A genetic profile of oxytocin receptor improves moral acceptability of outcome-maximizing harm in male insurance brokers.

In recent years, conflicting findings have been reported in the scientific literature about the influence of dopaminergic, serotonergic and oxytocinergic gene variants on moral behavior. Here, we utilized a moral judgment paradigm to test the potential effects on moral choices of three polymorphisms of the Oxytocin receptor (OXTR): rs53576, rs2268498 and rs1042770. We analyzed the influence of each single polymorphism and of genetic profiles obtained by different combinations of their genotypes in a sample of male insurance brokers (n = 129), as compared to control males (n = 109). Insurance brokers resulted significantly more oriented to maximize outcomes than control males, thus they expressed more than controls the utilitarian attitude phenotype. When analyzed individually, none of the selected variants influenced the responses to moral dilemmas. In contrast, a composite genetic profile that potentially increases OXTR activity was associated with higher moral acceptability in brokers. We hypothesize that this genetic profile promotes outcome-maximizing behavior in brokers by focusing their attention on what represents a greater good, that is, saving the highest number of people, even though at the cost of sacrificing one individual. Our data suggest that investigations in a sample that most expresses the phenotype of interest, combined with the analysis of composite genetic profiles rather than individual variants, represent a promising strategy to find out weak genetic influences on complex phenotypes, such as moral behavior.

Enterodiol and enterolactone modulate the immune response by acting on nuclear factor-kappaB (NF-kappaB) signaling.

Lignan-rich whole-grain cereals, beans, berries, and nuts show protective effects against a variety of chronic diseases, including cancer. Lignans are converted by intestinal microflora to enterolactone (EL) and its oxidation product enterodiol (ED). To investigate the immunomodulatory effect of EL and ED in human cells, peripheral blood lymphocytes were treated with increasing physiologically relevant concentrations of EL and ED (0-1000 microM) and stimulated with lipopolysaccharide (LPS) and anti-CD3 plus anti-CD28 monoclonal antibodies. A dose-related inhibition of cell proliferation and cytokine production was observed, with EL being the most active. Molecular investigations in THP-1 cells showed that both EL and ED prevented inhibitory-kappaB (I-kappaB) degradation and nuclear factor-kappaB (NF-kappaB) activation, which in turn resulted in decreased tumor necrosis factor-alpha (TNF-alpha) production. EL and ED were also able to pass the intestinal barrier and modulate cytokine production. The findings of the present study reveal potential mechanisms that could explain some in vivo beneficial effects of lignans.

Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens.

Assessment of allergenic potential of chemicals is performed using animal models, such as the murine local lymph node assay, which does not distinguish between respiratory and contact allergens. 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. The aim of the present study was to evaluate the possibility to use intracellular interleukin-18 (IL-18) production to assess in vitro the contact sensitization potential of low molecular weight chemicals. The human keratinocyte cell line NCTC2455 was used. Cells were exposed to contact allergens (cinnamaldehyde, dinitrochlorobenzene, glyoxal, isoeugenol, p-phenylediamine, resorcinol, tetramethylthiuram disulfide, 2-mercaptobenzothiazole, 4-nitrobenzylbromide), to proaptens (cinnamyl alcohol, eugenol), to respiratory allergens (diphenylmethane diisocyanate, trimellitic anhydride, ammonium hexachloroplatinate) and to irritants (sodium lauryl sulphate, salicylic acid, phenol). Cell associated IL-18 were evaluated 24 later. At not cytotoxic concentrations (cell viability higher of 75%, as assessed by MTT reduction assay), all contact sensitizers, including proaptens, induced a dose-related increase in IL-18, whereas both irritants and respiratory failed. Similar results were also obtained using primary human keratinocytes. Results were reproducible, and the method could be transferred to another laboratory, suggesting the potential use of the test in immunotoxicity testing strategies. Overall, results obtained indicated that cell-associated IL-18 may provide an in vitro tool for identification and discrimination of contact versus respiratory allergens and/or irritants.

Role of p38 MAPK in the selective release of IL-8 induced by chemical allergen in naive THp-1 cells.

