Caspase-8 is required for HSV-1-induced apoptosis and promotes effective viral particle release via autophagy inhibition.

Regulated cell death (RCD) plays an important role in the progression of viral replication and particle release in cells infected by herpes simplex virus-1 (HSV-1). However, the kind of RCD (apoptosis, necroptosis, others) and the resulting cytopathic effect of HSV-1 depends on the cell type and the species. In this study, we further investigated the molecular mechanisms of apoptosis induced by HSV-1. Although a role of caspase-8 has previously been suggested, we now clearly show that caspase-8 is required for HSV-1-induced apoptosis in a FADD-/death receptor-independent manner in both mouse embryo fibroblasts (MEF) and human monocytes (U937). While wild-type (wt) MEFs and U937 cells exhibited increased caspase-8 and caspase-3 activation and apoptosis after HSV-1 infection, respective caspase-8-deficient (caspase-8-/-) cells were largely impeded in any of these effects. Unexpectedly, caspase-8-/- MEF and U937 cells also showed less virus particle release associated with increased autophagy as evidenced by higher Beclin-1 and lower p62/SQSTM1 levels and increased LC3-I to LC3-II conversion. Confocal and electron microscopy revealed that HSV-1 stimulated a strong perinuclear multivesicular body response, resembling increased autophagy in caspase-8-/- cells, entrapping virions in cellular endosomes. Pharmacological inhibition of autophagy by wortmannin restored the ability of caspase-8-/- cells to release viral particles in similar amounts as in wt cells. Altogether our results support a non-canonical role of caspase-8 in both HSV-1-induced apoptosis and viral particle release through autophagic regulation.

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.

Natriuretic peptides are neuroprotective on in vitro models of PD and promote dopaminergic differentiation of hiPSCs-derived neurons via the Wnt/ß-catenin signaling.

Over the last 20 years, the efforts to develop new therapies for Parkinson's disease (PD) have focused not only on the improvement of symptomatic therapy for motor and non-motor symptoms but also on the discovering of the potential causes of PD, in order to develop disease-modifying treatments. The emerging role of dysregulation of the Wnt/ß-catenin signaling in the onset and progression of PD, as well as of other neurodegenerative diseases (NDs), renders the targeting of this signaling an attractive therapeutic opportunity for curing this brain disorder. The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), are cardiac and vascular-derived hormones also widely expressed in mammalian CNS, where they seem to participate in numerous brain functions including neural development/differentiation and neuroprotection. We recently demonstrated that ANP affects the Wnt/ß-catenin pathway possibly through a Frizzled receptor-mediated mechanism and that it acts as a neuroprotective agent in in vitro models of PD by upregulating this signaling. Here we provide further evidence supporting the therapeutic potential of this class of natriuretic hormones. Specifically, we demonstrate that all the three natriuretic peptides are neuroprotective for SHSY5Y cells and primary cultures of DA neurons from mouse brain, subjected to neurotoxin insult with 6-hydroxydopamine (6-OHDA) for mimicking the neurodegeneration of PD, and these effects are associated with the activation of the Wnt/ß-catenin pathway. Moreover, ANP, BNP, CNP are able to improve and accelerate the dopaminergic differentiation and maturation of hiPSCs-derived neural population obtained from two differed healthy donors, concomitantly affecting the canonical Wnt signaling. Our results support the relevance of exogenous ANP, BNP, and CNP as attractive molecules for both neuroprotection and neurorepair in PD, and more in general, in NDs for which aberrant Wnt signaling seems to be the leading pathogenetic mechanism.

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.

Diesel exhaust particles induce autophagy and citrullination in Normal Human Bronchial Epithelial cells.

