Special Issue "Cosmetic Contact Allergens"

In Europe, a cosmetic is defined as any substance or preparation intended to be placed in contact with the various external parts of the human body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance and/or correcting body odours and/or protecting them or keeping them in good condition.[...

Present and future of in vitro immunotoxicology in drug development.

The realization, that the immune system can be the target of many chemicals including environmental contaminants and drugs with potentially adverse effects on the host's health, has raised serious concerns within the public and the regulatory agencies. At present, assessment of immunotoxic effects relies on different animal models and several assays have been proposed to characterize immunosuppression and sensitization. The use of whole animals, however, presents many secondary issues, such as expense, ethical concerns, and eventual relevance to risk assessment for humans. Furthermore, due to the new policy on chemicals (REACH), in the European Union, in vitro methods will play a major role in the near future. In addition, there is still a lack of human cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical, and reliable way. Hypersensitivity and immunosuppression, for which animal models have been developed and validated, are considered the primary focus for developing in vitro methods in immunotoxicology. Nevertheless, in vitro assays, as well as in vivo models, to detect immunostimulation and autoimmunity are also needed. Even if no validated alternative in vitro tests to assess immunotoxicity exist, in the last decade, much progress has been made toward these assays. Such models can be, at least, used for the pre-screening and hazard identification of unintended immunosuppression and contact hypersensitivity of direct immunotoxicants. Following a brief introduction to immunotoxicology and to in vivo models use to assess immunotoxicity, this manuscript will review the state-of-the-art in the field of in vitro immunotoxicity

Erosione genetica e rischio di estinzione delle razze cunicole in Veneto: cause e possibili soluzioni

In Italia esistono 43 razze di coniglio riconosciute dall’ANCI (Associazione Nazionale Coniglicoltori Italiani) ma la maggior parte di esse è scomparsa dagli allevamenti italiani e sopravvive perlopiù grazie a pochi appassionati. In Veneto, la regione responsabile di circa il 40% della produzione nazionale di carne cunicola, rimane un numero esiguo di allevatori di razze pure dal momento che negli allevamenti intensivi vengono preferiti tipi genetici caratterizzati da prestazioni produttive più elevate. Il declino delle razze cunicole è esacerbato da molteplici fattori di diversa natura: in primo luogo il rischio di erosione genetica è elevato in quanto le popolazioni locali sono costituite da pochi individui, il che spinge gli allevatori a ricorrere ad inbreeding ed incrocio, impoverendo ulteriormente il pool genetico della razza. Al quadro generale si aggiunge un crescente disinteresse del consumatore per il prodotto carne, il quale presenta caratteristiche poco gradite, e un aumentato interesse per il benessere animale che fa sì che il consumatore non reputi più accettabile la modalità di allevamento intensivo attuale. Inoltre, il settore cunicolo è interessato da una grave crisi che ha portato alla chiusura del 40% degli allevamenti italiani negli ultimi dieci anni. Le difficoltà di gestione dell’allevamento, che vanno dall’aumento dei costi di produzione alla gestione delle patologie, contribuiscono ad aumentare il disinteresse per un settore ormai sempre meno redditizio. Infine, la mancata collaborazione tra associazioni ed allevatori fa sì che questi ultimi spesso non aderiscano a progetti di conservazione come l’iscrizione dei propri animali di razza al Registro Anagrafico. In questa tesi sono state esplorate possibili soluzioni ai problemi sopra riportati, ovvero la messa in atto di piani di conservazione delle risorse genetiche che prevedano l’adesione degli allevatori al Registro Anagrafico, la riduzione del tasso di inbreeding mediante accorgimenti che aumentino la numerosità effettiva degli animali, la limitazione dell’utilizzo dei riproduttori e l’istituzione di piani di accoppiamento. Inoltre, viene valutata la possibilità di utilizzare la crioconservazione del seme e l’istituzione degli allevatori custodi, entrambe misure già impiegate in altre specie ma non nel coniglio. Infine, vengono esaminate modalità di allevamento alternativo che consentano l’utilizzo (e di conseguenza la tutela) delle razze autoctone e che risultino più gradite al consumatore e meno costose per l’allevatore.In Italy there exist 43 rabbit breeds recognized by ANCI (Associazione Nazionale Coniglicoltori Italiani, National Association of Italian Rabbit Breeders), although the majority of them has completely disappeared from Italian farms, and is mostly enduring thanks to a few enthusiasts. In Veneto, the region that is responsible of around 40% of the national production of rabbit meat, there remains a small number of breeders of native varieties, since intensive farming prefers genetic types characterised by higher productive performances. The decline of the characteristic breeds is exacerbated by multiple factors of various nature: first of all, the risk of genetic erosion is high because local populations only count a small number of individuals, which compels breeders to resort to inbreeding and crossbreeding, thus further depleting the genetic pool of the variety itself. The general framework also includes an increasing lack of interest of consumers towards the rabbit meat, which presents according to them disagreeable characteristics, and an increased concern for the animal’s wellbeing, leading consumers to stop accepting the current intensive farming methods. Furthermore, the rabbit industry is affected by a serious crisis which has resulted in the closing of 40% of Italian farms during the last ten years. The management difficulties, which range from a growth in the costs of production to the handling of pathologies, contribute to the decrease of interest in a field now less and less profitable. Lastly, the failed collaboration between associations and breeders has led to the latter ones often not adhering to conservation projects like registering their purebred animals to the Registry Office. In this dissertation, possible solutions to the aforementioned problems have been explored, namely the implementing of plans for the conservation of genetic resources which involve the adhesion of breeders to the Registry Office, the mitigation of the inbreeding rate through arrangements resulting in an increased effective population size of the animals, the limitation of the use of reproducers and the establishment of guard breeders, both of which have already been employed for other species, but not yet for the rabbit. Finally, alternative breeding methods have been examined, which allow the use (and subsequently the safeguard) of autochthonous varieties while also making them more agreeable to the consumers and less expensive for the breeders

