Tumor Microenvironment Modulates Invadopodia Activity of Non-Selected and Acid-Selected Pancreatic Cancer Cells and Its Sensitivity to Gemcitabine and C18-Gemcitabine.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug. METHODS: For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pHe 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine. RESULTS: We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. CONCLUSIONS: We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.

Slightly viscous dispersions of mucoadhesive polymers as vehicles for nasal administration of dopamine and grape seed extract-loaded solid lipid nanoparticles.

With the aim to find an alternative vehicle to the most used thermosensitive hydrogels for efficient nanotechnology-based nose-to-brain delivery approach for Parkinson's disease (PD) treatment, in this work we evaluated the Dopamine (DA) and the antioxidant grape seed-derived pro-anthocyanidins (Grape Seed Extract, GSE) co-loaded solid lipid nanoparticles (SLNs) put in slight viscous dispersions (SVDs). These SVDs were prepared by dispersion in water at low concentrations of mucoadhesive polymers to which SLN pellets were added. For the purpose, we investigated two polymeric blends, namely Poloxamer/Carbopol (PF-127/Carb) and oxidized alginate/Hydroxypropylmethyl cellulose (AlgOX/HPMC). Rheological studies showed that the two fluids possess Newtonian behaviour with a viscosity slightly higher that water. The pH values of the SVDs were mainly within the normal range of nasal fluid as well as almost no osmotic effect was associated to both SVDs. All the SVDs were capable to provide DA permeation through nasal porcine mucosa. Moreover, it was found that PF-127/Carb blend possesses penetration enhancer capability better than the Alg OX/HPMC combination. Flow cytometry studies demonstrated the uptake of viscous liquids incorporating fluorescent SLNs by human nasal RPMI 2650 cell in time-dependent manner. In conclusion, the SVD formulations may be considered promising alternatives to thermosensitive hydrogels strategy. Moreover, in a broader perspective, such SVD formulations may be also hopeful for treating various neurological diseases beyond PD treatment.

Phenolic Compounds from Tropea Red Onion as Dietary Agents for Protection against Heavy Metals Toxicity.

The present study aims to highlight the cell protective effect of Tropea red onion (TRO) hydroalcoholic extract and some of its components against "non-essential" heavy metals. For this purpose, the cytoprotective roles of cyanidin, cyanidin-3-O-glucoside and quercetin against Cd, Hg and Pb and of TRO extract against Hg and Pb have been investigated, and data are reported here. To the best of our knowledge, this is the first detailed evaluation of the protective effect against cell damage induced by "non-essential" heavy metals through the simultaneous administration of cyanidin, cyanidin-3-O-glucoside and quercetin with CdCl2, HgCl2 or PbCl2 and the TRO extract against HgCl2 and PbCl2. Present data are also compared with our previous results from the TRO extract against Cd. The antioxidant capacity of the extract was also determined by the ferric reducing antioxidant power (FRAP) and the bovine brain peroxidation assay. Both of the assays indicated a good antioxidant capacity of the extract. Cell viability and the impact on necrotic cell death were examined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test and lactate dehydrogenase (LDH) release assay. After 24 h of exposure, Caco-2 cell viability decreased by approximately 50% at 0.25 µM for Cd, Hg and Pb and, after 72 h, the ranking order of "non-essential" heavy metal toxicity on cell viability was PbCl2 > CdCl2 > HgCl2. Cell viability was assessed by treating the cells with the biomolecules at doses of 25, 50 and 100 µg/mL for 24 and 72 h. The same analysis was carried out on Caco-2 cells treated with combinations of TRO extract, cyanidin, cyanidin-3-O-glucoside, or quercetin and "non-essential" heavy metals. Treatments with the bioactive metabolites did not significantly improve cell viability. The identical treatment of Caco-2 cells produced instead LDH release, suggesting a decrease in cell viability. Consistently with the finding that TRO extract showed a good antioxidant activity, we suggest that its higher cytotoxicity, compared to that of the individual assayed phytochemicals, may be derived by the combined antioxidant and chelating properties of all the molecules present in the extract. Therefore, from all the acquired experimental evidence, it appears that the TRO extract may be a better promising protective agent against the toxic effect of Cd, Hg and Pb compared to its bioactive metabolites.

Dopamine- and Grape-Seed-Extract-Loaded Solid Lipid Nanoparticles: Interaction Studies between Particles and Differentiated SH-SY5Y Neuronal Cell Model of Parkinson's Disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder, primarily associated with dopaminergic neuron depletion in the Substantia Nigra. Current treatment focuses on compensating for dopamine (DA) deficiency, but the blood-brain barrier (BBB) poses challenges for effective drug delivery. Using differentiated SH-SY5Y cells, we investigated the co-administration of DA and the antioxidant Grape Seed Extract (GSE) to study the cytobiocompability, the cytoprotection against the neurotoxin Rotenone, and their antioxidant effects. For this purpose, two solid lipid nanoparticle (SLN) formulations, DA-co-GSE-SLNs and GSE-ads-DA-SLNs, were synthesized. Such SLNs showed mean particle sizes in the range of 187-297 nm, zeta potential values in the range of -4.1--9.7 mV, and DA association efficiencies ranging from 35 to 82%, according to the formulation examined. The results showed that DA/GSE-SLNs did not alter cell viability and had a cytoprotective effect against Rotenone-induced toxicity and oxidative stress. In addition, this study also focused on the evaluation of Alpha-synuclein (aS) levels; SLNs showed the potential to modulate the Rotenone-mediated increase in aS levels. In conclusion, our study investigated the potential of SLNs as a delivery system for addressing PD, also representing a promising approach for enhanced delivery of pharmaceutical and antioxidant molecules across the BBB.

