Laura Cornaghi

An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17

BACKGROUND Among all cytokines involved in the pathogenesis and in the progression of psoriasis, Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role. OBJECTIVE The aim of the present study was to mimic a psoriatic microenvironment and to investigate the early effects of TNF-alpha and IL-17 in a three-dimensional model of organotypic normal human skin. METHODS Human skin explants were obtained from plastic aesthetic surgery of healthy young women 20-40years old (n=7). The study was approved by the Institutional Review Board and written informed consent was obtained from all subjects. Bioptic fragments were cultured at the air-liquid interface overnight in a Transwell system and further divided before adding either 50ng/ml IL-17 or 100ng/ml TNF-alpha or a combination of both cytokines. For each subject, a control sample was cultured without any cytokine. Samples were harvested 24 or 48h after cytokine incubation. At both time points and for all cytokine treatments a bioptic fragment obtained from each patient was processed. Epidermal proliferation, expressions of terminal differentiation (keratin 10, K10, and 14, K14) and of intercellular adhesion (occludin for tight junctions and E-cadherin for adherens junctions) biomarkers were investigated by indirect immunofluorescence. RESULTS IL-17 and TNF-alpha induced an early and statistically significant inhibition of keratinocyte proliferation (more than 80% compared with their respective controls). At 24h, the combination of both cytokines did not further reduce cell proliferation. Starting from 24h of incubation, a non-continuous occludin expression in the granular layer was observed after both IL-17 and TNF-alpha exposure. Immunolabelling for E-cadherin in adherens junctions, for K10 in the suprabasal layers, and for K14 in the basal layer was similar in all experimental groups and unaffected after cytokine treatment. CONCLUSIONS These results suggest that in this experimental model IL-17 and TNF-alpha induced an early alteration of the homeostasis of the inner proliferative layer and of the upper granular layer, as shown by cell proliferation inhibition and occludin expression.

Ranolazine prevents INaL enhancement and blunts myocardial remodelling in a model of pulmonary hypertension

AIMS Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current (INaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive INaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available INaL blocker, may prevent constitutive INaL enhancement and PAH-induced myocardial remodelling. METHODS AND RESULTS PAH was induced in rats by a single monocrotaline (MCT) injection [60 mg/kg intraperitoneally (i.p.)]; studies were performed 3 weeks later. RAN (30 mg/kg bid i.p.) was administered 48 h after MCT and washed-out 15 h before studies. MCT increased RV systolic pressure and caused RV hypertrophy and loss of left ventricular (LV) mass. In the RV, collagen was increased; myocytes were enlarged with T-tubule disarray and displayed myosin heavy chain isoform switch. INaL was markedly enhanced; diastolic Ca(2+) was increased and Ca(2+) release was facilitated. K(+) currents were down-regulated and APD was prolonged. In the LV, INaL was enhanced to a lesser extent and cell Ca(2+) content was strongly depressed. Electrical remodelling was less prominent than in the RV. RAN completely prevented INaL enhancement and limited most aspects of PAH-induced remodelling, but failed to affect in vivo contractile performance. RAN blunted the MCT-induced increase in RV pressure and medial thickening in pulmonary arterioles. CONCLUSION PAH induced remodelling with chamber-specific aspects. RAN prevented constitutive INaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect.

Effects of UV Rays and Thymol/Thymus vulgaris L. Extract in an ex vivo Human Skin Model: Morphological and Genotoxicological Assessment.

Ultraviolet (UV) radiation is the major environmental factor affecting functions of the skin. Compounds rich in polyphenols, such as Thymus vulgaris leaf extract and thymol, have been proposed for the prevention of UV-induced skin damage. We compared the acute effects induced by UVA and UVB rays on epidermal morphology and proliferation, cytotoxicity, and genotoxicity. Normal human skin explants were obtained from young healthy women (n = 7) after informed consent and cultured at the air-liquid interface overnight. After 24 h, the samples were divided in 2 groups: the former exposed to UVA (16 or 24 J/cm2) and the latter irradiated with UVB (0.24 or 0.72 J/cm2). One hour after the end of irradiation, supernatants were collected for evaluation of the lactate dehydrogenase activity. Twenty-four hours after UVB exposure, biopsies were processed for light and transmission electron microscopy analysis, proliferation, cytotoxicity, and genotoxicity. UVB and UVA rays induced early inhibition of cell proliferation and DNA damage compared to controls. In particular, UVB rays were always more cytotoxic and genotoxic than UVA ones. For this reason, we evaluated the effect of either T. vulgaris L. extract (1.82 µg/ml) or thymol (1 µg/ml) on all samples treated for 1 h before UVB irradiation. While Thymus had a protective action for all of the endpoints evaluated, the action of the extract was less pronounced on epidermal proliferation and morphological features. The results presented in this study could be the basis for investigating the mechanism of thymol and T. vulgaris L. extract against the damage induced by UV radiation.

