Clinical, histological, immunohistochemical, and biomolecular analysis of hyaluronic acid in early wound healing of human gingival tissues: A randomized, split-mouth trial.

BACKGROUND: Hyaluronic acid (HA) exerts a fundamental role in tissue repair. In vitro and animal studies demonstrated its ability to enhance wound healing. Nevertheless, in vivo human studies evaluating mechanisms involved in oral soft tissue repair are lacking. The aim of this study was to evaluate the in vivo effect of HA on early wound healing of human gingival (G) tissues. METHODS: In the present randomized, split-mouth, double-blind, clinical trial, G biopsies were obtained in eight patients 24 h post-surgery after HA application (HA group) and compared with those obtained from the same patients without HA application (no treatment; NT group). Clinical response was evaluated through the Early Wound Healing Score (EHS). Microvascular density (MVD), collagen content and cellular proliferation were evaluated through sirius red and Masson trichrome staining, and Ki-67 immunohistochemistry, respectively. To assess collagen turnover, MMP-1, MMP-2, MMP-9, TGF-ß1 protein levels and LOX, MMP1, TIMP1, TGFB1 gene expression were analyzed by western blot and real time polymerase chain reaction. RESULTS: Twenty-four hours after surgery, the EHS was significantly higher in the HA group. MVD, collagen content, and cell proliferation were not affected. LOX mRNA, MMP-1 protein, and TIMP1 gene expression were significantly upregulated in the HA compared to the NT group. CONCLUSIONS: The additional use of 0.8% HA gel does not modify new blood vessel growth in the early phase of gingival wound healing. Concerning the secondary outcomes, HA seems to enhance extracellular matrix remodeling and collagen maturation, which could drive early wound healing of G tissues to improve clinical parameters.

Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study.

Duchenne muscular dystrophy (DMD) is a rare genetic disease leading to progressive muscle wasting, respiratory failure, and cardiomyopathy. Although muscle fibrosis represents a DMD hallmark, the organisation of the extracellular matrix and the molecular changes in its turnover are still not fully understood. To define the architectural changes over time in muscle fibrosis, we used an mdx mouse model of DMD and analysed collagen and glycosaminoglycans/proteoglycans content in skeletal muscle sections at different time points during disease progression and in comparison with age-matched controls. Collagen significantly increased particularly in the diaphragm, quadriceps, and gastrocnemius in adult mdx, with fibrosis significantly correlating with muscle degeneration. We also analysed collagen turnover pathways underlying fibrosis development in cultured primary quadriceps-derived fibroblasts. Collagen secretion and matrix metalloproteinases (MMPs) remained unaffected in both young and adult mdx compared to wt fibroblasts, whereas collagen cross-linking and tissue inhibitors of MMP (TIMP) expression significantly increased. We conclude that, in the DMD model we used, fibrosis mostly affects diaphragm and quadriceps with a higher collagen cross-linking and inhibition of MMPs that contribute differently to progressive collagen accumulation during fibrotic remodelling. This study offers a comprehensive histological and molecular characterisation of DMD-associated muscle fibrosis; it may thus provide new targets for tailored therapeutic interventions.

Mechanical Cues, E-Cadherin Expression and Cell "Sociality" Are Crucial Crossroads in Determining Pancreatic Ductal Adenocarcinoma Cells Behavior.

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell lines, characterized by different EMT-related phenotypes, cultured in 2D monolayers and 3D spheroids. We observed that the YAP/p-YAP ratio was reduced in HPAC and MIA PaCa-2 cell lines and remained unchanged in BxPC-3 cells when cultured in a 3D setting. CTGF and CYR61 gene expression were down-regulated in all PDAC 3D compared to 2D cultures, without any significant effect following actin cytoskeleton inhibition by Cytochalasin B (CyB) treatment. Moreover, LATS1 mRNA, indicating the activation of the Hippo pathway, was not influenced by CyB and differed in all PDAC cell lines having different EMT-related phenotype but a similar pattern of CTGF and CYR61 expression. Although the role of YAP modulation in response to mechanical cues in cancer cells remains to be completely elucidated, our results suggest that cell arrangement and phenotype can determine variable outcomes to mechanical stimuli in PDAC cells. Moreover, it is possible to speculate that YAP and Hippo pathways may act as parallel and not exclusive inputs that, converging at some points, may impact cell behavior.

Evaluation of expression of Toll-Like Receptors 7 and 9, proliferation, and cytoskeletal biomarkers in plaque and guttate psoriasis: A pilot morphological study.

