Essential Oil from Eucalyptus globulus (Labill.) Activates Complement Receptor-Mediated Phagocytosis and Stimulates Podosome Formation in Human Monocyte-Derived Macrophages.

Eucalyptus essential oil and its major constituent eucalyptol are extensively employed in the cosmetic, food, and pharmaceutical industries and their clinical use has recently expanded worldwide as an adjuvant in the treatment of infective and inflammatory diseases. We previously demonstrated that essential oil from Eucalyptus globulus (Labill.) (EO) stimulates in vitro the phagocytic activity of human monocyte-derived macrophages and counteracts the myelotoxicity induced by the chemotherapeutic 5-fluorouracil in immunocompetent rats. Here we characterize some mechanistic aspects underlying the immunostimulatory ability exerted by EO on macrophages. The internalization of fluorescent beads, fluorescent zymosan BioParticles, or apoptotic cancer cells was evaluated by confocal microscopy. Pro-inflammatory cytokine and chemokine release was determined by flow cytometry using the BD cytometric bead array. Receptor involvement in EO-stimulated phagocytosis was assessed using complement- or IgG-opsonized zymosan particles. The localization and expression of podosome components was analyzed by confocal microscopy and western blot. The main results demonstrated that: EO-induced activation of a macrophage is ascribable to its major component eucalyptol, as recently demonstrated for other cells of innate immunity; EO implements pathogen internalization and clearance by stimulating the complement receptor-mediated phagocytosis; EO stimulates podosome formation and increases the expression of podosome components. These results confirm that EO extract is a potent activator of innate cell-mediated immunity and thereby increase the scientific evidence supporting an additional property of this plant extract besides the known antiseptic and anti-inflammatory properties.

Suspected adverse drug reactions (ADRs) trends in older Italian patients: an analysis from the National Pharmacovigilance Network.

BACKGROUND: The clinical management of older populations is a social and clinical challenge. These subjects are more vulnerable to adverse drug reactions (ADRs) due to multi morbidity, polypharmacy, and physio-pathological changes. METHODS: We performed a study using data from the National Network of Pharmacovigilance of Italy available from 2001 to 2012, which include all spontaneous reports of suspected ADRs. RESULTS: Our study showed that 29,036 reports of suspected ADRs were recorded in 2012. Those for patients aged ≥ 65 years were 11,426 that correspond to a reporting rate of 923.6 reports per million inhabitants. On the contrary, the reporting rate for total population was of 489 reports per million inhabitants. The evaluation of level of severity showed that 37% of reports of suspected ADRs in patients aged ≥ 65 years were serious compared to 30% in the general population. Furthermore, 27% of reports recorded for older patients showed that they required more hospitalization or long-term care in comparison with the 21% for the general population, with a relevant impact on National Health Service costs. CONCLUSION: These data suggest the need to optimize the clinical strategy for older patients due to the multiple factors that increase the susceptibility to ADRs. There is a high need to potentiate clinical trials on older patients to adopt new diagnostic-therapeutic pathways in real life.

Hovenia dulcis Thumberg: Phytochemistry, Pharmacology, Toxicology and Regulatory Framework for Its Use in the European Union.

Hovenia dulcis Thunberg is an herbal plant, belonging to the Rhamnaceae family, widespread in west Asia, USA, Australia and New Zealand, but still almost unknown in Western countries. H. dulcis has been described to possess several pharmacological properties, such as antidiabetic, anticancer, antioxidant, anti-inflammatory and hepatoprotective, especially in the hangover treatment, validating its use as an herbal remedy in the Chinese Traditional Medicine. These biological properties are related to a variety of secondary metabolites synthesized by the different plant parts. Root, bark and leaves are rich of dammarane-type triterpene saponins; dihydrokaempferol, quercetin, 3,3',5',5,7-pentahydroflavone and dihydromyricetin are flavonoids isolated from the seeds; fruits contain mainly dihydroflavonols, such as dihydromyricetin (or ampelopsin) and hovenodulinol, and flavonols such as myricetin and gallocatechin; alkaloids were found in root, barks (frangulanin) and seeds (perlolyrin), and organic acids (vanillic and ferulic) in hot water extract from seeds. Finally, peduncles have plenty of polysaccharides which justify the use as a food supplement. The aim of this work is to review the whole scientific production, with special focus on the last decade, in order to update phytochemistry, biological activities, nutritional properties, toxicological aspect and regulatory classification of H. dulcis extracts for its use in the European Union.