Tumor necrosis factor-alpha and interleukin-17 differently affects Langerhans cell distribution and activation in an innovative three-dimensional model of normal human skin.

Among the several cytokines involved in the psoriasis pathogenesis, tumor necrosis factor (TNF)-alpha and interleukin (IL)-17 play a central role. Many biomolecular steps remain unknown due to difficulty to obtain psoriatic models. To investigate the effect of TNF-alpha and IL-17 on the ultrastructure, immunophenotype, and number of epidermal Langerhans cells (LCs), human skin explants (n=7) were cultured air-liquid interface in a Transwell system. Four different conditions were used: medium alone (control), medium added with 100 ng/ml TNF-alpha or 50 ng/ml IL-17 or a combination of both cytokines. Samples were harvested 24 and 48 h after cytokine addition and were frozen. Samples harvested at 24h were also processed for transmission electron microscopy (TEM). By immunofluorescence analysis with anti-human Langerin antibody (three experiments/sample) we calculated the percentage of LCs/mm(2) of living epidermis after 24 and 48 h of incubation (considering control as 100%). At 24h LC number was significantly higher in samples treated with both cytokines (216.71+15.10%; p<0.001) and in TNF-alpha (125.74+26.24%; p<0.05). No differences were observed in IL-17-treated samples (100.14+38.42%). After 48 h, the number of epidermal Langerin-positive cells in IL-17- and TNF-alpha treated samples slightly decreased (94.99+36.79% and 101.37+23% vs. their controls, respectively). With the combination of both cytokines epidermal LCs strongly decreased (120+13.36%). By TEM, upon TNF-alpha stimulus LCs appeared with few organelles, mostly mitochondria, lysosomes, and scattered peripherical BGs. Upon IL-17 stimulus, LCs showed a cytoplasm with many mitochondria and numerous BGs close to the perinuclear space and Golgi apparatus, but also at the periphery, at the beginning of the dendrites. The addition of both cytokines did not affect LC ultrastructure. Our study showed that IL-17 induced significant changes in LC ultrastructure, while the combination of both cytokines seems to have a strong chemo-attractant effect on epidermal LCs, supporting the relevance of investigating the interplay between LCs and pro-inflammatory cytokines in the ongoing of the disease.

Interleukin 22 early affects keratinocyte differentiation, but not proliferation, in a three-dimensional model of normal human skin

Interleukin (IL)-22 is a pro-inflammatory cytokine driving the progression of the psoriatic lesion with other cytokines, as Tumor Necrosis Factor (TNF)-alpha and IL-17. Our study was aimed at evaluating the early effect of IL-22 alone or in combination with TNF-alpha and IL-17 by immunofluorescence on i) keratinocyte (KC) proliferation, ii) terminal differentiation biomarkers as keratin (K) 10 and 17 expression, iii) intercellular junctions. Transmission electron microscopy (TEM) analysis was performed. A model of human skin culture reproducing a psoriatic microenvironment was used. Plastic surgery explants were obtained from healthy young women (n=7) after informed consent. Fragments were divided before adding IL-22 or a combination of the three cytokines, and harvested 24 (T24), 48 (T48), and 72 (T72)h later. From T24, in IL-22 samples we detected a progressive decrease in K10 immunostaining in the spinous layer paralleled by K17 induction. By TEM, after IL-22 incubation, keratin aggregates were evident in the perinuclear area. Occludin immunostaining was not homogeneously distributed. Conversely, KC proliferation was not inhibited by IL-22 alone, but only by the combination of cytokines. Our results suggest that IL-22 affects keratinocyte terminal differentiation, whereas, in order to induce a proliferation impairment, a more complex psoriatic-like microenvironment is needed.

