Laura Lucchi

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.

Aging process affects a single class of dopamine receptors

[3H]Spiroperidol and [3H](-)-sulpiride specific binding have been used to assay for D1 and D2 dopaminergic recognition sites in striatal membranes of aged rats. While [3H]spiroperidol binding shows a decreased number of binding sites, no changes have been detected in [3H](-)-sulpiride binding, which is a marker for D2 dopaminergic receptors. Data obtained with GTP and DA-dependent adenylyl cyclase activity confirm the hypothesis that aging selectively affects in rats those dopaminergic receptors coupled to the formation of cyclic AMP (D1).

Protein kinase C activity, translocation, and conventional isoforms in aging rat brain.

Protein kinase C was studied in various brain areas in aging Wistar rats. Histone-directed kinase activity from the cortex, hippocampus and cerebellum did not change with aging. Using purified protein B-50 as a substrate, between 3 and 8 months a decrease in in vitro phosphorylation was detected in the membrane fraction of the cortex but after this age values remained stable. In hippocampal membranes, B-50 phosphorylation was increased in aged rats. PKC translocation was impaired in aged rats in both the cortex and the hippocampus. PKC alpha and beta mRNA decreased in the cortex between 3 and 8 months with no further decline in aged animals. Hippocampal mRNA for calcium-dependent PKC isoforms was not modified during aging, as assessed by Northern and in situ hybridization. Western blot analysis revealed a change in PKC gamma protein only, which was increased in hippocampal membranes from aged rats. The data indicate that the key PKC function that is impaired in aged rats is enzyme translocation irrespective of the brain area investigated.

Protein Kinase C Anchoring Deficit in Postmortem Brains of Alzheimer's Disease Patients

Protein kinase C (PKC) has been implicated in the pathophysiology of Alzheimers disease (AD). The levels of particular isoforms and the activation of PKC are reduced in postmortem brain cortex of AD subjects. Receptors for activated C kinase (RACK) are a family of proteins involved in anchoring activated PKCs to relevant subcellular compartments. Recent evidence has indicated that the impaired activation (translocation) of PKC in the aging brain is associated with a deficit in RACK1, the most well-characterized member of this family. The present study was conducted to determine whether alterations in RACK1 occurred in cortical areas where an impaired translocation of PKC has been demonstrated in AD. Here we report the presence of RACK1 immunoreactivity in human brain frontal cortex for the first time and demonstrate a decrease in RACK1 content in cytosol and membrane extracts in AD when compared with non-AD controls. By comparison, the levels of the RACK1-related PKCbetaII were not modified in the same membrane extracts. These observations add a new perspective in understanding the disease-associated defective PKC signal transduction and indicate that a decrease in an anchoring protein for PKC is an additional determinant of this deficit.

Further development of the NCTC 2544 IL-18 assay to identify in vitro contact allergens

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.

Effects of ethanol, given during pregnancy, on the offspring dopaminergic system

The fetal alcohol syndrome is characterized by a number of abnormalities consisting of a pre- and post-natal growth deficiency, microcephaly, areas of abnormal nerve cell migration in the brain, mental and psychomotor retardation in children of alcoholic women. These findings may be referred as a teratogenic effect of ethanol on the central nervous system. In order to investigate the above ethanol-neurotoxic effect the striatal dopaminergic transmission was studied. The dopaminergic turnover was measured by 3,4-dihyroxyphenilacetic acid content and 3H-Spiperone binding has been carried out to determine dopaminergic receptor alterations induced by chronic ethanol consumption during pregnancy. Our work demonstrates long-lasting modifications of dopaminergic neuronal function after exposure of the experimental animal to ethanol during fetal life. In particular, a decreased receptor function has been observed in rats exposed to ethanol only during the perinatal period. In the same group of rats, diminished receptor activity leads to an enhancement in DOPAC content still detectable after a long period from cessation of ethanol treatment. Neurochemical data are reinforced by behavioral observations. In fact, a significant decrease of spontaneous locomotor activity in the rats chronically treated with ethanol during fetal life was observed. In addition, the altered response of locomotor activity after drug administration may be ascribed to the modified dopaminergic function. With this experimental approach we assume that the action of ethanol on the central nervous system may be a marker of its teratogenic effect.

Brain neurotransmitter systems and chronic lead intoxication

Summary The neurotoxic effect of lead has been studied measuring the function of different neurotransmitters in various rat brain areas. Dopaminergic and GABAergic systems are modified at various steps: both at presynaptic and postsvnaptic (receptors) levels. The changes observed, together with those related with other brain neurotransmitters, suggest the hypothesis that the behavioural and clinical changes observed in humans exposed to lead may be explained on the bases of selective actions of lead on neurotransmitters in discrete brain areas.

Effect of chronic ethanol treatment on dopamine receptor subtypes in rat striatum.

Chronic exposure to ethanol (6% in the drinking water, 25 days) reduces the responsiveness of both the dopamine-stimulated and of the dopamine-inhibited adenylate cyclase in rat striatum. The changes in the adenylate cyclase activity are paralleled by alterations in dopamine recognition sites, in fact binding studies using selective ligands indicate that the number of both D1- and D2-receptors is reduced in striatal membranes of treated rats.

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 α (TNF‐α), increased PHA‐induced interleukin‐10 (IL‐10) release, and similar PHA‐induced interferon‐γ 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‐α 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.

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-α 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.