PDGF receptor alpha inhibition induces apoptosis in glioblastoma cancer stem cells refractory to anti-Notch and anti-EGFR treatment.

BACKGROUND: Cancer stem cells (CSC) represent a rare fraction of cancer cells characterized by resistance to chemotherapy and radiation, therefore nowadays there is great need to develop new targeted therapies for brain tumors and our study aim to target pivotal transmembrane receptors such as Notch, EGFR and PDGFR, which are already under investigation in clinical trials setting for the treatment of Glioblastoma Multiforme (GBM). METHODS: MTS assay was performed to evaluate cells response to pharmacological treatments. Quantitative RT-PCR and Western blots were performed to state the expression of Notch1, EGFR and PDGFRα/ß and the biological effects exerted by either single or combined targeted therapy in GBM CSC. GBM CSC invasive ability was tested in vitro in absence or presence of Notch and/or EGFR signaling inhibitors. RESULTS: In this study, we investigated gene expression and function of Notch1, EGFR and PDGFR to determine their role among GBM tumor core- (c-CSC) vs. peritumor tissue-derived cancer stem cells (p-CSC) of six cases of GBM. Notch inhibition significantly impaired cell growth of c-CSC compared to p-CSC pools, with no effects observed in cell cycle distribution, apoptosis and cell invasion assays. Instead, anti-EGFR therapy induced cell cycle arrest, sometimes associated with apoptosis and reduction of cell invasiveness in GBM CSC. In two cases, c-CSC pools were more sensitive to simultaneous anti-Notch and anti-EGFR treatment than either therapy alone compared to p-CSC, which were mostly resistant to treatment. We reported the overexpression of PDGFRα and its up-regulation following anti-EGFR therapy in GBM p-CSC compared to c-CSC. RNA interference of PDGFRα significantly reduced cell proliferation rate of p-CSC, while its pharmacological inhibition with Crenolanib impaired survival of both CSC pools, whose effects in combination with EGFR inhibition were maximized. CONCLUSIONS: We have used different drugs combination to identify the more effective therapeutic targets for GBM CSC, particularly against GBM peritumor tissue-derived CSC, which are mostly resistant to treatments. Overall, our results provide the rationale for simultaneous targeting of EGFR and PDGFR, which would be beneficial in the treatment of GBM.

Detection of high levels of Survivin-immunoglobulin M immune complex in sera from hepatitis C virus infected patients with cirrhosis.

AIM: The identification and surveillance of patients with liver dysfunctions and the discovering of new disease biomarkers are needed in the clinical practice. The aim of this study was to investigate on Survivin-immunoglobulin (Ig)M immune complex (IC) as a potential biomarker of chronic liver diseases. METHODS: Serum levels of Survivin-IgM were measured using an enzyme-linked immunoassay that had been standardized and validated in our laboratory in 262 individuals, including healthy subjects and patients with chronic viral hepatitis, cirrhosis and hepatocellular carcinoma (HCC). RESULTS: Survivin-IgM IC was lower in healthy subjects (median, 99.39 AU/mL) than in patients with chronic viral hepatitis (median, 148.03 AU/mL; P = 0.002) or with cirrhosis (median, 371.00 AU/mL; P < 0.001). Among patients with cirrhosis, those with hepatitis C virus (HCV) infection showed the highest level of Survivin-IgM IC (median, 633.71 AU/mL; P < 0.001). The receiver-operator curve analysis revealed that Survivin-IgM accurately distinguishes HCV correlated cirrhosis from chronic viral hepatitis (area under the curve [AUC], 0.738; sensitivity, 74.5%; specificity, 70.7%). A multivariate logistic regression model, including Survivin-IgM IC, aspartate aminotransferase (AST) and AST/alanine aminotransferase (ALT) ratio increased the prediction accuracy for the identification of the cirrhotic HCV patients (AUC, 0.818; sensitivity, 87.2%; specificity, 65.9%). Conversely, Survivin-IgM IC significantly decreased in HCC patients (median, 165.72 AU/mL; P = 0.022). CONCLUSION: Our results suggest that Survivin-IgM immune complex may be used as a potential biomarker for liver damage, particularly for the identification of the HCV-related cirrhotic population.