At present, the assessment of the allergenic potential of chemicals is carried out using animal models. Over the last decade, several in vitro methods mainly using primary dendritic cells have been proposed to identify the potential of chemicals to induce skin sensitization to meet current animal welfare and public opinions. The major limitations of such tests are the donor-to-donor variability, the low levels in the source, and a possible shortage of human sources. The aim of the present investigation was to establish an in vitro test to identify chemical allergens using the human promyelocytic cell line THP-1 in order to avoid some of these difficulties. We investigated whether the chemokine interleukin-8 or CXCL8 (IL-8) production could provide a methodology for the detection of both respiratory and contact allergens. THP-1 cells were exposed to contact allergens (cinnamaldehyde, dinitrochlorobenzene, nickel sulfate, penicillin G, p-phenylenediamine, tetramethylthiuram disulfide), to respiratory allergens (ammonium hexachloroplatinate, diphenylmethane diisocyanate, trimellitic anhydride) and to irritants (salicylic acid, phenol, sodium lauryl sulphate). Following 48 h of incubation, the release of IL-8 was evaluated by sandwich ELISA. IL-8 production was significantly increased after stimulation with all allergens tested, with the exception of trimellitic anhydride, whereas irritants exposure failed to induce IL-8 release. The lack of IL-8 production by trimellitic anhydride can be explained by the rapid hydrolysis of this chemical in water to trimellitic acid, which is not an allergen. In contrast to IL-8 release, CD54 and CD86 expression did not provide a sensitive method failing to correctly identify approximately 30% of the tested compounds. Although CD86 appears to be a more sensitive marker than CD54 when discriminating allergens from irritants neither of these markers provided robust methodology. We also investigated if a common activation pathway in allergen-induced IL-8 production involving p38 mitogen-activated protein kinase could be identified. By Western blot analysis we could indeed demonstrate p38 activation by all chemical allergens tested and, using the selective p38 MAPK inhibitor SB203580, a significant modulation of allergen-induced IL-8 release could be achieved in all cases. Our data suggests that production of IL-8 by naïve THP-1 cells may represent a promising in vitro model for the screening of potential chemical allergens and activation of p38 MAPK represents a common pathway triggered by allergens.

Immunomodulatory effects of the herbicide propanil on cytokine production in humans: In vivo and in vitro exposure.

Propanil, 3,4-dichloropropionanilide, a commonly used herbicide, has been shown to induce effects on the mouse immune system. The aim of this study was to assess the immunotoxicity of propanil in occupationally exposed agricultural workers and to characterize its molecular mechanism of action. Seven agricultural workers intermittently exposed to propanil and 7 healthy matched controls entered the study. Data were collected through physical examination, and laboratory investigations addressed at the main serum, cellular, and functional immune parameters. The levels of exposure were assessed by determining the urine concentration of the major propanil metabolite, 3,4-dichloroaniline. The investigation of serum, cellular, and functional immune parameters suggested that propanil exposure results in a modest immunomodulatory effect, characterized by an increase in the plasma level of IgG(1) and in LPS-induced IL-6 release and, by a reduction in PHA-induced IL-10 and IFN release, associated with a reduced IFN/IL-4 ratio. As observed, following in vivo exposure, in vitro treatment of human peripheral blood leukocytes with propanil resulted in a dose-dependent reduction in PHA-induced IFN-gamma and IL-10 production, while LPS-induced TNF-alpha production was not affected indicating a direct effect of propanil on selected immune parameters. We demonstrated that propanil interfering with PHA-induced intracellular calcium increase modulated IL-10 and IFN-gamma transcription and translation, which indicates that propanil acts on early events triggered by PHA. Overall, our results suggest that human exposure to propanil has slight immunomodulatory effects, and point out that the inhibition of the PHA-induced intracellular calcium rise is an important target of propanil. These findings improve our understanding of the mechanism underlying propanil-induced immunotoxicity.

Role of SP-1 in SDS-induced adipose differentiation related protein synthesis in human keratinocytes.