A variety of environmental agents has been found to influence the development of autoimmune diseases; in particular, the studies investigating the potential association of systemic autoimmune rheumatic diseases with environmental micro and nano-particulate matter are very few and contradictory. In this study, the role of diesel exhaust particles (DEPs), one of the most important components of environment particulate matter, emitted from Euro 4 and Euro 5 engines in altering the Normal Human Bronchial Epithelial (NHBE) cell biological activity was evaluated. NHBE cells were exposed in vitro to Euro 4 and Euro 5 particle carbon core, sampled upstream of the typical emission after-treatment systems (diesel oxidation catalyst and diesel particulate filter), whose surfaces have been washed from well-assessed harmful species, as polycyclic aromatic hydrocarbons (PAHs) to: (1) investigate their specific capacity to affect cell viability (flow cytometry); (2) stimulate the production of the pro-inflammatory cytokine IL-18 (Enzyme-Linked ImmunoSorbent Assay -ELISA-); (3) verify their specific ability to induce autophagy and elicit protein citrullination and peptidyl arginine deiminase (PAD) activity (confocal laser scanning microscopy, immunoprecipitation, Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis -SDS-PAGE- and Western blot, ELISA). In this study we demonstrated, for the first time, that both Euro 4 and Euro 5 carbon particles, deprived of PAHs possibly adsorbed on the soot surface, were able to: (1) significantly affect cell viability, inducing autophagy, apoptosis and necrosis; (2) stimulate the release of the pro-inflammatory cytokine IL-18; (3) elicit protein citrullination and PAD activity in NHBE cells. In particular, Euro 5 DEPs seem to have a more marked effect with respect to Euro 4 DEPs.

Synergistic antiproliferative and differentiating effect of 2,4-monofurfurylidene-tetra-O-methylsorbitol and 4,6-dimethyl-2-(3,4,5-trimethoxyphenylamino)pyrimidine on primary and immortalized keratinocytes.

Psoriasis is one of the most common chronic autoinflammatory skin disease, associated with hyperproliferation and abnormal differentiation of keratinocytes, inflammation, and angiogenesis. The available treatments for psoriasis are not curative and may have numerous side effects, and topical administration is preferred over systemic therapy due to the reduced systemic burden of the drug. Thus, novel and more efficacious formulations of anti-inflammatory and/or differentiating compounds for topical application could be very useful for the disease management and for improving the quality of life of the patients. Here we evaluated the potential as anti-psoriatic of an equimolar mixture of two compounds, 2,4-Monofurfurylidene-tetra-O-methylsorbitol (Compound A) and 4,6-dimethyl-2-(3,4,5-trimethoxyphenylamino)pyrimidine (Compound B), that, used individually, are known to possess immunomodulating properties (Compound A) and keratolitic and anti-inflammatory activity (Compound B). Human immortalized keratinocyte cell line (HaCaT cells) and primary human keratinocyte cells from adult donor (HEKa) were used as in vitro experimental models. We show that the mix A + B exhibits antiproliferative activity and induces terminal differentiation more efficiently than compounds A and B used individually. We confirm that the compound B is the active ingredient of the mixture and the mainly responsible for anti-psoriatic activity, but the mix A + B is more effective and possesses lower cytotoxicity than the compound B alone. This could be ascribable to the association with compound A, that is known to possess, in addition to the immunomodulating ability, antioxidant and antiradical action. Our results indicate that mix A + B could be a suitable candidate for a new cosmeceutical formulation for topical treatment of psoriasis.

Atrial Natriuretic Peptide Acts as a Neuroprotective Agent in in Vitro Models of Parkinson's Disease via Up-regulation of the Wnt/ß-Catenin Pathway.

In the last decades increasing evidence indicated a crucial role of the Wnt/ß-catenin signaling in development of midbrain dopaminergic (mDA) neurons. Recently dysregulation of this pathway has been proposed as a novel pathomechanism leading to Parkinson's disease (PD) and some of the molecules participating to the signaling have been evaluated as potential therapeutic targets for PD. Atrial natriuretic peptide (ANP) is a cardiac-derived hormone having a critical role in cardiovascular homeostasis. ANP and its receptors (NPRs) are widely expressed in mammalian central nervous system (CNS) where they could be implicated in the regulation of neural development, synaptic transmission and information processing, as well as in neuroprotection. Until now, the effects of ANP in the CNS have been mainly ascribed to the binding and activation of NPRs. We have previously demonstrated that ANP affects the Wnt/ß-catenin signaling in colorectal cancer cells through a Frizzled receptor-mediated mechanism. The purpose of this study was to investigate if ANP is able to exert neuroprotective effect on two in vitro models of PD, and if this effect could be related to activation of the Wnt/ß-catenin signaling. As cellular models of DA neurons, we used the proliferating or RA-differentiated human neuroblastoma cell line SH-SY5Y. In both DA neuron-like cultures, ANP is able to positively affect the Wnt/ß-catenin signaling, by inducing ß-catenin stabilization and nuclear translocation. Importantly, activation of the Wnt pathway by ANP exerts neuroprotective effect when these two cellular systems were subjected to neurotoxic insult (6-OHDA) for mimicking the neurodegeneration of PD. Our data support the relevance of exogenous ANP as an innovative therapeutic molecule for midbrain, and more in general for brain diseases for which aberrant Wnt signaling seems to be involved.