Anti-proliferative effect of atrial natriuretic peptide on colorectal cancer cells: evidence for an Akt-mediated cross-talk between NHE-1 activity and Wnt/ß-catenin signaling.

Acidic tumor microenvironment and Wnt/ß-catenin pathway activation have been recognized as two crucial events associated with the initiation and progression of cancer. The aim of this study was to clarify the molecular mechanisms underlying the anti-proliferative effects of atrial natriuretic peptide (ANP) as well as to investigate the relationship between the cellular pH and the Wnt/ß-catenin signaling in cancer cells.To pursue our aims, we conducted investigations in DHD/K12/Trb rat colon adenocarcinoma cells. Intracellular pH was measured by Confocal Laser Scanning Microscopy (CLSM) using the lysosensor Green DND-189 probe. Expression of crucial molecules in the Wnt/ß-catenin signaling pathway was analyzed by CLSM, western blot, and real time PCR. Measurements of activation (phosphorylation state) of Akt, ERK1/2, and p38MAPKinase were performed by Reverse-Phase Protein Microarray Analysis (RPMA).We showed that ANP triggered a NHE-1-mediated increase of the intracellular acidity, inhibiting the Wnt/ß-catenin signaling simultaneously. Moreover, we observed that the Wnt1a, a Wnt signaling activator, affected the intracellular pH in an opposite fashion. Results from the comparative analysis of ANP and EIPA (a NHE-1 specific inhibitor) showed that these two molecules affect both the intracellular acidification and the Wnt/ß-catenin signaling cascade. Specifically, ANP acts on the upstream of the cascade, through a Frizzled-mediated activation, while EIPA does on the downstream.We show for the first time that the Akt activity might be a relevant molecular event linking the NHE-1-regulated intracellular pH and the Wnt/ß-catenin signaling. This provides evidence for a cross-talk between the intracellular alkalinization and the Wnt signaling in tumor cells.

TCTP is a critical survival factor that protects cancer cells from oxidative stress-induced cell-death.

The translationally controlled tumor protein (TCTP) displays growth-promoting and antiapoptotic properties. To gain information on the role of TCTP in cancer disease, we studied the modulation of TCTP and cell survival under stress conditions on tumor cell lines of different origins. When cancer cells were exposed to a mild oxidative stress, such low doses of Arsenic trioxide (ATO) or hydrogen peroxide (H(2)O(2)), up-regulation of TCTP was observed in cells survived to the treatment. Differently, a strong oxidative hit provided by ATO combined with glutathione (GSH) depletion or condition of glucose deprivation caused a down-modulation of TCTP followed by cell death. Clones with a forced expression of TCTP or with silenced TCTP were obtained from the breast cancer cell line MDA-MB-231. The sensitivity to oxidative stress was strongly enhanced in down-modulated TCTP cells while decreasing in cells with high levels of TCTP. Together these results indicate that TCTP is a survival factor that protects cancer cells from oxidative stress-induced cell-death. We propose TCTP as a "stress hallmark" that may be exploited as a therapeutic target to decrease the resistance of cancer cells to anticancer therapy.