alternative approach for potency assessment in vitro methods

Over the last decade, incredible progress has been made in the development of non-animal tests to assess contact hypersensitivity. Four methods have been successfully validated and Organisation for Economic Co-operation and Development (OECD) guidelines are available or soon will be. Currently validated methods are useful for hazard identification, classification and labeling. However, to achieve a complete replacement of animals in skin sensitization assessment, dose-response information and evaluation of relative skin sensitizing potency to support effective risk assessment are necessary. In this context, potency is based on the concentration of chemicals needed to induce a positive response. This will require a better understanding of the mechanisms determining potency, including pathway analysis and marker signature identification (selection of an appropriate immune-mediated response to serve as the basis), together with quantitative and qualitative correlations between marker signatures and potency of chemicals in relation with T cell responses. This review aims to discuss the state-of-the-art in the field of in vitro assessment of the no induction sensitization level of contact sensitizers

Role of Mitochondria in Tributyltin-Induced Interleukin-1α Production in Murine Keratinocytes

Tributyltin (TBT) salts are well known skin irritants in rodents and humans. TBT induced both the intracellular production of Interleukin-1α (IL-1α) and its release into culture medium in a murine keratinocyte cell line (HEL30). Here, we report that mitochondria are important for TBT-induced IL-1α production.Confluent cells were treated with increasing concentrations of TBT (0–2.5 μM) or dimethylsulfoxide as vehicle control. At different times thereafter (0–24 h), nuclear extracts were analyzed for nuclear factor-κB (NF-κB) binding activity by electrophoretic mobility shift assay, and the released and cell-associated IL-1α was measured by enzyme-linked immunosorbent assay. TBT induced a direct and concentration-related activation of NF-κB, which peaked at 2h and was blocked by pyrrolidinedithiocarbamate, a potent NF-κB inhibitor, and rotenone, an inhibitor of the electron entry from complex I to ubiquinone. Rotenone also induced a concentration-related inhibition of IL-1α synthesis induced by TBT, but rotenone did not completely abrogate TBT-induced IL-1α production, which suggests that other transcription factors may be involved in IL-1α production.Prolongod treatment with ethidium bromide, an inhibitor of mitochondrial DNA and RNA synthesis, was used to partially deplete cells of functional mitochondria. After 5 d of treatment, mitochondria conversion of tetrazolium bromide to formazan was reduced by 50%, and IL-1α release was decreased by 65%, whereas no induction of intracellular IL-1α was observed. This effect was not due to inhibition prot in synthesis because identical incorporation of [3H]leucine into protein in control and ethidium bromide–treated cells was identical. This impairment of mitochondrial metabolism inhibited NF-κB activation by TBT. These findings indicate that mitochondria may be the source of second messenger molecules important for TBT-induced IL-1α production

Establishment of an In Vitro Photoallergy Test Using NCTC2544 Cells and IL-18 Production

Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of eratinocytes 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 demonstrated the possibility to use the human keratinocyte cell line NCTC2455 and the production of interleukin-18 (IL-18) to screen low molecular weight sensitizers. The purpose of this work was to explore the possibility to use the NCTC2544 assay to identify photoallergens and discriminate from phototoxic chemicals. First, we identified suitable condition of UV-irradiation (3.5 J/cm2) by investigating the effect of UVAirradiation on intracellular IL-18 on untreated or chloropromazine (a representative phototoxic compound)- treated NCTC2544 cells. Then, the effect of UVA-irradiation over NCTC2544 cells treated with increasing concentrations of 15 compounds including photoallergens (benzophenone, 4-ter-butyl-4-methoxydibenzoylmethane, 2-ethylexyl-p-methoxycinnamate, ketoprofen, 6-methylcumarin); photoirritant and photoallergen (4-aminobenzoic acid, chlorpromazine, promethazine); photoirritants (acridine, ibuprofen, 8-methoxypsoralen, retinoic acid); and negative compounds (lactic acid, SDS and p-phenilendiamine) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay or LDH leakage, while ELISA was used to measure the production of IL-18. At the maximal concentration assayed with non-cytotoxic effects (CV80 under irradiated condition), all tested photoallergens induced a significant and a dose-dependent increase of intracellular IL-18 following UVA irratiation, whereas photoirritants failed. We suggest that this system may be useful for the in vitro evaluation of the photoallergic potential of chemicals

