Botanical drugs are complementary therapies in the management of diabetes mellitus. In this work, we studied the effects of chronic treatment of diabetic rats with A. indica (neem) on blood glucose, pancreatic islet histopathology, and oxidative status of the pancreas. Fifty-four Wistar rats (5-8 weeks old) were randomly assigned to 5 treatment groups. Hyperglycemia was induced in 34 fasted rats with a single i.p. injection of STZ (70 mg/kg bw/d). Ethanolic extract of A. indica leaves (500 mg/kg bw/d) was given orally to diabetic rats (n=12) for 50d. Glibenclamide was given (p.o) at 600 μg/ kg bw/d. In each group, blood glucose, islet histopathology, and pancreatic oxidative status, were assessed. All hyperglycemic rats in the neem-treated group had become normoglycemic at the end of week 2. By 50d, the number of viable b cells was highest in the neem-treated diabetic rats (compared with the diabetic and glibenclamide groups). Similarly, islet histology showed marked improvement in this group, in addition to improved oxidative stress. Our findings confirmed the hypoglycemic effect of neem. Besides, the improved islet morphology and oxidative status in neem-treated diabetic rats suggest the potential of this herb at improving lesions of the pancreatic islet in diabetes mellitus.

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SCs) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SCs biology remains the ability to identify stem cells in situ and in vitro. To date, not so much markers exist for the identification of different phenotypes. CESCs (corneal epithelial stem cells) isolated from limbal biopsies were maintained in primary culture for 14 days and stained with Hoechst and a panel of FITC-conjugated lectins. All lectins, with the exception of Lycopersicon esculentum, labelled CESCs irrespective of the degree of differentiation. Lycopersicon esculentum, that binds N-acetylglucosamine oligomers, labelled intensely only the surface of TACs (single corneal epithelial stem cells better than colonial cells). These results suggest that Lycopersicon esculentum lectin is a useful and easy-to-use marker for the in vitro identification of TACs (transient amplifying cells) in cultures of isolated CESCs.

This study aims to elucidate the processes underlying neuroprotection of kaempferol in models of rotenone-induced acute toxicity. We demonstrate that kaempferol, but not quercetin, myricetin or resveratrol, protects SH-SY5Y cells and primary neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species (ROS) levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenyilpiridinium, paraquat) used to reproduce the typical features of Parkinson’s disease (PD), but is inefficient against apoptotic stimuli not directly affecting mitochondria (H2O2, 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in Parkinson’s disease. Overall, the data provide further evidence for kaempferol to be identified as an autophagic enhancer with potential therapeutic capacity.

This study aims to elucidate the processes underlying neuroprotection of kaempferol in models of rotenone-induced acute toxicity. We demonstrate that kaempferol, but not quercetin, myricetin or resveratrol, protects SH-SY5Y cells and primary neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species (ROS) levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenyilpiridinium, paraquat) used to reproduce the typical features of Parkinson's disease (PD), but is inefficient against apoptotic stimuli not directly affecting mitochondria (H2O2, 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in Parkinson's disease. Overall, the data provide further evidence for kaempferol to be identified as an autophagic enhancer with potential therapeutic capacity.