Treatment of doxorubicin-resistant MCF7/Dx cells with nitric oxide causes histone glutathionylation and reversal of drug resistance.

Acquired drug resistance was found to be suppressed in the doxorubicin-resistant breast cancer cell line MCF7/Dx after pre-treatment with GSNO (nitrosoglutathione). The effect was accompanied by enhanced protein glutathionylation and accumulation of doxorubicin in the nucleus. Among the glutathionylated proteins, we identified three members of the histone family; this is, to our knowledge, the first time that histone glutathionylation has been reported. Formation of the potential NO donor dinitrosyl-diglutathionyl-iron complex, bound to GSTP1-1 (glutathione transferase P1-1), was observed in both MCF7/Dx cells and drug-sensitive MCF7 cells to a similar extent. In contrast, histone glutathionylation was found to be markedly increased in the resistant MCF7/Dx cells, which also showed a 14-fold higher amount of GSTP1-1 and increased glutathione concentration compared with MCF7 cells. These results suggest that the increased cytotoxic effect of combined doxorubicin and GSNO treatment involves the glutathionylation of histones through a mechanism that requires high glutathione levels and increased expression of GSTP1-1. Owing to the critical role of histones in the regulation of gene expression, the implication of this finding may go beyond the phenomenon of doxorubicin resistance.

Multi-approach LC-MS methods for the characterization of species-specific attributes of monoclonal antibodies from plants.

In this work, an analytical platform based on the use of chromatography and mass spectrometry (MS), has been applied to the characterization of Rituximab (RTX) obtained from two plant expression systems (rice and tobacco) in comparison to the mammalian cell-derived reference monoclonal antibody (mAb). Different chromatographic approaches, hyphenated to high resolution MS (HRMS), were applied to RTX structural investigation both at middle- and peptide level. In particular, cation exchange chromatography (CEX), size exclusion chromatography (SEC), reversed phase (RPLC) and hydrophilic interaction liquid chromatographic (HILIC) methods were developed and applied on intact mAbs, IdeS-, and trypsin digests in order to address critical attributes such as primary structure, glycan composition, species-related heterogeneity, glycosylation degree, charge variants, aggregation tendency and enzymatic stability. All the collected data highlight the features and criticalities of each production approach. Production in rice results in a heterogeneous but stable product over time, suggesting the absence of proteases in seeds; while tobacco expression system leads to more homogeneous glycosylation, but protein stability seems to be a critical issue probably due to the presence of proteases. This analytical strategy represents a robust support to scientists in the selection and optimization of the best plant expression system to produce recombinant humanized mAbs.

Genetic immunization with CDR3-based fusion vaccine confers protection and long-term tumor-free survival in a mouse model of lymphoma.

Therapeutic vaccination against idiotype is a promising strategy for immunotherapy of B-cell malignancies. We have previously shown that CDR3-based DNA immunization can induce immune response against lymphoma and explored this strategy to provide protection in a murine B-cell lymphoma model. Here we performed vaccination employing as immunogen a naked DNA fusion product. The DNA vaccine was generated following fusion of a sequence derived from tetanus toxin fragment C to the V(H)CDR3(109-116) epitope. Induction of tumor-specific immunity as well as ability to inhibit growth of the aggressive 38C13 lymphoma and to prolong survival of vaccinated mice has been tested. We determined that DNA fusion vaccine induced immune response, elicited a strong protective antitumor immunity, and ensured almost complete long-term tumor-free survival of vaccinated mice. Our results show that CDR3-based DNA fusion vaccines hold promise for vaccination against lymphoma.

Nonalcoholic Fatty Liver Disease Is Associated With Low Skeletal Muscle Mass in Overweight/Obese Youths.