Skin irritation is a complex phenomenon, and keratinocytes play an important role in it. We have recently characterized the expression and protective role of adipose differentiation related protein (ADRP) in skin irritation. In particular, ADRP expression is induced to recover functional cell membrane following the cell damage caused by skin irritants. The purpose of this study was to characterize in a human keratinocyte cells line (NCTC 2544) the biochemical events that lead to ADRP expression following SDS treatment, and in particular, to investigate the role of transcription factor SP-1. Analysis of ADRP promoter region revealed the presence of a potential binding site for the transcription factor SP-1 close to the start site. Evaluated by measuring the DNA binding activity, we found that SDS induced a dose and time related SP-1 activation, which was correlated with SDS-induced ADRP mRNA expression. Furthermore, SDS-induced SP-1 activation, ADRP mRNA expression and lipid droplets accumulation could be modulated by mithramycin A, an antibiotic that selectively binds to the GC box preventing SP-1 binding and gene expression. This demonstrated that SDS-induced ADRP expression was mediated in part through the transcription factor SP-1. In addition, SDS-induced SP-1 activation and ADRP expression could be modulated by the calcium chelator BAPTA, indicating a role of calcium in ADRP-induction. Thus, every time an irritant perturbs the membrane barrier, it renders the membrane leaky and allows extracellular calcium to enter the cells, an event that provides the upstream mechanisms initiating the signaling cascade that triggers the activation of SP-1 and culminates in the enhancement of ADRP expression, which helps to restore the normal homeostasis and ultimately repairs the to membrane.

High interleukin-10 production is associated with low antibody response to influenza vaccination in the elderly.

The present study was designed to determine the correlation among dehydroepiandrosterone (DHEA), cortisol plasma levels, and immune functionality at the time of vaccination with antibody response to influenza vaccination in young and old, healthy volunteers. Fifty-two elderly subjects, ages 63-85 years, and 14 young subjects, ages 26-41 years, entered the study. Plasma levels of DHEA and cortisol and in vitro cytokine production in response to lipopolysaccharide (LPS) and phytohaemagglutinin (PHA) by peripheral blood leukocytes were assessed at the time of vaccination, and antibody titer was measured before and 18 days after influenza virus vaccination. Elderly subjects were characterized by an increase in the cortisol:DHEA ratio, mainly as a result of a decrease in DHEA. A decrease in LPS-induced tumor necrosis factor alpha (TNF-alpha), increased PHA-induced interleukin-10 (IL-10) release, and similar PHA-induced interferon-gamma production were observed in elderly subjects compared with young volunteers. Lower antibody titer to influenza A virus was observed in elderly individuals, and the seroconversion factor was found to be correlated inversely with IL-10 production and correlated directly with TNF-alpha production and to a lesser extent, with the plasma level of DHEA. These results suggest that altered cytokine production in elderly subjects at the moment of vaccination can be predictive of a low response to influenza vaccination and warrant the study of strategies to improve protection afforded by the use of vaccines.

Molecular mechanisms underlying mancozeb-induced inhibition of TNF-alpha production.

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 microg/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-kappaB (NF-kappaB) tightly regulates TNF transcription. We could demonstrate that mancozeb, modulating LPS-induced reactive oxygen species generation, prevented IkappaB degradation and NF-kappaB 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-kappaB promoter-luciferase construct, and the effect of mancozeb on luciferase activity was measured. Cells transfected with promoter constructs containing kappaB site showed decreased LPS-induced luciferase activity relative to control after mancozeb treatment, confirming NF-kappaB binding as an intracellular target of mancozeb. Overall, this study contributes to our understanding of the mechanism underlying mancozeb-induced immunotoxicity.

Increased carrageenan-induced acute lung inflammation in old rats.