Developing drugs that target the Wnt pathway: recent approaches in cancer and neurodegenerative diseases.

INTRODUCTION: Wnt/ß-catenin signaling is an evolutionarily conserved pathway that has a crucial role in embryonic and adult life. Dysregulation of Wnt/ß-catenin pathway has been associated with various diseases, including cancer and neurodegenerative disorders, including Parkinson's disease (PD). Several molecular components of the signaling have been proposed as innovative targets for cancer therapy, and very recently, some of them have been also evaluated as potential therapeutic targets for PD. Areas covered: This review focuses on the role of Wnt/ß-catenin pathway in the pathogenensis of cancer and PD, examining some recent therapeutic approaches that are ongoing in preclinical and clinical studies. The possibilities that this signaling offers for diagnosis and prognosis of neoplastic diseases, and the concerns of targeting this pathway are also discussed. Expert opinion: Despite the stimulating results obtained in preclinical studies on cancer and other disease models, the clinical experience with Wnt modulators is still in its infancy, and is mainly restricted to anticancer therapy. Even with concerns of the safety of drugs targeting Wnt signaling, the attention of researchers worldwide is increasing to this issue in terms of their therapeutic potential for diseases such as PD, for which no cure exists.

Thymosin α1 modifies podosome architecture and promptly stimulates the expression of podosomal markers in mature macrophages.

BACKGROUND AND AIMS: The immunomodulatory activity of thymosin α1 (Tα1) on innate immunity has been extensively described, but its mechanism of action is not completely understood. We explored the possibility that Tα1-stimulation could affect the formation of podosomes, the highly dynamic, actin-rich, adhesion structures involved in macrophage adhesion/chemotaxis. METHODS: The following methods were used: optical and scanning electron microscopy for analyzing morphology of human monocyte-derived macrophages (MDMs); time-lapse imaging for visualizing the time-dependent modifications induced at early times by Tα1 treatment; confocal microscopy and Western blot for analyzing localization and expression of podosome components; and Matrigel Migration Assay and zymography for testing MDM invasive ability and metalloproteinase secretion. RESULTS: We obtained data to support that Tα1 could affect MDM motility, invasion and chemotaxis by promptly stimulating assembly and disassembly of podosomal structures. At very early times after its addition to cell culture medium and within 1 h of treatment, Tα1 induces modifications in MDM morphology and in podosomal components that are suggestive of increased podosome turnover. CONCLUSIONS: Since impairment of podosome formation leads to reduced innate immunity and is associated with several immunodeficiency disorders, we confirm the validity of Tα1 as a potent activator of innate immunity and suggest possible new clinical application of this thymic peptide.

WNT-pathway components as predictive markers useful for diagnosis, prevention and therapy in inflammatory bowel disease and sporadic colorectal cancer.