Background: Recent studies in adult non-elderly and elderly individuals have reported a link between nonalcoholic fatty liver disease (NAFLD) and sarcopenia. Nonetheless, whether this relationship would be found outside these populations it is still unknown. Hence, we evaluated the relationship between NAFLD and skeletal muscle mass in children and adolescents with overweight/obesity. Methods: Two-hundred and thirty-four overweight/obese youths were enrolled. NAFLD was diagnosed by ultrasononography, after exclusion of infectious and metabolic disorders. Forty of the patients with NAFLD had also liver biopsy. Total and regional lean body mass and total fat mass measurements were obtained by dual-energy X-ray absorptiometry. The relative muscle mass (RMM) was defined as the percent of muscle mass (kg) relative to the sum of muscle and fat (kg) mass. Appendicular skeletal muscle mass (ASM) was calculated by the sum of muscle masses of the four limbs (kg), and expressed as percent of body weight. Results: Subjects were stratified according to tertiles of RMM. The prevalence of abdominal obesity, dyslipidemia, insulin resistance, metabolic syndrome, NAFLD as well as biopsy-proven nonalcoholic steatohepatitis (NASH) was significantly increased in the lowest tertile of RMM. After controlling for age, sex and Tanner stage, children in the lowest tertile of RMM had an increased risk for NAFLD (OR= 2.80, 95% CI=1.57-5.02) compared to those in the other two tertiles. This association persisted after additional adjustments for clinical and metabolic variables. Similarly, the risk of NAFLD in the lowest tertile of ASM/weight index was significantly higher compared to those in the other two tertiles after adjustment for the above confounders. Conclusions: This is the first study to establish an independent association between low muscle mass and NAFLD/NASH in overweight/obese youths. Considering the worldwide increase of pediatric obesity, measurements of muscle mass may serve as useful method of identifying among obese children those at high metabolic risk who may need intensive lifestyle interventions to prevent NAFLD and its progression.

Nature-derived compounds modulating Wnt/ ß -catenin pathway: a preventive and therapeutic opportunity in neoplastic diseases.

The Wnt/ß-catenin signaling is a conserved pathway that has a crucial role in embryonic and adult life. Dysregulation of the Wnt/ß-catenin pathway has been associated with diseases including cancer, and components of the signaling have been proposed as innovative therapeutic targets, mainly for cancer therapy. The attention of the worldwide researchers paid to this issue is increasing, also in view of the therapeutic potential of these agents in diseases, such as Parkinson's disease (PD), for which no cure is existing today. Much evidence indicates that abnormal Wnt/ß-catenin signaling is involved in tumor immunology and the targeting of Wnt/ß-catenin pathway has been also proposed as an attractive strategy to potentiate cancer immunotherapy. During the last decade, several products, including naturally occurring dietary agents as well as a wide variety of products from plant sources, including curcumin, quercetin, berberin, and ginsenosides, have been identified as potent modulators of the Wnt/ß-catenin signaling and have gained interest as promising candidates for the development of chemopreventive or therapeutic drugs for cancer. In this review we make an overview of the nature-derived compounds reported to have antitumor activity by modulating the Wnt/ß-catenin signaling, also focusing on extraction methods, chemical features, and bio-activity assays used for the screening of these compounds.

Carotid Extra-Media Thickness in Children: Relationships With Cardiometabolic Risk Factors and Endothelial Function.