CACO2/HT-29 coculture : an in vitro intestinal cell model to study nutrients-gut interaction

Introduction: In the last years and decades more and more new plants came to the market as food or old crops have been rediscovered. Based on the presence of secondary plant metabolites specific effects are often attributed to these plants and/or derived preparations. Depending on the amount and the accompanying substances in extracts and preparations these substances can also be toxic. In many cases, plants and plant preparations are not adequately tested for their safety before marketing. In the European Union, authorization procedures and health assessments are only required for foods that are considered novel foods or produced from genetically modified organisms. A working group of federal and state governments, the BfR has worked with, has drawn up a list of nearly 600 plants and plant parts, which should facilitate the assessment of these plants (or plant parts) and derived preparations by food inspection bodies and food industry. Objectives: 18 plants or parts of plants were selected for an evaluation because of their known pharmacological or psychotropic effects or due to possible health risks. Method / Design: The risk assessments of these plants or parts of plants, including for example goji berries and yohimbe bark, were performed using the “Guidance on Safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements” of the European Food Safety Authority (EFSA). Results: Nine of the 18 plants or parts of plants pose a risk to consumers and should not be used in food. Five plants or parts of plants might pose risks when used in food. For four plants or plant parts, no risks were seen. Conclusions: The health assessments are intended to be a first step in the process of harmonization at European level. The opinions are published in a booklet and accessible on the web(04/08/2019) Organic food for sustainable and healthy diets lessons from the nordic diet? Introduction: The New Nordic Diet (NND) was developed in 2004 by chefs and food professionals from the five Nordic countries. The goal for the NND was that it should be based on traditional regional food products but healthier than the traditional eating habits. The NND builds on four key principles: Nordic identity, health, gastronomic potential and sustainability.Objectives: Can the NND be used as a model for a sustainable diet in other geographical regions?Methods/design: The NND can be described by a few overall guidelines: 1) more calories from plant foods and fewer from meat; 2) more foods from the wild countryside and 3) more foods from sea and lakes. In many ways, the New NND is very similar to a Mediterranean diet but relies on rapeseed (canola) oil instead of olive oil and ramson instead of garlic. The diets differ in their types of produce due to regional differences in climate, soil and water.Results: The health effects and sustainability of the NND has been tested in a number of scientific studies, including the OPUS project (Optimal Well-Being, Development and Health for Danish Children through a Healthy New Nordic Diet) supported by the Nordea foundation (http://foodoflife.ku.dk/opus/english/nyheder/publikationer/) in which the NND was compared to the Average Danish Diet (ADD). The use of mostly local products and reduction of the meat intake were of both socioeconomic and environmental advantage. Including organic produce increased environmental impact of the NND.Conclusion: In line with the Mediterranean diet the NND is a predominantly plant-based diet, and although the two have not been directly compared, it would be fairly safe to assume that they are equally healthy. Overall, the NND is just a regional interpretation of the tenets of healthy eating. Basically the principles of the NND could be incorporated into any regional diet.Who are we eating with? There are always companions to relate to in a meal, both to those who are present and they who are on another place or even in a past history. The choice of food and beverag ...Objectives : The aim of the present work was to compare the effect of ALA, EPA and DHA on the development of adipose tissue and its metabolism during a high fat-high sucrose (HFHS) challenge.Ann Nutr Metab 2015; 67(suppl 1) 435 BMD (BL: -2.4±0.7, PI: -0.9±0.44), serum calcium (BL: 9.5±0.6, PI: 10.5±0.52) and vitamin D (BL: 17.63±4.9, PI: 42.72±8.9) was observed. Locomotor problems were reduced among 44.06% subjects in group B compared to 11.11% in group A. Post interventional BMD (t: -2.16, P≤0.05), serum calcium (t: -4.05, P≤0.001) and vitamin D (t: -4.20, P≤0.001) of group B was significantly higher compared to group A.Affiliation: (1) PhD Student in Food Science. Human Nutrition Unit. Department of Food Science. University of Parma. Italy; (2) Associate Professor. Department of Food Science. University of Parma. Parma. Italy; (3) Full Professor. Department of Food Science, University of Parma. Parma. Italy; (4) Post-Doc Research Assistant. Department of Agricultural, Forest and Food Science, University of Turin. Torino. Italy; (5) Assistant Professor. Department of Soil, Plant and Food Science. University of Bari Aldo Moro. Bari. Italy; (6) Post-Doc Research Assistant. Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna. Bologna. Italy; (7) Full Professor. Department of Soil, Plant and Food Science. University of Bari Aldo Moro. Bari. Italy; (8) Nutritionist. Department of Food Science. University of Parma. Parma Italy.Effect on attention of a vegetable smoothie, rich in berries, fruitsand vegetables, served at the school mid-morning brake. ViktoriaOhlsson. Kristianstad UniversityPurpose:The purpose of this pilot ...Introduction: Since appetite control works differently in fluid and solid intake we wanted to analyse the energy contribution from those two types of energy sources.Objectives: The objective of thi ...Introduction: Infrequent fruit and vegetable (FV) intake is especially common among children with low parental educational level (PEL) and among boys.Objectives: Our aim was to examine whether a school-based intervention was efficient in increasing children’s FV intake especially among those whose FV intake is the lowest and which factors could explain the the group differences in the associations.Method / Design: In Finland 11-year-old (at baseline) children participated in the PRO GREENS intervention in winter 2009. In control schools were 424 and in intervention schools 386 children (response rate 77%). Children filled in validated food frequency questionnaire assessing FV intake (times/day) and a validated questionnaire about factors influencing FV intake (availability of FV, liking for FV, preferences, self-efficacy to eat FV, attitudes towards FV and knowledge of the recommendations) both at baseline May 2009 and follow-up May 2010. Parental educational level (low, middle, high) was reported by the parents. Associations were examined with linear regression and mediation analyses.Results: The intervention increased fruit intake among girls but not among boys. Intervention increased also children’s knowledge of the recommendations. Since knowledge had no impact on boys’ fruit intake, the increase in knowledge mediated only intervention’s effect on girls’ fruit intake. Intervention increased children’s fruit intake similarly in all PEL groups.Intervention increased vegetable intake only in the middle PEL group but no intervention effect was noted among children with low or high PEL. Knowledge, the only factor which mediated the intervention’s effect on children’s vegetable intake, could not explain PEL differences in the effectivity of the intervention.Conclusions: Increase in knowledge was not a sufficient prerequisite to increase FV intake among boys or the lowest PEL group. More in depth analyses are needed to find out which factors to target in interventions to reach an effect in the target groups.

