Mechanistic understanding of dendritic cell activation in skin sensitization: additional evidences to support potency classification

Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to chemical allergens. In the EU, it has been calculated that 4 % of animals are used in toxicity test for the assessment of skin sensitization (Peiser et al., 2012). To come a complete replacement of animals, evaluation of relative skin sensitization potency is necessary. The identification of mechanisms influencing allergen potency requires a better understanding of molecular events that trigger cell activation. Therefore, (i) the effects of selected allergens on surface markers expression and cytokines release in contact allergen-induced cell activation were assessed, and (ii) the role of Protein Kinase C (PKC) beta activation in contact allergen-induced cell activation was investigated. The human pro-myelocytic cell line THP-1 was used as experimental model surrogate of dendritic cells. Cells were exposed to select contact allergens of different potency and cell surface marker expression (CD80, CD86, HLA-DR) was determined by flow cytometry analysis. Cytokines production (IL-6, IL-8, IL-10, IL-12p40, IL-18) was evaluated with specific sandwich ELISA. The effective contribution of PKC beta in chemical allergen-induced cell activation was assessed by Western Blot analysis (PKC beta activation) and using a specific PKC beta inhibitor (PKC beta pseudosubstrate). In addition, to investigate if contact allergens are able to induce indeed dendritic cells (DCs) maturation, THP-1 cells were differentiated to immature DC and then exposed to contact allergen of different potency. Overall, our finding provides insights into the process of sensitization and strength of cell activation associated with allergens of different potency. Results obtained suggest that contact allergens of different potency are able to induce a different degree of activation of dendritic cells maturation involved in the process of ACD