Background: Emerging evidence suggests that structural adventitial modifications and perivascular adipose tissue (PAT) may have a role in early atherogenesis. In a cohort of children and adolescents, we explored (1) the association of carotid extra-media thickness (cEMT), an ultrasound measure whose main determinants are arterial adventitia and PAT, with obesity and its cardiometabolic complications; and (2) the interplay between cEMT and endothelial function. Methods: The study participants included 286 youths (age, 6-16 years; 154 boys, and 132 girls). Anthropometric and laboratory parameters, liver ultrasound, vascular structure measures [cEMT and carotid intima-media thickness (cIMT)], endothelial function [brachial artery flow-mediated dilation (FMD)] were obtained in all subjects. Non-alcoholic fatty liver disease (NAFLD) was diagnosed in the presence of hepatic fat on ultrasonography, in the absence of other causes of liver disease. Diagnosis of metabolic syndrome (MetS) was established on the basis of three or more of the following cardiovascular disease (CVD) risk variables: abdominal obesity, high triglycerides, low high-density lipoprotein cholesterol, elevated blood pressure (BP), and impaired fasting glucose. Results: cEMT demonstrated significant associations with body-mass index (BMI) and waist circumference (WC), BP, insulin resistance, NAFLD, and inflammation. No association was found between cEMT and lipid values, and between cEMT and MetS. A stepwise multivariate linear regression analysis indicated that WC (ß coefficient, 0.35; P < 0.0001) was the only determinant of cEMT, independently of other major cardiometabolic risk factors. Further adjustment for cIMT did not significantly alter this association. FMD was correlated to age, Tanner stage, total and abdominal obesity, BP, NAFLD, and cEMT. The association between FMD and cEMT was independent of age, sex, Tanner stage, WC, and BMI (ß coefficient, -0.14; P = 0.027). After controlling for CVD risk factors and basal brachial artery diameter, cEMT remained associated with FMD (ß coefficient, -0.11; P = 0.049). Conclusions: In youths, cEMT is associated with abdominal fat, a well-established body fat depot with important implications for cardiovascular diseases. Furthermore, cEMT is related to FMD, suggesting that arterial adventitia and PAT may be involved in the early changes in endothelial function.

Preclinical Validation of SilkBridgeTM for Peripheral Nerve Regeneration.

Silk fibroin (Bombyx mori) was used to manufacture a nerve conduit (SilkBridgeTM) characterized by a novel 3D architecture. The wall of the conduit consists of two electrospun layers (inner and outer) and one textile layer (middle), perfectly integrated at the structural and functional level. The manufacturing technology conferred high compression strength on the device, thus meeting clinical requirements for physiological and pathological compressive stresses. As demonstrated in a previous work, the silk material has proven to be able to provide a valid substrate for cells to grow on, differentiate and start the fundamental cellular regenerative activities in vitro and, in vivo, at the short time point of 2 weeks, to allow the starting of regenerative processes in terms of good integration with the surrounding tissues and colonization of the wall layers and of the lumen with several cell types. In the present study, a 10 mm long gap in the median nerve was repaired with 12 mm SilkBridgeTM conduit and evaluated at middle (4 weeks) and at longer time points (12 and 24 weeks). The SilkBridgeTM conduit led to a very good functional and morphological recovery of the median nerve, similar to that observed with the reference autograft nerve reconstruction procedure. Taken together, all these results demonstrated that SilkBridgeTM has an optimized balance of biomechanical and biological properties, which allowed proceeding with a first-in-human clinical study aimed at evaluating safety and effectiveness of using the device for the reconstruction of digital nerve defects in humans.

The Iterative Development of Medicines Through the European Medicine Agency's Adaptive Pathway Approach.

The development of novel regulatory tools such as adaptive clinical trial design and utilization of real-world evidence are topics of high interest. Recently, the European Medicines Agency (EMA) introduced the Adaptive Pathways (AP) that represents an innovative tool in healthcare systems allowing the early dialogue with multiple stakeholders on promising and innovative medicinal products in areas with an high unmet medical need. The innovative aspect in the AP is the early involvement of several stakeholders such as pharmaceutical industry, the Academia, Health Technology Assessment (HTA) bodies, and patient representatives bringing their real experience with the disease and their expectations about the treatment. AP is not a new licensing tool but an opportunity for a very early discussions, before starting the phase II studies, among all stakeholders, including regulators, companies, HTA bodies, and patient representatives on a new potential medicine in areas of high unmet medical need. The aim of this paper is to describe the evolution of the AP approach from the beginning of the pilot project to date, highlighting major advances, and achievement at European level.

Association between Vitamin D Levels and Nonalcoholic Fatty Liver Disease: Potential Confounding Variables.