The key role of the Wnt/ß-catenin signaling in colorectal cancer (CRC) insurgence and progression is now recognized and several therapeutic strategies targeting this pathway are currently in developing. Wnt/ß-catenin signaling not only dominates the early stages of sporadic colorectal cancer (SCC), but could also represent the connection between inflammatory bowel diseases (IBD) and increased risk of developing SCC. The knowledge on the sequential molecular events of Wnt-signaling cascade in IBD and during colorectal carcinogenesis, might provide new diagnostic/prognostic markers and could be helpful for optimizing the treatment protocols, thus improving the efficacy of Wnt-targeting therapies. We performed a comparative evaluation of the expression of some crucial molecules participating to Wnt signaling in an animal model of chemically-induced CRC and in human tissues obtained from patients suffering from IBD or at sequential stages of SCC. Specifically, we analyzed upstream events of Wnt signaling including ß-catenin nuclear translocation and loss of E-cadherin and APC functions, and downstream events including c-Myc and Cyclin-D1 expression. We demonstrated that these crucial components of the Wnt/ß-catenin pathway, when evaluated by immunohistochemistry using a multiparametric approach that includes the analyses of both expression and localization, could be potent markers for diagnosis, prevention and therapy in IBD and SCC, also possessing a predictive value for responsiveness to Wnt-targeting therapies. Furthermore, we showed that the animal model of chemically-induced CRC mimics the molecular events of Wnt signaling during IBD and SCC development in humans and may therefore be suitable for testing chemopreventive or therapeutic drugs targeting this pathway.

Thymosin α1 activates complement receptor-mediated phagocytosis in human monocyte-derived macrophages.

Thymosin α1 (Tα1) is a naturally occurring thymic peptide used worldwide in clinical trials for the treatment of infectious diseases and cancer. The immunomodulatory activity of Tα1 on innate immunity effector cells has been extensively described, but its mechanism of action is not completely understood. We report that Tα1-exposed human monocyte-derived macrophages (MDMs) assume the typical activated morphology also exhibited by lipopolysaccharide-activated MDMs, but show a comparatively higher ability of internalizing fluorescent beads and zymosan particles. Tα1 exposure also promptly and dramatically stimulates MDM phagocytosis and killing of Aspergillus niger conidia starting as soon as 30 min after challenge. The effect is dose dependent and early coupled to low transcription of the proinflammatory cytokines tumor necrosis factor α and interleukin-6 and unmodified Toll-like receptor expression. The Tα1-stimulated phagocytosis is strictly dependent on the integrity of the microtubule network and protein kinase C activity and occurs by a variation in the classic zipper model, with recruitment of vinculin and actin at the phagosome exhibiting a punctate distribution. These findings indicate that, in human mature MDMs, Tα1 implements pathogen internalization and killing via the stimulation of the complement receptor-mediated phagocytosis. Our observations document that Tα1 is an early and potent activator of innate immunity and reinforce the concept of its pleiotropy.

Increased expression and copy number amplification of LINE-1 and SINE B1 retrotransposable elements in murine mammary carcinoma progression.

In higher eukaryotic genomes, Long Interspersed Nuclear Element 1 (LINE-1) retrotransposons and endogenous retroviruses represent large families of repeated elements encoding reverse transcriptase (RT) proteins. Short Interspersed Nuclear Element B1 (SINE B1) retrotrasposons do not encode RT, but use LINE-1-derived RT for their retrotransposition. We previously showed that many cancer types have an abundant endogenous RT activity. Inhibition of that activity, by either RNA interference-dependent silencing of active LINE-1 elements or by RT inhibitory drugs, reduced proliferation and promoted differentiation in cancer cells, indicating that LINE-1-encoded RT is required for tumor progression. Using MMTV-PyVT transgenic mice as a well-defined model of breast cancer progression, we now report that both LINE-1 and SINE B1 retrotransposons are up-regulated at a very early stage of tumorigenesis; LINE-1-encoded RT product and enzymatic activity were detected in tumor tissues as early as stage 1, preceding the widespread appearance of histological alterations and specific cancer markers, and further increased in later progression stages, while neither was present in non-pathological breast tissues. Importantly, both LINE-1 and SINE B1 retrotransposon families undergo copy number amplification during tumor progression. These findings therefore indicate that RT activity is distinctive of breast cancer cells and that, furthermore, LINE-1 and SINE B1 undergo copy number amplification during cancer progression.

Thymosin α1 as a stimulatory agent of innate cell-mediated immune response.