ASSESSMENT OF THE ALLERGENIC POTENTIAL OF XENOBIOTICS: IN VIVO IN VITRO A BACK-AND-FORTH APPROACH

Allergic contact dermatitis (ACD) is an important occupational and environmental disease caused by topical exposure to low molecular weight chemical allergens. The development of ACD requires the activation of innate immune cells, such as keratinocytes (KC), necessary for the maturation and the migration of dendritic cells (DC), which in turn are required for the activation of specific T cells. Human KC constitutively express several cytokines, including pro IL-1 alpha, pro IL-1 beta and pro IL-18. In vivo it has been demonstrated that IL-18 plays a key proximal role in the induction of allergic contact sensitization, favoring Th-1 type immune response by enhancing the secretion of pro-inflammatory mediators such as TNF-\u3b1, IL-8 and IFN-\u3b3 (Shornick et al., 1996; Wang et al., 1999, Cumberbatch et al., 2001). Toxicologists have the responsibility of identifying and characterizing the skin and respiratory allergenic potential of chemicals, and estimating the risk they pose to human health. Growing political and practical resistance to toxicity testing in animals has driven the development of animal-free methods for screening and prioritization of toxicants, including those causing allergic hypersensitivity. The purpose of this thesis was to develop an alternative in vitro test based on the keratinocytes and IL-18 to characterize the allergenic potential of low molecular weight chemicals, and to understand the molecular mechanism(s) underlying chemical allergen-induced IL-18 production. In addition to human keratinocytes cell lines (NCTC2544, HaCaT, HPKII), commercially available reconstituted human epidermis 3D-epidermal models were also used as experimental model. Due to their anatomical location and critical role in skin inflammatory and immunological reactions, the use of the KC and skin organotypic culture as a simplified in vitro model to evaluate the potential toxicity of chemicals destined for epicutaneous application is amply justified. To perform these studies 22 contact allergens, 12 photoallergens/photoirritant compounds, 3 respiratory allergens and 9 irritants chemicals were used. The choice of chemicals was dictated by the SENS-IT-IV programme as relevant and representative of the \u2018universe\u2019 of irritants, respiratory and contact allergens. Phototoxic chemicals were selected based on compounds used in similar published studies and reported to cause phototoxicity. Results obtained indicate that the NCTC2544 IL-18 assay is able to discriminate contact allergens and photoallergens from irritants/photoirritants and respiratory allergens. Important factors including compound solubility, chemical reactivity and metabolic activation, which may mask the potential allergenicity of some chemicals, must be considered in the development of in vitro tests. Submerged cell culture may be unfavourable for many of the respiratory sensitizers, due to chemical instability; for this reason we have tested IL-18 production also in reconstituted human epidermis, which allows application in organic solvent, i.e. acetone: olive oil. The lack of metabolic activation may be a relevant problem in case of proaptens. However, NCTC 2544 cells posses both phase I and II metabolic activation capacity (Gelardi et al., 2001), and positive results were indeed obtained with the proaptens tested (eugenol and cinnamic alcohol). A sensitivity of 87%, specificity of 95% and an accuracy of 90% was obtained (Corsini et al., 2009; Galbiati et al., 2011). In addition to being able to determine whether or not a chemical is a sensitizer (labelling) it is also equally important to determine the potency of a sensitizer (classification) in order to identify a maximum safe concentration for human exposure (risk assessment). The combination of the epidermal equivalent potency assay with the release of IL-18 lead to the development of an in vitro model able to identify contact allergens and rank them according to their potency. One other objective of this thesis was to study the signal transduction pathways involved in PPD, DNCB and citral-induced IL-18. For such purpose several inhibitors were used. To investigate the intracellular source of ROS, specific inhibitors of the three main cellular sources of ROS, namely DPI, a NADPH synthetase inhibitor; rotenone, a mitochondrial electron transport inhibitor; allopurinol, a xanthine oxidase inhibitor, were used. Z-VAD-FMK, a cell-permeant pan caspase inhibitor, that irreversible binds to the catalytic site of caspases, and a neutralizing anti-TLR4 antibody were used to investigate the role of the inflammasome and TLR4. Glycirrizic acid, a direct inhibitor of HMGB1 protein, was used to establish the role of HMGB1 as possible DAMP associated with allergen-induced IL-18. To specifically investigate the signal transduction pathway involved in PPD-induced IL-18 production selective inhibitors were used: GF109203X to inhibit PKC, PDTC and Bay 11-70-85 to inhibit NF-\u3baB, and SB203580, as p38 MAPK inhibitor. The results obtained during this three year of research activity have clearly shown that the in vitro methods based on NCTC2544 and IL-18 production are able to discriminate contact/photocontact allergens from irritants/photoirritants and respiratory allergens. Furthermore, the combined use of the epidermis in vitro model with the IL-18 production, beside the ability to identify sensitising compounds, is able to rank them according to their potency. With respect to the molecular mechanisms behind skin sensitization I could demonstrate that different intracellular sources of ROS are triggered by different contact allergens. Allergens-induced IL-18 production is dependent upon NF-\u3baB and p38 MAPK activation and TLR4 and inflammasome activation. Among the DAMPs, the evolutionarily conserved non-hystone chromatin-binding protein HMGB1 is released into the extracellular space following exposure to contact allergens, resulting in TLR4 activation and IL-18 neosynthesis. Even if more studies are necessary to elucidate the mechanisms that are involved in chemical allergens-induced oxidative stress, the signalling pathways activated and their role in contact allergy, data clearly indicated a pivotal role of ROS in chemical allergy. Consequently, the redox state of the cell becomes imbalanced, with the activation of several pathways, including MAPK, such as SAPK/JNK, ERK1/2 and p38, NF-\u3baB, Akt/ASK1 or Keap1/Nrf2 pathways, resulting in a inflammatory and cytotoxic response with the production of costimulatory molecules, cytokines, chemokines, and phase 1 detoxifying enzymes. On the basis of the results obtained, the following scenario could be imagined: chemical sensitisers can induce oxidative stress owing to their elecrophilicity, which in turn activates the inflammasome and HMGB1 release (and possible other DAMPs), which can activate TLR4. Activation of TLR4 will results in NF-\u3baB and p38 MAPK activation and in the neosynthesis of IL-18

Quantum Circuits for the Unitary Permutation Problem

We consider the Unitary Permutation problem which consists, given $n$ unitary gates $U_1, \ldots, U_n$ and a permutation $\sigma$ of $\{1,\ldots, n\}$, in applying the unitary gates in the order specified by $\sigma$, i.e. in performing $U_{\sigma(n)}\ldots U_{\sigma(1)}$. This problem has been introduced and investigated by Colnaghi et al. where two models of computations are considered. This first is the (standard) model of query complexity: the complexity measure is the number of calls to any of the unitary gates $U_i$ in a quantum circuit which solves the problem. The second model provides quantum switches and treats unitary transformations as inputs of second order. In that case the complexity measure is the number of quantum switches. In their paper, Colnaghi et al. have shown that the problem can be solved within $n^2$ calls in the query model and $\frac{n(n-1)}2$ quantum switches in the new model. We refine these results by proving that $n\log_2(n) +\Theta(n)$ quantum switches are necessary and sufficient to solve this problem, whereas $n^2-2n+4$ calls are sufficient to solve this problem in the standard quantum circuit model. We prove, with an additional assumption on the family of gates used in the circuits, that $n^2-o(n^{7/4+\epsilon})$ queries are required, for any $\epsilon >0$. The upper and lower bounds for the standard quantum circuit model are established by pointing out connections with the permutation as substring problem introduced by Karp.Comment: 8 pages, 5 figure