High-density lipoprotein deficiency in genetically modified mice deeply affects skin morphology: A structural and ultrastructural study.

Cutaneous lipids, endogenously synthetized and transported by lipoproteins, play a pivotal role in maintaining skin barrier. An impairment of extracutaneous lipid trafficking leads to the development of xanthomas, mostly arising in hyperlipidemic patients, but also in subjects with high-density lipoprotein (HDL) deficiency. The aim of this work was to evaluate, in a genetically modified mouse model, lacking two protein components of HDL particles, apolipoprotein(apo)E and apoA-I, the effect of HDL deficiency on skin morphology. Control mice (C57BL/6), apoE deficient mice (EKO), apoA-I deficient mice (A-IKO) and apoA-I/apoE double knockout mice (A-IKO/EKO) were maintained on a low-fat/low-cholesterol diet up to 30 weeks of age. At sacrifice, skin biopsies were processed for light (LM) and transmission electron microscopy (TEM). Whereas the skin of EKO, A-IKO, and C57BL/6 mice was comparable, LM analysis in A-IKO/EKO mice showed an increase in dermal thickness and the presence of foam cells and T lymphocytes in reticular dermis. TEM analysis revealed the accumulation of cholesterol clefts in the papillary dermis and of cholesterol crystals within foam cells. In conclusion, A-IKO/EKO mice represent an experimental model for investigating the cutaneous phenotype of human HDL deficiency, thus mimicking a condition in which human xanthomatous lesions can develop.

In vitro assessment of silver nanoparticles immunotoxicity

This study aimed to characterize unwanted immune effects of nanoparticles (NP) using THP-1 cells, human whole blood and enriched peripheral blood monocytes. Commercially available silver NP (AgNP < 100 nm, also confirmed by Single Particle Extinction and Scattering) were used as prototypical NP. Cells were treated with AgNP alone or in combination with classical immune stimuli (i.e. LPS, PHA, PWM) and cytokine assessed; in addition, CD54 and CD86 expression was evaluated in THP-1 cells. AgNP alone induced dose-related IL-8 production in all models, with higher response observed in THP-1 cells, possibly connected to different protein corona formation in bovine versus human serum. AgNP potentiated LPS-induced IL-8 and TNF-α, but not LPS-induced IL-10. AgNP alone induced slight increase in IL-4, and no change in IFN-γ production. While responses to PHA in term of IL-4 and IFN-γ production were not affected, increased PWM-induced IL-4 and IFN-γ production were observed, suggesting potentiation of humoral response. Reduction in PHA-induced IL-10 was observed. Overall, results indicate immunostimulatory effects. THP-1 cells work as well as primary cells, representing a useful and practical alternative, with the awareness that from a physiological point of view the whole blood assay is the one that comes closest to reality.