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

Role of hormones in the regulation of RACK1 expression as a signaling checkpoint in immunosenescence

Immunosenescence defines the decline in immune function that occurs with aging. This has been associated, at least in part, with defective cellular signaling via protein kinase C (PKC) signal transduction pathways. Our data suggest reduced PKC activation and consequently reduced response to lipopolysaccharide (LPS) stimulation and cytokine release. The lack of PKC activation seems to be dependent on the reduced expression of the receptor for activated C kinase 1 (RACK1), a scaffolding protein involved in multiple signal transduction cascades. The defective expression of RACK1 may be dependent on age-related alteration of the balance between the adrenal hormones cortisol and dehydroepiandrosterone (DHEA). DHEA levels reduce with aging, while cortisol levels remain substantially unchanged, resulting in an overall increase in the cortisol:DHEA ratio. These hormonal changes are significant in the context of RACK1 expression and signaling function because DHEA administration in vivo and in vitro can restore the levels of RACK1 and the function of the PKC signaling cascade in aged animals and in human cells. In contrast, there is evidence that cortisol can act as a negative transcriptional regulator of RACK1 expression. The rack1 gene promoter contains a glucocorticoid responsive element that is also involved in androgen signaling. Furthermore DHEA may have an indirect influence on the post-transcriptional regulation of the functions of the glucocorticoid receptor. In this review, we will examine the role of the hormonal regulation of rack1 gene transcriptional regulation and the consequences on signaling and function in immune cells and immunosenescence

A long pentraxin-3-derived pentapeptide for the therapy of FGF8b-driven steroid hormone-regulated cancers

Fibroblast growth factor-8b (FGF8b) affects the epithelial/stromal compartments of steroid hormone-regulated tumors by exerting an autocrine activity on cancer cells and a paracrine pro-angiogenic function, thus contributing to tumor progression. The FGF8b/FGF receptor (FGFR) system may therefore represent a target for the treatment of steroid hormone-regulated tumors. The soluble pattern recognition receptor long pentraxin-3 (PTX3) binds various FGFs, including FGF2 and FGF8b, thus inhibiting the angiogenic and tumorigenic activity of androgen-regulated tumor cells. Nevertheless, the complex/proteinaceous structure of PTX3 hampers its pharmacological exploitation. In this context, the acetylated pentapeptide Ac-ARPCA-NH2 (ARPCA), corresponding to the N-terminal amino acid sequence PTX3(100-104), was identified as a minimal FGF2-binding peptide able to antagonize the biological activity of FGF2. Here, we demonstrate that ARPCA binds FGF8b and inhibits its capacity to form FGFR1-mediated ternary complexes with heparan sulphate proteoglycans. As a FGF8b antagonist, ARPCA inhibits FGFR1 activation and signalling in endothelial cells, hampering the angiogenic activity exerted in vitro and in vivo by FGF8b. Also, ARPCA suppresses the angiogenic and tumorigenic potential of prototypic androgen/FGF8b-dependent Shionogi 115 mammary carcinoma cells and of androgen/FGF8b/FGF2-dependent TRAMP-C2 prostate cancer cells. In conclusion, ARPCA represents a novel FGF8b antagonist with translational implications for the therapy of steroid hormone-regulated tumor

Toxicological profile of PM from different sources in the bronchial epithelial cell line BEAS-2B

The toxicity of particulate matter (PM) is strictly associated with its physical-chemical characteristics, such as size or chemical composition. While these properties depend on the origin of the particles, the study of the toxicological profile of PM from single sources has rarely been highlighted. Hence, the focus of this research was to investigate the biological effects of PM from five relevant sources of atmospheric PM: diesel exhaust particles, coke dust, pellet ashes, incinerator ashes, and brake dust. Cytotoxicity, genotoxicity, oxidative, and inflammatory response were assessed in a bronchial cell line (BEAS-2B). BEAS-2B cells were exposed to different concentrations (25, 50, 100, and 150 g/mL medium) of particles suspended in water. The exposure lasted 24 h for all the assays performed, except for reactive oxygen species, which were evaluated after 30 min, 1 h, and 4 h of treatment. The results showed a different action of the five types of PM. All the tested samples showed a genotoxic action on BEAS-2B, even in the absence of oxidative stress induction. Pellet ashes seemed to be the only ones able to induce oxidative stress by boosting the formation of reactive oxygen species, while brake dust resulted in the most cytotoxic. In conclusion, the study elucidated the differential response of bronchial cells to PM samples generated by different sources. The comparison could be a starting point for a regulatory intervention since it highlighted the toxic potential of each type of PM tested