrial impairment and the relevance of mitochondria in gene-regulatory signalling pathways. The results obtained indicate the pivotal role of Ca2+ as the starting event following TBT treatment and confirm the important role of mitochondria as the source of second messenger molecules essential for TBT-induced NF-κB activation and IL-1α production.

Cytokines may be useful tools to discriminate between irritant and allergic contact dermatitis. In the mouse only, it has been demonstrated by other, that contact sensitizers up-regulated keratinocytes-derived interleukin-1alpha (IL-1), macrophage inflammatory protein-2 and interferon induced protein 10 mRNAs. The purpose of this study was to investigate the possibility to use in vitro IL-1 production by a murine keratinocyte cell line for preliminary screening of chemicals for their irritant and/or allergic potential. We investigated the effects of five relevant skin allergens (dinitrochlorobenzene, oxazolone, nickel sulfate, penicillin G and eugenol), two skin irritants (benzalkonium chloride, and methylsalicilate) and two compounds with no sensitizing activity (glycerol and ethanol) on IL-1 production in HEL30 cells. Twenty four hours following treatment, both IL-1 release in conditioned media and cell-associated IL-1 were measured by a specific sandwich ELISA. Under our experimental conditions, only contact sensitizers were able to increase in a dose dependent fashion cell-associated IL-1, confirming the in vivo findings. Both skin irritants and allergens induced the release of IL-1, because of the irritative properties of both chemicals, while ethanol and glycerol failed to induce changes in IL-1 production, confirming the specificity of the proposed test. Taken together, these data indicate that it may be realistic to consider potential skin allergens those chemicals which are able to increase cell-associated IL-1, to consider skin irritants those chemicals which induce only IL-1 release, and to exclude as potential allergens or irritants those chemicals which fail to induce changes in IL-1 production.

Skin irritant reactions are under the control of a network of cytokines and lipid mediators. This study characterized the production of tumor necrosis factor-alpha (TNF) induced by a skin irritant treatment, tributyltin (TBT), in mice through transcription factor activation and its pharmacologic modulation by anti-inflammatory agents. The ears of BALB/c mice were painted with different amounts of TBT (67-536 nmol in acetone) or with acetone alone. At different times thereafter, TNF production was analyzed both at the mRNA and protein level, by semiquantitative RT-PCR and L929 cytotoxicity assay, respectively. TBT induced rapid (1 h) TNF gene expression and protein synthesis. Maximal TNF production was observed 2 h after treatment. The production of TNF was paralleled by accumulation of skin water; this was partially prevented by intraperitoneal injection of antibody against murine TNF. These data indicate that skin irritation induced by TBT is attributable, in addition to the actions of other inflammatory mediators, to the action of keratinocyte-derived TNF. TNF production was preceded by a rapid (5 min) activation of nuclear factor-kappaB (NF-kappaB), which was also maximal 30 min after treatment. TBT-induced accumulation of skin water and TNF production were significantly reduced by topical treatment with dexamethasone and pentamidine, two anti-inflammatory agents. Interestingly, dexamethasone, but not pentamidine, decreased TBT-induced NF-kappaB activation, confirming in vivo that the glucocorticoid receptor interacts functionally within the nucleus with other transcription factors opposing one another's activity.

Trisubstituted organotin pesticides are lethal for different cell types. In this study we investigated whether triphenyltin chloride (TPT) causes apoptosis in HL-60 promyelocytic cells and, if so, by what mechanisms. We report that 5 microM TPT increased intracellular Ca2+ in HL-60 cells within seconds; concomitantly actin depolymerization was detected 30 s and 1 min after the treatment. This was followed 15 min later by NF-kappaB activation, and apoptotic bodies and DNA fragmentation were evident after 3 and 6 h, respectively. At these times TPT also induced the release of tumor necrosis factor-alpha (TNF-alpha). Prior treatment of the cells with a polyclonal antibody to human TNF-alpha abolished TPT-induced DNA fragmentation, which suggests that the ultimate effect of TPT may be mediated by TNF-alpha. Prior treatment of the cells with 100 microM pyrrolidine dithiocarbamate, an antioxidant and potent inhibitor of NF-kappaB activation, prevented actin depolymerization, NF-kappaB activation, and DNA fragmentation, although it did not affect TPT-induced Ca2+ mobilization. These findings suggest that TPT increases intracellular Ca2+, alters actin polymerization and the cytoskeleton, and induces NF-kappaB activation, TNF-alpha synthesis, DNA degradation, and apoptosis. Reactive oxygen species seem to be essential to NF-kappaB activation, TNF-alpha synthesis, and the subsequent steps.

A full-length cDNA clone for human thyroid peroxidase inserted into the mammalian cell expression vector pECE was stably transfected into the rat thyroid cell line FRTL5. Clones expressed immunologically and enzymatically assessed human thyroid peroxidase protein. Methimazole (25 microM) inhibited the thyroid peroxidase activity dose-dependently and this effect was completely antagonised by 100 microM NaI. Ethylenethiourea, metabolite of dithiocarbamate pesticides, inhibited the enzyme at 50 microM. Thus, we have obtained thyroidal cells stably expressing enzymatically active human thyroid peroxidase which can be pharmacologically modulated and studied.