The innate immune response and its cellular effectors-peripheral blood mononuclear cells and differentiated macrophages-play a crucial role in detection and elimination of pathogenic microorganisms. Chemotherapy and some immunosuppressive drugs used after organ transplantation and for treatment of autoimmune diseases have, as main side effect, bone marrow suppression, which can lead to a reduced response of the innate immune system. Hence, many immune-depressed patients have a higher risk of developing bacterial and invasive fungal infections compared with immune-competent individuals. Thymosin α1 (Tα1) immunomodulatory activity on effector cells of the innate immunity has been extensively described, even if its mechanism of action is not completely understood. Here, we report some of the main knowledge on this topic, focusing on our in vitro and in vivo work in progress that reinforce the validity of Tα1 as a stimulatory agent for detection and elimination of pathogens by differentiated macrophages and for restoring immune parameters after chemotherapy-induced myelosuppression.

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.

Highly electroconductive multiwalled carbon nanotubes as potentially useful tools for modulating calcium balancing in biological environments.

Aiming to explore the mechanisms modulating cell-carbon nanotube interactions, we investigated whether Ca(2+) ion balancing between intra- and extracellular environments could be affected by multiwalled carbon nanotubes (MWCNTs). We analyzed the effects induced by two different kinds of MWCNTs (as prepared and annealed at 2400°C) on the intracellular Ca(2+) ion levels in rat electrically sensitive cells and on the intercellular junction integrity of rat adenocarcinoma colon cells and platelet aggregation ability, which depend on the Ca(2+) concentration in the medium. MWCNTs, purified by annealing and more electroconductive as compared to nonannealed MWCNTs, affected Ca(2+) ion balancing between extra- and intracellular environments and induced changes on Ca(2+) ion-dependent cellular junctions and platelet aggregation, behaving as the calcium chelator ethylene glycol tetraacetic acid. This could be due to the sorption of cationic Ca(2+) ions on CNTs surface because of the excess of negatively charged electrons on the aromatic units formed on MWCNTs after annealing. From the ClinicAL Editor: The authors investigated whether Ca(2+) ion balance between intra- and extracellular space can be modulated by multiwalled carbon nanotubes (MWCNTs). Annealed nanotubes induced changes on Ca(2+) dependent cellular junctions and platelet aggregation, behaving similary to ethylene glycol tetraacetic acid, an established calcium chelator.

CD44-targeting for antitumor drug delivery: a new SN-38-hyaluronan bioconjugate for locoregional treatment of peritoneal carcinomatosis.

An innovative approach for cancer therapy implies the use of drugs covalently conjugated to macromolecular carriers that specifically target molecules over-expressed on tumor cells. This drug delivery strategy may allow a controlled release of the drug and a high targeting selectivity on tumor cells, increasing drug cytotoxicity and decreasing its undesirable side effects. We provide in vitro and in vivo preclinical data on the antitumor efficacy of ONCOFID™-S, a new bioconjugate of hyaluronic acid (HA) with SN-38 (the CPT11 active metabolite), that support the validity of the drug delivery strategy implying the use of HA as macromolecular carrier of antineoplastic drugs, an approach based on the over-expression of its target CD44 (the receptor for HA-mediated motility) in a wide variety of cancers. We show that ONCOFID™-S exerts a strong in vitro anti-proliferative activity on CD44 over-expressing rat DHD/K12/trb colon adenocarcinoma cells, as well as on gastric, breast, oesophageal, ovarian and lung human cancer cells, higher than that exerted by unconjugated SN-38. We also demonstrated the in vivo anti-tumor efficacy of locoregional treatment with ONCOFID™-S on two pre-clinical models of colorectal cancer (CRC) in BDIX rats: a) syngeneic model of subcutaneous tumor; b) syngeneic model of metastatic tumor induced by injection of cells into the peritoneal cavity, mimicking the clinical situation of peritoneal carcinomatosis. Specifically, in the latter model ONCOFID™-S is able to dramatically reduce all parameters indicative of a poor prognosis in peritoneal metastatization of CRC without any myelotoxicity or mesothelial inflammation. We propose this CD44-targeted therapeutic strategy for locoregional treatment of peritoneal carcinomatosis from CRC, against which systemic chemotherapy results almost inefficient.