Ageing is associated with increased susceptibility to lung infections and delayed resolution of pulmonary infiltrates. The purpose of this study was to investigate the effect of age on the onset of carrageenan-induced lung inflammation. When compared with carrageenan-treated young rats (3 months old), old rats (>18 months old) exhibited a preponderance of pleural exudation and polymorphonuclear cell infiltration. Lung myeloperoxidase activity, an index of neutrophil infiltration and activation, was significantly increased in old rats in comparison with young rats. Consistent with the biochemical markers of inflammation, increased lung damage, as assessed by nitrosative stress and lipid peroxidation, was observed in carrageenan-treated old rats. In the lung exudate obtained from old rats, a significant reduction in interleukin-10 (IL-10) was observed, while similar expression of monocyte chemotactic protein-1 was induced, suggesting that a decrease in IL-10 rather than increased chemotaxis may account for the preponderance of the inflammatory cellular infiltrate in old rats. Similar to the in vivo situation, freshly isolated alveolar macrophages obtained from old rats produced less IL-10. This defective IL-10 production could be explained by a reduction in the cAMP-dependent signalling pathway, which mediates IL-10 production. Indeed, we found decreased cAMP-responsive element binding protein (CREB) and phosphorous-CREB (P-CREB) expression in old rats, which may account for reduced IL-10 production in old rats.

Age-related decline in RACK-1 expression in human leukocytes is correlated to plasma levels of dehydroepiandrosterone.

Aging is associated with remodeling of the immune system, contributing to increased incidence of infections, autoimmune diseases, and cancer among the elderly. Alterations in several signal transduction pathways have been reported to play an important role in immunosenescence. We show that peripheral blood leukocytes obtained from old donors (> or =65 years) have a significantly reduced expression of receptor for activated C kinase 1 (RACK-1), a protein required for protein kinase C (PKC)-beta signaling, as compared with young donors (< or =40 years), both in males and females. The decline in RACK-1 immunoboth in reactivity was age-related (Spearman correlation, r=-0.278, P=0.012). All leukocyte subpopulations, namely lympho-monocytes, granulocytes, and B and T cells, showed a similar defect. We also observed a direct correlation between circulating dehydroepiandrosterone (DHEA) and RACK-1 expression in leukocytes (Spearman correlation, r=0.388, P=0.001). Furthermore, in vitro treatment with DHEA resulted in increased RACK-1 expression in leukocytes and lymphocyte proliferation, confirming the role of this hormone in the modulation of its expression and immune functions. A relevant consequence of RACK-1-reduced expression was the observation that release of tumor necrosis factor alpha following lipopolysaccharide challenge and mitogen-induced lymphocye proliferation, which involves PKC-beta activation, was significantly reduced in elderly subjects. Overall, our findings contribute to the understanding of the complex process of immunosenescence and identify age-related loss in immunological responses as partially associated with decreased RACK-1 expression.

Resistance to acute silicosis in senescent rats: role of alveolar macrophages.

We have previously demonstrated in alveolar macrophages that aging is associated with a decline in lipopolysaccharide-induced tumor necrosis factor-alpha production. The purpose of the present study was to investigate the immunotoxicological consequences of this defective activation in an experimental model of acute silicosis. Young (3 months old) and old (>18 months old) rats were intratracheally instilled with silica or saline as control. In young animals, as expected, silica induced a significant increase in bronchoalveolar lavage fluid of tumor necrosis factor-alpha, lactate dehydrogenase, and cell numbers, which correlated with increased collagen deposition and silicotic nodule formations. On the contrary, in old rats, no changes in bronchoalveolar lavage fluid or lung parameters were observed, indicating that senescent rats are resistant to the acute effects of silica. These in vivo results were confirmed in vitro, where silica-induced tumor necrosis factor-alpha release was drastically reduced in alveolar macrophages obtained from old animals. This could be explained with a defective protein kinase C betaII translocation in aged macrophages, due to decreased expression of its anchoring protein RACK-1. Furthermore, a decrease in FAS-L expression and silica-induced apoptosis in old macrophages was observed, supporting the idea that age-associated alterations in signal transduction pathways contribute to decreased sensitivity to silica-induced acute lung fibrosis in old animals.

Induction of adipose differentiation related protein and neutral lipid droplet accumulation in keratinocytes by skin irritants.