The use of nanocarriers, which respond to different stimuli controlling their physicochemical properties and biological responsivness, shows a growing interest in pharmaceutical science. The stimuli are activated by targeting tissues and biological compartments, e.g., pH modification, temperature, redox condition, enzymatic activity, or can be physically applied, e.g., a magnetic field and ultrasound. pH modification represents the easiest method of passive targeting, which is actually used to accumulate nanocarriers in cells and tissues. The aim of this paper was to physicochemically characterize pH-sensitive niosomes using different experimental conditions and demonstrate the effect of surfactant composition on the supramolecular structure of niosomes. In this attempt, niosomes, made from commercial (Tween21) and synthetic surfactants (Tween20 derivatives), were physicochemically characterized by using different techniques, e.g., transmission electron microscopy, Raman spectroscopy, and small-angle X-ray scattering. The changes of niosome structure at different pHs depend on surfactants, which can affect the supramolecular structure of colloidal nanocarriers and their potential use both in vitro and in vivo. At pH 7.4, the shape and structure of niosomes have been maintained; however, niosomes show some differences in terms of bilayer thicknesses, water penetration, membrane coupling, and cholesterol dispersion. The acid pH (5.5) can increase the bilayer fluidity, and affect the cholesterol depletion. In fact, Tween21 niosomes form large vesicles with lower curvature radius at acid pH; while Tween20-derivative niosomes increase the intrachain mobility within a more interchain correlated membrane. These results demonstrate that the use of multiple physicochemical procedures provides more information about supramolecular structures of niosomes and improves the opportunity to deeply investigate the effect of stimuli responsiveness on the niosome structure.

It is becoming evident that failure in the removal of dying cells causes and/or promotes the onset of chronic diseases. Impairment of phagocytosis of apoptotic cells can be due not only to genetic or molecular malfunctioning but also to external/environmental factors. Two of these environmental factors have been recently reported to down regulate the clearance of apoptotic cells: cigarette smoke and static magnetic fields. Cigarette smoke (CS) contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. Human macrophages interacting with carbonyl or cigarette smoke modified extracellular matrix (ECM) proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. It was postulated that changes in the ECM environment as a result of CS affect the ability of macrophages to remove apoptotic cells. This decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion. Downregulation of the phagocytosis of apoptotic cells was also described when performed in presence of static magnetic fields (SMFs) of moderate intensity. SMFs have been reported to perturb distribution of membrane proteins and glycoproteins, receptors, cytoskeleton and trans-membrane fluxes of different ions, especially calcium [Ca2+]i, that in turn, interfere with many different physiological activities, including phagocytosis. The effects of CS and SMF on the phagocytosis of dying cells will be here discussed.

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFβ. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

Cell deaths in Photodynamic therapy, that is an anticancer therapy requiring exposure of cells or tissue to photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, occur by the efficient induction of apoptotic as well as non-apoptotic cell deaths, like necrosis and autophagy, or by a combination of the three mechanisms. However, the exact role of autophagy in Photodynamic therapy is still a matter of debate. To this purpose, we investigated the induction of autophagy in HeLa cells photosensitized with Rose Bengal Acetate (RBAc). After incubation with Rose Bengal Acetate (10-5 M), HeLa cells were irradiated for 90 seconds (green LED DPL 305, emitting at 530 ± 15 nm in order to obtain 1.6 J/cm2 as total light dose) and allowed to recover for 72 h. Induction of autophagy and apoptosis was observed with peaks at 8 h and 12 h after irradiation respectively for autophagy and apoptosis. Autophagy was detected by biochemical (Western Blot of LC3B protein) and morphological criteria (TEM, cytochemistry). In addition, the pan-caspases inhibitor z-VAD was not able to completely prevent cell deaths. The simultaneous onset of apoptosis and autophagy following Rose Bengal Acetate Photodynamic therapy is of remarkable interest in consideration of the findings that autophagy can result in class II presentation of antigens and thus explain why low dose Photodynamic therapy can yield anti-tumour vaccines.