Nonalcoholic fatty liver disease (NAFLD), historically considered to be the hepatic component of the metabolic syndrome, is a spectrum of fat-associated liver conditions, in the absence of secondary causes, that may progress to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Disease progression is closely associated with body weight or fatness, dyslipidemia, insulin resistance, oxidative stress, and inflammation. Recently, vitamin D deficiency has been linked to the pathogenesis and severity of NAFLD because of vitamin D "pleiotropic" functions, with roles in immune modulation, cell differentiation and proliferation, and regulation of inflammation. Indeed, several studies have reported an association between vitamin D and NAFLD/NASH. However, other studies have failed to find an association. Therefore, we sought to critically review the current evidence on the association between vitamin D deficiency and NAFLD/NASH, and to analyze and discuss some key variables that may interfere with this evaluation, such as host-, environment-, and heritability-related factors regulating vitamin D synthesis and metabolism; definitions of deficient or optimal vitamin D status with respect to skeletal and nonskeletal outcomes including NAFLD/NASH; methods of measuring 25(OH)D; and methods of diagnosing NAFLD as well as quantifying adiposity, the cardinal link between vitamin D deficiency and NAFLD.

Bioassay-Guided Isolation of Nigracin, Responsible for the Tissue Repair Properties of Drypetes Klainei Stem Bark.

Drypetes klainei Pierre ex Pax is used in Cameroon by Baka people in the wound healing process and for the treatment of burns. In a previous paper we demonstrated the ability of both water (WE) and defatted methanol (DME) extracts to accelerate scratch wound closure in fibroblast cultures, thus validating the traditional use of D. klainey stem bark in the treatment of skin lesions. In this work we carried out a bioassay-guided fractionation of the most active DME, which exhibited in vitro efficacy in accelerating wound healing process, in order to isolate and identify the compound/s responsible for the assessed biological activity. HPLC was used for the metabolite profiling of DME and fractions (analytical) and for the isolation of the bioactive compound (semi-preparative). MS analyses and NMR spectroscopy were used for identifying the isolated compound. The abilities of treatments in accelerating wound healing were studied on murine fibroblasts in terms of cell viability and cell migration (scratch wound-healing assay). The results obtained allowed to unambiguously identify the isolated bioactive compound as nigracin, a known phenolic glycoside firstly isolated and characterized from bark and leaves of Populus nigra in 1967. However, this is the first time that nigracin is identified in the Drypetes genus and that a wound healing activity is demonstrated for this molecule. Specifically, we demonstrated that nigracin significantly stimulates fibroblast growth and improves cell motility and wound closure of fibroblast monolayer in a dose-dependent manner, without any toxicity at the concentrations tested, and is still active at very low doses. This makes the molecule particularly attractive as a possible candidate for developing new therapeutic options for wound care.

Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests.

Silk fibroin (SF) is an eligible biomaterial for the development of small caliber vascular grafts for substitution, repair, and regeneration of blood vessels. This study presents the properties of a newly designed multi-layered SF tubular scaffold for vascular grafting (SilkGraf). The wall architecture consists of two electrospun layers (inner and outer) and an intermediate textile layer. The latter was designed to confer high mechanical performance and resistance on the device, while electrospun layers allow enhancing its biomimicry properties and host's tissues integration. In vitro cell interaction studies performed with adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) demonstrated that the electrospun layers favor cell adhesion, survival, and growth. Once cultured in vitro on the SF scaffold the three cell types showed an active metabolism (consumption of glucose and glutamine, release of lactate), and proliferation for up to 20 days. HAAF cells grown on SF showed a significantly lower synthesis of type I procollagen than on polystyrene, meaning a lower fibrotic effect of the SF substrate. The cytokine and chemokine expression patterns were investigated to evaluate the cells' proliferative and pro-inflammatory attitude. Interestingly, no significant amounts of truly pro-inflammatory cytokines were secreted by any of the three cell types which exhibited a clearly proliferative profile. Good hemocompatibility was observed by complement activation, hemolysis, and hematology assays. Finally, the results of an in vivo preliminary pilot trial on minipig and sheep to assess the functional behavior of implanted SF-based vascular graft identified the sheep as the more apt animal model for next medium-to-long term preclinical trials.