Premature loss of primary molars in children: space recovery through molar distalisation. A literature review

Aim: The integrity of primary dentition is essential in the development of the jaws and permanent occlusion. The consequences of a premature loss of primary molars are: space loss, crowding, risk of impaction of the permanent teeth, ectopic eruption, anomalous inclination of the teeth adjacent to the loss molar, reduction of arch length. The mesial displacement of the posterior permanent teeth during eruption can cause a loss of space, which can be regained with orthodontic appliances. Therefore, a careful diagnosis is of great importance to be able to decide what appliance is indicated to recover from this situation. Molar distalisation consists in displacing permanent molars distally, allowing them to reach class I relationship and to recover the correct space for the second bicuspids when the second deciduous molar has been lost early. Methods: The aim of this study is to carry out a narrative literature review regarding the different appliances and their effectiveness in regaining space after premature loss of the upper primary molars. Conclusion: The paediatric dentist should be aware of the advantages and disadvantages related to each device and select the most appropriate distalisation appliance based on an individual plan of diagnosis and a careful treatment. The distalisation of the upper molar must be adequately stabilised and so it is important to consider also some retainers such as Nance's appliance, the Palatal Plate, the extraoral traction, the utility arch, or II Class elastic bands

Study on the differences of BMI between olms found in cave and the ones sampled in spring

The olm (Proteus anguinus) is a blind amphibian, depigmented and with an extremely reduced metabolism. These are adaptations to cave life that allow the animals to exploit this hostile (from a human perspective) habitat. Adaptations to cave life, should prevent successful exploitation of the external environment. However, although the Proteus is a cave animal, it has been reported by some authors in the spring environment, Our previous results showed that this presence, would seem not to be random, in but linked to very specific factors such as hydroperiod the absence of potential predators. In the light of these results, we have conducted a study with the aim of verifying whether the Proteus benefits from exploiting the trophic resources occurring in springs. Since June 2020 we have sampled 64 springs and 12 caves in the Monfalcone province, each olm found during the sampling was photographed, measured and weighed. A total of 73 different olms were analyzed and the Body Mass Index (BMI) was calculated for each individual. Olms found in springs had a significantly higher BMI than those found in caves. The BMI of springs’ olms was significantly related to amount of the trophic available, evidencing that they took advantages from exploiting the external environment. These results contribute to support the hypothesis of the non-random presence of the Proteus in the spring environment, the olm, in fact, seem to benefit from this type of habitat taking advantage of the greater number of resources present in it

La tecnologia del forno intelligente (iEAF®): concetti di base, descrizione generale e dettagli della prima installazione

L’iEAF® di Tenova è un innovativo sistema di automazione per il controllo dinamico e l’ottimizzazione olisticadel forno elettrico ad arco. I modelli matematici del processo vengono utilizzati, unitamente alla composizionechimica dei fumi ( acquisita tramite il sistema di analisi proprietario EFSOP™ ), ed insieme anche ad altreinformazioni relative allo stato del processo ottenute tramite sensori innovativi sviluppati appositamente perl’applicazione sull’EAF, al fine di calcolare i bilanci di massa ed energia in modo dinamico ed in tempo reale.L’insieme dei modelli e dei sensori permette all’operatore di disporre di informazioni critiche per la produzionequali l’energia netta fornita alla carica metallica, la percentuale di fusione del rottame, la temperaturae composizione del bagno e della scoria

In vitro Models to evaluate drug-induced hypersensitivity : potential test based on activation of dendritic cells

Hypersensitivity drug reactions (HDRs) are the adverse effect of pharmaceuticals that clinically resemble allergy. HDRs account for approximately 1/6 of drug-induced adverse effects, and include immune-mediated ("allergic") and non-immune-mediated ("pseudo allergic") reactions. In recent years, the severe and unpredicted drug adverse events clearly indicate that the immune system can be a critical target of drugs. Enhanced prediction in preclinical safety evaluation is, therefore, crucial. Nowadays, there are no validated in vitro or in vivo methods to screen the sensitizing potential of drugs in the pre-clinical phase. The problem of non-predictability of immunologically-based hypersensitivity reactions is related to the lack of appropriate experimental models rather than to the lack of -understanding of the adverse phenomenon. We recently established experimental conditions and markers to correctly identify drug associated with in vivo hypersensitivity reactions using THP-1 cells and IL-8 production, CD86 and CD54 expression. The proposed in vitro method benefits from a rationalistic approach with the idea that allergenic drugs share with chemical allergens common mechanisms of cell activation. This assay can be easily incorporated into drug development for hazard identification of drugs, which may have the potential to cause in vivo hypersensitivity reactions. The purpose of this review is to assess the state of the art of in vitro models to assess the allergenic potential of drugs based on the activation of dendritic cells