The new concept of Immunogenic Cell Death (ICD), associated with Damage Associated Molecular Patterns (DAMPs) exposure and/or release, is recently becoming very appealing in cancer treatment. In this context, PhotoDynamic Therapy (PDT) can give rise to ICD and to immune response upon dead cells removal. The list of PhotoSensitizers (PSs) able to induce ICD is still short and includes Photofrin, Hypericin, Foscan and 5-ALA. The goal of the present work was to investigate if Rose Bengal Acetate (RBAc), a powerful PS able to trigger apoptosis and autophagy, enables photosensitized HeLa cells to expose and/or release pivotal DAMPs, i.e. ATP, HSP70, HSP90, HMGB1, and calreticulin (CRT), that characterize ICD. We found that apoptotic HeLa cells after RBAc-PDT exposed and released, early after the treatment, high amount of ATP, HSP70, HSP90 and CRT; the latter was distributed on the cell surface as uneven patches and co-exposed with ERp57. Conversely, autophagic HeLa cells after RBAc-PDT exposed and released HSP70, HSP90 but not CRT and ATP. Exposure and release of HSP70 and HSP90 were always higher on apoptotic than on autophagic cells. HMGB1 was released concomitantly to secondary necrosis (24 h after RBAc-PDT). Phagocytosis assay suggests that CRT is involved in removal of RBAc-PDT generated apoptotic HeLa cells. Altogether, our data suggest that RBAc has all the prerequisites (i.e. exposure and/or release of ATP, CRT, HSP70 and HSP90), that must be verified in future vaccination experiments, to be considered a good PS candidate to ignite ICD. We also showed tha CRT is involved in the clearance of RBAc photokilled HeLa cells. Interestingly, RBAc-PDT is the first cancer PDT protocol able to induce the translocation of HSP90 and plasma membrane co-exposure of CRT with ERp57.

Silver nanostructures were successfully synthesized through a simple and “green” method using saccharides as reducing and capping agent. Transmission electron microscopy (TEM) and UV–Vis absorption were used to certify the quality of the silver nanoparticles obtained: first, size and dispersion. In this work Silver NanoParticles (AgNPs) cytotoxicity related to saccharides capping (Glucose and Glucose-Sucrose) was explored. Human epitheloid cervix carcinoma cells (HeLa) were used for cytotoxicity test. The cells were incubated with increasing AgNPs number/cell and HeLa cells viability was monitored for a period of 48 h compared with the positive and negative controls. We observed that the toxicity increases in a incubation time and AgNPs number/cell related manner. In addition, the AgNP-G are more toxic than AgNP-GS, suggesting that AgNPs citotoxicity could depend on the capping agent. in HeLa cells the highest AgNP-G number/cell induces , cell deathsoon after 1 hr of incubation; conversely the lowest AgNP-GS number/cell induces cell proliferation.

The targeting of BCR-ABL, a hybrid oncogenic tyrosine (Y) kinase, does not eradicate chronic myeloid leukemia (CML)-initiating cells. Activation of β-catenin was linked to CML leukemogenesis and drug resistance through its BCR-ABL-dependent Y phosphorylation and impaired binding to GSK3β (glycogen synthase kinase 3β). Herein, we show that GSK3β is constitutively Y(216) phospho-activated and predominantly relocated to the cytoplasm in primary CML stem/progenitor cells compared with its balanced active/inactive levels and cytosolic/nuclear distribution in normal cells. Under cytokine support, persistent GSK3β activity and its altered subcellular localization were correlated with BCR-ABL-dependent and -independent activation of MAPK and p60-SRC/GSK3β complex formation. Specifically, GSK3β activity and nuclear import were increased by imatinib mesylate (IM), a selective ABL inhibitor, but prevented by dasatinib that targets both BCR-ABL- and cytokine-dependent MAPK/p60-SRC activity. SB216763, a specific GSK3 inhibitor, promoted an almost complete suppression of primary CML stem/progenitor cells when combined with IM, but not dasatinib, while sparing bcr-abl-negative cells. Our data indicate that GSK3 inhibition acts to prime a pro-differentiative/apoptotic transcription program in the nucleus of IM-treated CML cells by affecting the β-catenin, cyclinD1, C-EBPα, ATF5, mTOR, and p27 levels. In conclusion, our data gain new insight in CML biology, indicating that GSK3 inhibitors may be of therapeutic value in selectively targeting leukemia-initiating cells in combination with IM but not dasatinib.