Risk Assessment in Immonotoxicology. II. Relationships between Immune and Host Resistance Tests. Luster, M. I., Portier, C., Pait, D. G., Rosenthal, G. J., Germolec, D. R., Corsini, E., Blaylock, B. L., Pollock, P., Kouchi, Y., Craig, W., White, K. L., Munson, A. E., and Comment, C. E. (1993). Fundam. Appl. Toxicol. 21, 71-82. We have reported on the design and content of a screening battery using a "tier" approach for detecting potential immunotoxic compounds in mice (Luster et al., Fundam. Appl. Toxicol. , 10, 2-19, 1988). The data base generated from these studies, which consists of over 50 selected compounds, has been collected and analyzed in an attempt to improve future testing strategies and provide information to aid in developing future quantitative risk assessment for immunotoxicity. In a recent study it was shown that as few as two or three immune parameters were needed to predict immunotoxicants in mice (Luster et al., Fundam. Appl. Toxicol., 18, 200-210, 1992). In particular, enumeration of lymphocyte populations and quantitation of the T-dependent antibody response were particularly beneficial. Furthermore, commonly employed apical measures (e.g., leukocyte counts, lymphoid organ weights) were fairly insensitive. The present analyses focus on the use of this data base to develop statistical models that examine the qualitative and quantitative relationship(s) between the immune function and host resistance tests. The conclusion derived from these analyses are: (1) A good correlation exists between changes in the immune tests and altered host resistance in that there were no instances where host resistance was altered without affecting an immune test(s). However, in some instances immune changes occurred without corresponding changes in host resistance. (2) No single immune test could be identified which was fully predictive for altered host resistance, although most assays were relatively good indicators (i.e., >70%). Several others, such as proliferative response to lipopolysaccharide and leukocyte counts, were found to be relatively poor indicators for host resistance changes. (3) The ability to resist infectious agent challenge is dependent upon the degrees of immunosuppression and the quantity of infectious agent administered. (4) Logistic and standard regression modeling using one extensive chemical data set from the immunosuppressive agent, cyclophosphamide, indicated that most immune function-host resistance relationships followed linear rather than linear-quadratic (threshold-like) models. For most of the relationships this could not be confirmed using a large chemical data set and, thus, a more mechanistically based approach for modeling will need to be developed. (5) Using this limited data set, methods were developed for modeling the precise quantitative relationships between changes in selected immune tests and host resistance tests.

Pneumocystis carinii , and the inflammatory response it provokes, together contribute to irreversible lung damage in immunocompromised patients. P. carinii cysts were found to be capable of inducing tumor necrosis factor-α (TNF) release from alveolar macrophages in a concentration-dependent manner. At physiologically achievable concentrations, pentamidine isethionate (pentamidine) substantially reduces such production. Pretreatment of alveolar macrophages (AMO) with interferon-γ (IFN-γ) synergizes with P. carinii to produce increased levels of TNF, a condition which pentamidine was also able to antagonize. Pentamidine treatment did not interfere with the phagocytic ability of AMO. Considering clinical reduction of TNF could lessen P. carinii pneumonia (PCP) induced inflammation, the efficacy of pentamidine in the treatment of PCP may be partially associated with its ability to inhibit the release of inflammatory mediators such as TNF.

Pentamidine isethionate, an antiprotozoal agent with therapeutic value against Pneumocystis carinii pneumonia, has been used for over 30 years without a precise understanding of its mechanism of pharmacologic action. We have previously reported that pentamidine has the capacity to inhibit the release of cytokines from macrophages through a post-translational processing event. The present studies were undertaken to assess the ability of pentamidine to modulate the detrimental effects of murine endotoxemia, a disease with a pathophysiology clearly linked to host-produced cytokines. Under conditions where normal B6C3F1 mice succumbed to the lethal effects of endotoxin, mice pretreated with pentamidine were significantly protected from both mortality and loss of thermoregulatory control. The EC50 for protection from mortality by pentamidine was approximately 11.4 mg/kg. These observations correlated with decreased serum levels of tumor necrosis factor (TNF) and interleukin 6. Inhibition of cytokines was not manifested as part of a generalized inhibition of protein synthesis as demonstrated by the lack of significant modulation of serum albumin in pentamidine-treated animals. In addition to decreased serum concentrations of cytokines, lungs isolated from mice treated with both pentamidine and endotoxin exhibited a decreased release of TNF compared to lungs isolated from mice treated with vehicle and endotoxin. The lower levels of TNF released from lung tissue in pentamidine-treated mice correlated with a lesser degree of alveolar deterioration than was observed in vehicle-treated mice. These data indicate that following endotoxin administration, pentamidine has a protective and antiinflammatory role both systemically and in the lung and suggest that inhibition of inflammatory cytokines may be one mechanism operable in the therapeutic activity of the drug against P carinii pneumonia.