Multiple Roles of Transforming Growth Factor Beta in Amyotrophic Lateral Sclerosis

Transforming growth factor beta (TGFB) is a pleiotropic cytokine, known to be dysregulated in many neurodegenerative disorders and particularly in amyotrophic lateral sclerosis (ALS). This motor neuronal disease is non-cell autonomous, as it affects not only motor neurons, but also their surrounding glial cells, and their target skeletal muscle fibers. Here, we analyze the multiple roles of TGFB in these cell types, and how TGFB signaling is altered in ALS tissues. Data reported support a crucial involvement of TGFB in the etiology and progression of ALS, leading us to hypothesize that an imbalance of TGFB signaling, diminished at the pre-symptomatic stage and then increased with time, could be linked to ALS progression. A reduced stimulation of the TGFB pathway at the beginning of disease blocks its neuroprotective effects and promotes glutamate excitotoxicity. At later disease stages, the persistent activation of the TGFB pathway promotes an excessive microglial activation and strengthens muscular dysfunction. The therapeutic potential of TGFB is discussed here, in order to foster new approaches to treat ALS

The role of extracellular vesicles in the removal of aggregated TDP43 responsible for ALS/FTD diseases

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are two related neurodegenerative diseases. ALS is caused by the death of both upper and lower motoneurons, while FTD is characterized predominantly by circumscribed atrophy of the frontal and temporal lobes. ALS and FTD overlap each other. This is demonstrated by the presence of cognitive and behavioral dysfunction in up to 50% of ALS patients and by the presence of frontotemporal atrophy in patients with ALS. Moreover, these diseases are both characterize by the presence of TAR DNA binding protein 43 (TDP43) inclusions in affected cells. These inclusions, observed in 97% of patients with ALS and 50% of patients with FTD, are composed by TDP43 and its C-terminal fragments of 35 kDa (TDP35) and 25 kDa (TDP25). These fragments are highly aggregation-prone and probably neurotoxic. Thus, their removal is protective for cells. The mechanism responsible for the clearance of aggregates and misfolded proteins is the intracellular protein quality control (PQC) system. It consists of molecular chaperones/co- chaperones and the degradative pathways. PQC controls the folding status of proteins and prevents the aggregation of misfolded proteins by refolding them or degrading. Recent data demonstrated that also extracellular secretory pathway, represented especially by exosomes (EXOs) and microvesicles (MVs), might be involved in the removal of misfolded proteins from affected cells. Thus, we evaluated the role of EXOs and MVs in the secretion of TDP43 and its C-terminal fragments, using neuronal cell models. We used ultracentrifugation, that allowed us to separate MVs from EXOs on the basis of their dimension. Then we analyzed them through i) Nanoparticle Tracking Analysis (NanoSight) to establish their number and sizes, and ii) western blot analysis, to characterize their protein content. Our preliminary results show that TDP43, TDP35 and TDP25 are all secreted, mainly by MVs. In particular, we found that MVs are enriched of insoluble forms of TDPs and also of superoxide dismutase 1 (SOD1), another ALS-related protein. Finally, both in EXOs and in MVs, we observed the presence of some important PQC-components, suggesting an interplay between the two pathways. GRANTS: Fondazione Cariplo, Italy (n. 2017_0747); Universit\ue0 degli Studi di Milano e piano di sviluppo UNIMI - linea B

Activity of the olm (Proteus anguinus) in surface habitats: ecological and evolutionary insights

The olm is considered as a classic example of troglobiont organism. However, in the past different observations of individuals of the typical troglomorphic populations have been reported for springs of Venetia Giulia. The aim of this work is to point out the non-random active use of surface habitats by the olm, providing a comparison with the occurrence observed in caves and performing an assessment of factors favouring ecotone habitats exploitation. Since 2020 we started multiple day and night surveys of olms in both springs and caves. Each spring and cave habitat monitored has been characterised by respect to abiotic and biotic features, including planktonic and benthic prey availability. We detected the olm at least once in 10 springs, with a maximum of 9 individuals occurring together. Detection probability in springs and caves was similar. Spring habitats provided higher density of potential prey available. Olms seems to prefer springs without predator fish and temporary hydroperiod. We recorded in one spring a larva of 3.5 cm which could be the smallest ever recorded in the field. We suggest that epigean habitats and borders with surface may have an underestimated importance for animals adapted to subterranean environments, including the olm. Our results stimulate for testing if exploitation of ecotones between surface and groundwater can lead to differentiation in populations/subpopulations of stygobiont animals