Keratinocytes play an important role in skin irritation. In an attempt to investigate mechanistic bases of human skin irritation response, we recently identified the upregulation by skin irritants of adipose differentiation related protein (ADRP) in reconstituted human epidermis. ADRP is a lipid-storage-droplet-associated protein, governing deposition and release of lipids from droplets. The purpose of this study was to characterize, in a human keratinocyte cell line (NCTC 2544), sodium-dodecyl-sulfate-induced ADRP expression, to identify the biochemical events that lead to ADRP expression, and to understand its function in sodium dodecyl sulfate cytotoxicity. Sodium dodecyl sulfate induced a concentration- and time-related production of ADRP that was associated with lipid droplet accumulation. Lipid accumulation following sodium dodecyl sulfate treatment was due to intracellular redistribution rather than lipid neosynthesis, as indicated by equivalent 14C-oleate and 14C-acetate incorporations. Other skin irritants, namely benzalkonium chloride, tributyltin, and 12-O-tetradecanoylphorbol 13-acetate, also induce lipid droplet accumulation. Sodium-dodecyl-sulfate-induced ADRP expression and lipid droplet accumulation were modulated by the calcium chelator BAPTA, indicating a role of calcium in ADRP induction. Decrease of sodium-dodecyl-sulfate-induced ADRP expression by specific ADRP antisense oligonucleotide resulted in increased cytotoxicity, indicating a protective role of ADRP and lipid accumulation in the process of cell damage induced by skin irritants. ADRP expression was also induced in vivo following treatment with sodium dodecyl sulfate in an experimental model of skin irritation, indicating that the in vitro model represents irritation.

The anti-inflammatory activity of estrogen in glial cells is regulated by the PKC-anchoring protein RACK-1.

It has recently been suggested that estrogen inhibits glial activation and the release of neurotoxic mediators. The mechanisms involved in this anti-inflammatory effect are unclear. We found that an nM concentration of 17-beta estradiol inhibits protein kinaseC-betaII translocation induced by lipopolysaccharide in primary astrocytes. Estradiol treatment did not change the total content of kinaseC-betaII or of lipopolysaccharide receptor, but dose-dependently reduced the levels of receptors for activated C kinases-1 (RACK-1), the anchoring protein involved in protein kinase C (PKC) shuttling. This decrease could thus account for the defective protein kinaseC-betaII activation. Pre-treatment with 1 nmbeta-estradiol, which reduced by approximately 35% the expression of RACK-1, prevented the lipopolysaccharide-induced expression of tumour necrosis factor-alpha mRNA and of the inducible form of nitric oxide (NO) synthase. As a consequence, the production of tumour necrosis factor-alpha and NO were decreased. An antisense oligonucleotide for RACK-1 also reduced tumour necrosis factor-alpha and nitric oxide production on lipopolysaccharide stimulation. These results demonstrate that estrogen reduction of the RACK-1 expression, leading to a defective protein kinase-C activation counteracts the inflammatory response in astrocytes.

In vivo dehydroepiandrosterone restores age-associated defects in the protein kinase C signal transduction pathway and related functional responses.

Elderly subjects are at increased risk of pneumonia, influenza, and tuberculosis. Besides the known age-related decrease in mechanisms for mechanical clearance of the lungs, impaired alveolar macrophage function contributes to the increased risk of illness in the elderly. We have previously shown that age-induced macrophage immunodeficiencies are associated with a defective system for anchoring protein kinase C. Castration of young male rats produces effects on alveolar macrophages similar to those of aging, suggesting a relationship between circulating sex hormones, particularly androgens, and the decreases in the receptor for activated C kinase (RACK-1) and macrophage function observed. The aging process in humans and rats is associated with a decline in the plasma concentrations of dehydroepiandrosterone (DHEA) and its sulfate, among other steroid hormones. We report here that in vitro and in vivo administration of DHEA to rats restores the age-decreased level of RACK-1 and the LPS-stimulated production of TNF-alpha in alveolar macrophages. DHEA in vivo also restores age-decreased spleen mitogenic responses and the level of RACK-1 expression. These findings suggest that the age-related loss in immunological responses, linked to defective pathways of signal transduction, are partially under hormonal control and can be restored by appropriate replacement therapy.