In a previous work we showed that 6mT SMF interferes with monocyte/macrophage 12-Otetradecanoylphorbol- 13-acetate (TPA) -induced differentiation of promonocytes (U937 cells) and monocytes (THP-1 cells), in this work we investigate whether in the same cells and under the same conditions phagocytosis of apoptotic cells is influenced by 6 mT SMF exposure. Fluid phase endocytosis and phagocytosis of latex particles were also analysed for comparison. The results indicate that SMF exposure, whose influence was greater at the late stages of the macrophage differentiation (THP1 > U937 cells), has effects on phagocytosis but not on fluid phase endocytosis. Phagocytosis index and rate decreased under SMF exposure while the number of latex particles bound to the plasma membrane of TPA-differentiated U937 and THP1 cells increased. Conversely, phagocytosis rate of apoptotic cells increased under SMF exposure while the number of apoptotic cells bound to the plasma membrane of TPA-differentiated U937 and THP1 cells and Raw 264.7 macrophages decreased. Even in conditions of inability to phagocytose, i.e. non-differentiated U937 and THP-1 cells, the SMF exposure enhanced the number of cell surface bound apoptotic cells and latex beads.

Rose Bengal Acetate PhotoDynamic Therapy (RBAc-PDT) induced multiple cell deaths pathways in HeLa cells through ROS generation. The onset of apoptosis, autophagy and of the different apoptotic pathways were timed by determining the levels of caspases, Bcl 2 family, Hsp 70, LC3, Grp78 and phospho-eIF2α proteins. Four different apoptotic pathways plus autophagy were sequentially initiated by RBAc-PDT: intrinsic, extrinsic, caspase 12- dependent and caspase independent as reflected by peaks of the relative caspases, 9, 8, 3 and 12. Autophagy, revealed by the formation of acidic autophagosomes and by increased Light Chain 3-II (LC3BII) expression was conspicuous at 8 h post-PDT. In our system, autophagy had a pro-death role, since its inhibitor, 3-MethylAdenine (3-MA), significantly augmented cell viability. The increase of cleaved caspase 12 was consequent to the increase of Grp78 and phospho-eIF2α proteins, suggesting Endoplasmic Reticulum. Regulation of the intrinsic pathway of apoptosis was under the control of Bcl-2 family (i.e. soon after irradiation Bcl2 decreased, Bax and tBid increased) and of Hsp70 proteins (peak at 12-18h post-PDT). Interestingly, inhibition of one pathway, i.e.caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting the independent onset of the different apoptotic and autophagic pathways in a not subordinated fashion.

In base alla procedura di ozonizzazione dell’olio extravergine d’oliva, appena descritta, si ottiene un prodotto di alta qualità e stabile nel tempo. Le basse temperature e il campo magnetico oltre a migliorare l’efficienza del processo produttivo, permettono di accorciare i tempi di lavorazione e di conseguenza abbassare i costi di produzione. Inoltre, la scelta di utilizzare come materia prima l’olio extravergine d’oliva ottenuto dalle cultivar salentine Ogliarola Leccese e Cellina di Nardò, consente di ridurre ulteriormente i tempi di lavorazione in quanto, questa tipologia di olio produce ozonidi in quantità più elevate rispetto ad altri oli extravegini d’oliva. Pertanto, la presente invenzione, Bioxoil, avendo una ottima stabilità nel tempo può rappresentare oltre che un valido prodotto a basso costo da impiegare nella cosmesi e nella parafarmacia, anche una interessante alternativa all’ozonoterapia, correntemente usata in medicina per scopi terapeutici. In altri termini, con l’utilizzo di Bioxoil si potrebbero abbattere i costi sanitari, poiché il paziente effettuerebbe la terapia nel proprio domicilio senza recarsi presso il centro medico, eliminando, oltremodo i rischi (come embolie) di una terapia, come l’ozonoterapia, troppo invasiva e dolorosa