Recombinant BCG-Rv1767 amount determines, in vivo, antigen-specific T cells location, frequency, and protective outcome.

One possibility to improve the efficacy of BCG vaccine against TB is to create a recombinant BCG (r-BCG), increasing the expression of mycobacterial antigens, to ameliorate the response to BCG. Here we describe a new r-BCG expressing the gene Rv1767, induced by Mycobacterium tuberculosis during its survival in human macrophages. The r-BCG elicited a specific T cells response in Balb/c mice higher than wt BCG. The r-BCG amount used to immunise mice determined diverse Th1/Th2 equilibriums, which was not the same in spleen and Lymph Nodes. Differences in cytokines production were found for IL-10, IL-4, TNF-alpha, and Arginase-1, which, in some conditions, resulted higher in r-BCG as compared to wt BCG-immunised mice. The immunisation with r-BCG-Rv1767 induced a lesser protective activity than wt BCG in a mouse model of TB. This reduction might likely be explained by the specific T cells phenotype and setting existing before MTB challenge, induced by either the single or the triple dose of r-BCG. The use of this model may help to highlight the capacity of different M. tuberculosis antigens to induce a protective immune response, actually not necessarily embodied by an increased frequency of Antigen-specific effector memory T cells.

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.

Nerve growth factor involvement in liver cirrhosis and hepatocellular carcinoma.

AIM: To define NGF (nerve growth factor) and its high-affinity receptor trkA(NGF) presence and distribution in fibrotic liver and in HCC, and to verify if NGF might have a role in fibrosis and HCC. METHODS: Intracellular distribution of NGF and trkA(NGF) were assessed by immunohistochemistry and immuno-electron microscopy in liver specimens from HCC, cirrhosis or both. ELISA was used to measure circulating NGF levels. RESULTS: NGF and trkA(NGF) were highly expressed in HCC tissue, mainly localized in hepatocytes, endothelial and some Kupffer cells. In the cirrhotic part of the liver they were also markedly expressed in bile ducts epithelial and spindle-shaped cells. Surprisingly, in cirrhotic tissue from patients without HCC, both NGF and trkA(NGF) were negative. NGF serum levels in cirrhotic and/or HCC patient were up to 25-fold higher than in controls. CONCLUSION: NGF was only detected in liver tissue with HCC present. Intracellular distribution suggests paracrine and autocrine mechanisms of action. Better definition of mechanisms may allow for therapeutic and diagnostic/prognostic use of NGF.

Differentiation of human melanoma cells induced by cyanidin-3-O-beta-glucopyranoside.

Great attention has been recently given to a flavonoid of the anthocyanin class, cyanidin-3-O-beta-glucopyranoside (C-3-G), which is widely spread throughout the plant kingdom, and is present in both fruits and vegetables of human diets. In this study, we investigated the effect of C-3-G on proliferation and differentiation of human melanoma cells. Both morphological and functional parameters were evaluated, using electron and confocal microscopy, cytofluorometric analysis, HPLC assay, Western blot analysis, and enzymatic assay, as appropriate. A treatment with a single dose of C-3-G decreased cell proliferation without affecting cell viability and without inducing apoptosis or necrosis. The mitotic index and cell percentage in S phase were significantly lower in C-3-G treated cells compared with untreated control. C-3-G treatment induced, in a dose- and time-dependent manner, melanoma cell differentiation characterized by a strong increase in dendrite outgrowth accompanied with a remodeling of the microtubular network, a dramatic increase of focal adhesion and an increased expression of "brain specific" cytoskeletal components such as NF-160 and NF-200 neurofilament proteins. C-3-G treatment also induced increase of cAMP levels and up-regulation of tyrosinase expression and activity resulting in an enhanced melanin synthesis and melanosome maturation. Up-regulation of the melanoma differentiation antigen Melan-A/MART-1 in treated cells respect to the untreated control was also recorded. Data obtained provide evidence that a single treatment with C-3-G is able to revert the human melanoma cells from the proliferating to the differentiated state. We conclude that C-3-G is a very promising molecule to include in the strategies for treatment of melanoma; also because of its nutritional relevance.