Stimulatory effect of Eucalyptus essential oil on innate cell-mediated immune response.

BACKGROUND: Besides few data concerning the antiseptic properties against a range of microbial agents and the anti-inflammatory potential both in vitro and in vivo, little is known about the influence of Eucalyptus oil (EO) extract on the monocytic/macrophagic system, one of the primary cellular effectors of the immune response against pathogen attacks. The activities of this natural extract have mainly been recognized through clinical experience, but there have been relatively little scientific studies on its biological actions. Here we investigated whether EO extract is able to affect the phagocytic ability of human monocyte derived macrophages (MDMs) in vitro and of rat peripheral blood monocytes/granulocytes in vivo in absence or in presence of immuno-suppression induced by the chemotherapeutic agent 5-fluorouracil (5-FU). METHODS: Morphological activation of human MDMs was analysed by scanning electron microscopy. Phagocytic activity was tested: i) in vitro in EO treated and untreated MDMs, by confocal microscopy after fluorescent beads administration; ii) in vivo in monocytes/granulocytes from peripheral blood of immuno-competent or 5-FU immuno-suppressed rats, after EO oral administration, by flow cytometry using fluorescein-labelled E. coli. Cytokine release by MDMs was determined using the BD Cytometric Bead Array human Th1/Th2 cytokine kit. RESULTS: EO is able to induce activation of MDMs, dramatically stimulating their phagocytic response. EO-stimulated internalization is coupled to low release of pro-inflammatory cytokines and requires integrity of the microtubule network, suggesting that EO may act by means of complement receptor-mediated phagocytosis. Implementation of innate cell-mediated immune response was also observed in vivo after EO administration, mainly involving the peripheral blood monocytes/granulocytes. The 5-FU/EO combined treatment inhibited the 5-FU induced myelotoxicity and raised the phagocytic activity of the granulocytic/monocytic system, significantly decreased by the chemotherapic. CONCLUSION: Our data, demonstrating that Eucalyptus oil extract is able to implement the innate cell-mediated immune response, provide scientific support for an additional use of this plant extract, besides those concerning its antiseptic and anti-inflammatory properties and stimulate further investigations also using single components of this essential oil. This might drive development of a possible new family of immuno-regulatory agents, useful as adjuvant in immuno-suppressive pathologies, in infectious disease and after tumour chemotherapy.

Anti-tumor immunity induced by CDR3-based DNA vaccination in a murine B-cell lymphoma model.

The idiotypic structure present on B-cell neoplasms is a tumor-specific antigen and an attractive target for immunotherapy. Here, the tumor protective effects recruited by CDR3-based DNA vaccines in the poorly immunogenic, highly aggressive 38C13 murine B-cell lymphoma model were evaluated. The regions belonging to the idiotypic V(H) and V(L) CDR3 sequences were chosen for the design of two synthetic mini-genes and arranged in high-level expression plasmids. Syngeneic C3H/HeN mice were immunized by intramuscular electroporation with pV(H)CDR3-IL-2 and pV(L)CDR3-IL-2 naked DNAs. This approach provided protection in about 60% of animals challenged with a 2-fold lethal dose of tumor cells, as opposed to non-survivors in control groups. Furthermore, a long-term survival was induced in these mice since they were still alive and tumor-free 4 months following tumor challenge. Analysis of the humoral immunity revealed the presence of antibodies reactive with the peptides encompassing the CDR3 sequences in the sera of vaccinated mice. Moreover, immune sera specifically reacted with the parental 38C13 tumor cells in flow cytometry assays, indicating that such immunization elicited anti-idiotypic antibodies. These findings provide a basis for exploring the use of CDR3-based DNA vaccines against B-cell lymphoma.