This pilot study was aimed at comparing TLR7/TLR9 expression, cytoskeletal arrangement, and cell proliferation by indirect immunofluorescence in parallel lesional and non lesional skin samples of guttate psoriasis (PG) and psoriasis vulgaris (PV) in five male patients for each group (n=10). TLR7 expression was detected throughout all the epidermal compartment in PV samples, while in PG skin was restricted to the granular layer. TLR9 was present in the granular layer of non lesional skin and in the suprabasal layers of PV/PG lesional skin. Cell proliferation was localized in all the epidermal layers in lesional PG and PV, consistently with the immunopositivity for the "psoriatic keratin" K16. In the suprabasal layers of lesional PG and PV skin, a similar K17 expression was detected and K10 exhibited a patchy distribution. The present results suggest that TLR7 expression can be considered an intrinsic and differential histomorphological feature of PV.

The psoriatic shift induced by interleukin 17 is promptly reverted by a specific anti-IL-17A agent in a three-dimensional organotypic model of normal human skin culture.

Interleukin 17A (IL-17A), mainly produced by the T helper subclass Th17, plays a key role in the psoriatic plaque formation and progression. The clinical effectiveness of anti-IL-17A agents is documented, but the early and specific mechanisms of their protection are not identified yet. The challenge of the present study is to investigate the possible reversal exerted by a specific anti-IL-17A agent on the psoriatic events induced by IL-17A in a three-dimensional organotypic model of normal human skin. Bioptic skin fragments obtained after aesthetic surgery of healthy women (n=5) were incubated with i) IL-17A biological inhibitor (anti-IL-17A), ii) IL-17A, iii) a combination of IL-17A and its specific IL-17A biological inhibitor (COMBO). A Control group was in parallel cultured and incubation lasted for 24 and 48 h epidermal-side-up at the air-liquid interface. All subjects were represented in all experimental groups at all considered time-points. Keratinocyte proliferation and the presence of epidermal Langerhans cells were quantitatively estimated. In parallel with transmission electron microscopy analysis, immunofluorescence studies for the epidermal distribution of keratin (K)10, K14, K16, K17, filaggrin/occludin, Toll-like Receptor 4, and Nuclear Factor kB were performed. IL-17A inhibited cell proliferation and induced K17 expression, while samples incubated with the anti-IL-17A agent were comparable to controls. In the COMBO group the IL-17A-induced effects were almost completely reverted. Our study, for the first time, elucidates the most specific psoriatic cellular events that can be partially affected or completely reverted by a specific anti-IL-17A agent during the early phases of the plaque onset and progression. On the whole, this work contributes to expand the knowledge of the psoriatic tableau.

Study on the inflammasome nlrp3 and blimp-1/nlrp12 after keratinocyte exposure to contact allergens.

We previously demonstrated that based on their potency, contact allergens differently modulate Blimp-1/NLRP12 expression in human keratinocytes, with the extreme allergen 2,4-dinitrochlorobenzene (DNCB) more rapidly upregulating Blimp-1, leading to downregulation of NLRP12, and to the production of interleukin-18 (IL-18). The purpose of this study was to further investigate the effects of DNCB and para-phenylenediamine (PPD) on the expression of the proteins of the inflammasome, namely NLRP3, ASC and caspase 1 by western blot analysis; to define the intracellular localization and co-localization of NLRP3 and NLPR12 by immunoprecipitation and immunohistochemistry; and to define the role of NF-κB in Blimp-1 induction by pharmacological inhibition. The human keratinocyte cell line NCTC2544 was used for all experiments. Dose and time course experiments were performed to evaluate the effect of the selected contact allergens on the parameters investigated. Results indicate, that consistent with previous finding, DNCB more rapidly (3 h) induces NLRP3, ASC protein expression and caspase-1 activation compared to PPD. Immunoprecipitation studies show the recruitment of ASC to the inflammasome following exposure to both allergens, while high level of NLRP12 and less ASC protein were found associated in control cells. By immunohistochemistry, we found increased NLRP3 expression following exposure to contact allergens, and observed a nuclear co-localization of the two proteins, indicating the NLRP12 likely acts preventing the cytosolic localization of NLRP3 and inflammasome assembly. Finally, contact allergen-induced Blimp-1 mRNA and protein expression can be completely blocked by inhibiting NF-κB activation, confirming the central role of NF-κB in contact allergen-induced keratinocyte activation.

Excess of nutrient-induced morphofunctional adaptation and inflammation degree in a Caco2/HT-29 in vitro intestinal co-culture.