Morphofunctional properties of a differentiated Caco2/HT-29 co-culture as an in vitro model of human intestinal epithelium

An intestinal 70/30 Caco2/HT-29 co-culture was set up starting from the parental populations of differentiated cells to mimic the human intestinal epithelium. Co-culture was harvested at confluence 0 (T0) and at 3, 6, 10, and 14 days post confluence after plating (T3, T6, T10, and T14, respectively) for morphological and functional analysis. Transmission electron microscopy revealed different features from T0 to T14: microvilli and a complete junctional apparatus from T6, mucus granules from T3, as also confirmed by PAS/Alcian Blue staining. The specific activity of alkaline phosphatase (ALP), aminopeptidase N (APN), and dipeptidyl peptidase IV (DPPIV) progressively increased after T0, indicating the acquirement of a differentiated and digestive phenotype. Transepithelial electrical resistance (TEER), indicative of the barrier properties of the monolayer, increased from T0 up to T6 reaching values very similar to the human small intestine. The apparent permeability coefficient for Lucifer Yellow (LY), along with morphological analysis, reveals a good status of the tight junctions. At T14, HT-29 cells reduced to 18.4% and formed domes, indicative of transepithelial transport of nutrients. This Caco2/HT-29 co-culture could be considered a versatile and suitable in vitro model of human intestinal epithelium for the presence of more than one prevalent intestinal cell type, by means of a minimum of 6 to a maximum of 14 post-confluence days obtained without the need of particular inducers of subclones and growth support to reach an intestinal differentiated phenotype.

Modulation of epidermal proliferation and terminal differentiation in a promising ex vivo human skin model mimicking a psoriatic microenvironment

Epidermal keratinocyte hyperproliferation is one of the key features involved in the formation/progression of psoriatic lesions and is driven by cytokines, among which TNF-α, IL-17, IL-22 and IL-23, secreted by both activated resident immune cells and keratinocytes (1). The network orchestrated by these cytokines is essential for the communication between resident cells and infiltrating cells and is due to redun- dancy, synergism and, sometimes, the reciprocal antagonism of cytokines. The aims of our study were to investigate whether the exposure of normal human skin to four main psoriatic cytokines, i.e. TNF-α, IL-17, IL-22, and IL-23 (cytokine mix) induced i) a modulation of epidermal proliferation and ii) a modification of keratinocyte terminal differentiation (TD). Human skin samples (n=5) obtained from healthy 20-40 years- old women after plastic surgery, were exposed to the cytokine mix in a Transwell sys- tem at air-liquid interface as previously described (2). For each patient a control (ctr) group was not exposed to cytokine mix. Samples were harvested 5 (T5), 24 (T24), 48 (T48) and 72 (T72) hours after cytokine stimulation, processed for paraffin embedding and immunofluorescence analysis for the quantitative analysis of epidermal proliferation and the expression of the TD biomarkers, keratin (K) 10 and 17. A decrease of cell proliferation was evident starting from T5 in samples exposed to cytokine mix and was progressively more marked at later time points (T5 ctr 47.44 ± 6.90 vs mix 23.07 ± 8.84; T24 ctr 41.12 ± 12.78 vs mix 12.16 ± 1.53; T48 ctr 25.88 ± 10.21 vs mix 2.19 ± 2.44; T72 ctr 10.49 ± 2.52 vs mix 0.65 ± 1.02) (p<0.05). K17 expression was evi- dent in samples exposed to the cytokine mix. Altogether the present results suggest that cell proliferation inhibition and K17 expression could be regarded as the basis for a later response to injury leading to psoriatic lesion formation/progression. In conclu- sion, this model allows to investigate the intimate interplay among different psoriatic cytokines and new insights may be of potential value for future clinical treatments.