OBJECTIVES: The intestinal cell function can be modulated by the type and quantity of nutrients. The aim of this study was to evaluate the effects of an excess of nutrients on intestinal morphofunctional features and a possible association of inflammation in a 70/30 Caco2/HT-29 intestinal in vitro co-culture. METHODS: An excess of nutrients (EX) was obtained by progressively increasing the medium change frequency with respect to standard cell growth conditions (ST) from confluence (T0) to 15 d after confluence (T15). RESULTS: In comparison with the ST group, the EX group revealed a maintenance in the number of microvilli, an increase in follicle like-structures and mucus production, and a decrease in the number of tight junction. The specific activity of markers of intestinal differentiation, alkaline phosphatase and aminopeptidase N, and of the enterocyte differentiation specific marker, dipeptidyl peptidase-IV, were progressively raised. The transepithelial electrical resistance, indicative of the co-culture barrier properties, decreased, whereas Lucifer yellow Papp evaluation, an index of the paracellular permeability to large molecules, showed an increase. Reactive oxygen species and nitric oxide production, indicative of an oxidative status, together with interleukin-6, interleukin-8, indicative of a low-grade inflammation, and peptide YY secretion were higher in the EX group than in the ST group. The differences between ST and EX were particularly evident at T15. CONCLUSION: These data support the suitability of our in vitro gut model for obesity studies at the molecular level and the necessity to standardize the medium frequency change in intestinal culture.

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.

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.

Epidermal barrier reaction to an in vitro psoriatic microenvironment.

Keratinocytes (KCs) and Langerhans cells (LCs) contribute to create the epidermal barrier. To form a functional epidermis, KCs express filaggrin and Toll-like Receptors (TLRs). LCs are the first line of epidermal defence and can be activated by interleukin (IL)-17 and Tumor Necrosis Factor (TNF)-alpha. In psoriasis, an alteration of TLR expression, a defective expression of filaggrin, and LC activation occur. In organotypic cultures of human skin we investigated the interplay between IL-17 and TNF-alpha on i) expression of filaggrin, TLR2, 7 and 9, and Nuclear Factor (NF)-kB localization by immunofluorescence and ii) LC ultrastructural features by transmission electron microscopy. Normal human skin was obtained after aesthetic surgery (n=7), overnight incubated in a Transwell system, and exposed to TNF-alpha and/or IL-17 for 24 (T24), 48 (T48), and 72 (T72) hours. Cytokines always influenced the expression of filaggrin. TNF-alpha alone activated LCs only starting from T48. TLR2 and TLR7 expressions were affected at T24 by IL-17 and the combination of cytokines, but not by TNF-alpha. TLR9-positive cells were detectable in the granular layer after cytokine exposure. A nuclear localization of NF-kB was always observed after cytokine incubation. In conclusion, each cytokine possess an intrinsic activity on the different components of the epidermal barrier.

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.

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.

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.

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.

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.

Mice lacking mitochondrial ferritin are more sensitive to doxorubicin-mediated cardiotoxicity.

UNLABELLED: Mitochondrial ferritin is a functional ferritin that localizes in the mitochondria. It is expressed in the testis, heart, brain, and cells with active respiratory activity. Its overexpression in cultured cells protected against oxidative damage and reduced cytosolic iron availability. However, no overt phenotype was described in mice with inactivation of the FtMt gene. Here, we used the doxorubicin model of cardiac injury in a novel strain of FtMt-null mice to investigate the antioxidant role of FtMt. These mice did not show any evident phenotype, but after acute treatment to doxorubicin, they showed enhanced mortality and altered heart morphology with fibril disorganization and severe mitochondrial damage. Signs of mitochondrial damage were present also in mock-treated FtMt(-/-) mice. The hearts of saline- and doxorubicin-treated FtMt(-/-) mice had higher thiobarbituric acid reactive substance levels, heme oxygenase 1 expression, and protein oxidation, but did not differ from FtMt(+/+) in the cardiac damage marker B-type natriuretic peptide (BNP), ATP levels, and apoptosis. However, the autophagy marker LC3 was activated. The results show that the absence of FtMt, which is highly expressed in the heart, increases the sensitivity of heart mitochondria to the toxicity of doxorubicin. This study represents the first in vivo evidence of the antioxidant role of FtMt. KEY MESSAGE: Mitochondrial ferritin (FtMt) expressed in the heart has a protective antioxidant role. Acute treatment with doxorubicin caused the death of all FtMt(-/-) and only of 60 % FtMt(+/+) mice. The hearts of FtMt(-/-) mice showed fibril disorganization and mitochondrial damage. Markers of oxidative damage and autophagy were increased in FtMt(-/-) hearts. This is the first in vivo evidence of the antioxidant role of FtMt.