Evaluation of the Potential Protective Effect of Ellagic Acid against Heavy Metal (Cadmium, Mercury, and Lead) Toxicity in SH-SY5Y Neuroblastoma Cells.

Ellagic acid (EA), a polyphenolic constituent of plant origin, has been thoroughly investigated for its hypothesised pharmacological properties among which antioxidant and neuroprotective activities are included. The present study was designed to explore whether EA could attenuate heavy metal (cadmium, mercury, and lead)-induced neurotoxicity in SH-SY5Y cells, which were utilized as a model system for brain cells. MTT and LDH assays were performed to examine the viability of the SH-SY5Y cells after exposure to Cd, Hg, and Pb (either individually or in combination with EA) as well as the effects of necrotic cell death, respectively. Furthermore, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), a cell-based assay, was performed to determine whether EA could protect SH-SY5Y from heavy metal-induced oxidative stress. Results allowed us to assess the capability of EA to enhance the number of viable SH-SY5Y cells after exposure to heavy metal toxicity. Pre-treatment with EA showed a considerable, concentration-dependent, cytoprotective effect, particularly against Cd2+-induced toxicity. This effect was confirmed through the reduction of LDH release after the simultaneous cell treatment with Cd2+ and EA compared with Cd2+-treated cells. Furthermore, a significant, concentration-dependent decrease in reactive oxygen species (ROS) production, induced by H2O2 or heavy metals, was observed in the same model. Overall, the obtained results provide further insight into the protective role of EA against heavy metal-induced neurotoxicity and oxidative stress, thus indicating the potential beneficial effects of the consumption of EA-rich foods. However, to confirm its effects, well-designed human randomized controlled trials are needed to fill the existing gap between experimental and clinical research.

Solid Lipid Nanoparticles Containing Dopamine and Grape Seed Extract: Freeze-Drying with Cryoprotection as a Formulation Strategy to Achieve Nasal Powders.

(1) Background: DA-Gelucire® 50/13-based solid lipid nanoparticles (SLNs) administering the neurotransmitter dopamine (DA) and the antioxidant grape-seed-derived proanthocyanidins (grape seed extract, GSE) have been prepared by us in view of a possible application for Parkinson's disease (PD) treatment. To develop powders constituted by such SLNs for nasal administration, herein, two different agents, namely sucrose and methyl-ß-cyclodextrin (Me-ß-CD), were evaluated as cryoprotectants. (2) Methods: SLNs were prepared following the melt homogenization method, and their physicochemical features were investigated by Raman spectroscopy, Scanning Electron Microscopy (SEM), atomic force microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS). (3) Results: SLN size and zeta potential values changed according to the type of cryoprotectant and the morphological features investigated by SEM showed that the SLN samples after lyophilization appear as folded sheets with rough surfaces. On the other hand, the AFM visualization of the SLNs showed that their morphology consists of round-shaped particles before and after freeze-drying. XPS showed that when sucrose or Me-ß-CD were not detected on the surface (because they were not allocated on the surface or completely absent in the formulation), then a DA surfacing was observed. In vitro release studies in Simulated Nasal Fluid evidenced that DA release, but not the GSE one, occurred from all the cryoprotected formulations. Finally, sucrose increased the physical stability of SLNs better than Me-ß-CD, whereas RPMI 2650 cell viability was unaffected by SLN-sucrose and slightly reduced by SLN-Me-ß-CD. (4) Conclusions: Sucrose can be considered a promising excipient, eliciting cryoprotection of the investigated SLNs, leading to a powder nasal pharmaceutical dosage form suitable to be handled by PD patients.

Potential Protective Effects of Spirulina (Spirulina platensis) against In Vitro Toxicity Induced by Heavy Metals (Cadmium, Mercury, and Lead) on SH-SY5Y Neuroblastoma Cells.

Spirulina, a filamentous microalga, is used all over the world as a nutraceutical dietary supplement. Recent studies have focused on examining its chelating activity and antioxidant properties, especially as a candidate for protection against neurotoxicity caused by heavy metals. The MTT test and LDH assay were used to examine the viability of the SH-SY5Y cells for 24, 48, and 72 h, to Cd, Hg, and Pb, individually or in combination with Spirulina, and the effects of necrotic cell death. In comparison to the control group, the viability of SH-SY5Y cells decreased after 24 h of exposure, with Cd being more toxic than Hg and Pb being less lethal. The effects of heavy metal toxicity on cell survival were ranked in order after 72 h under identical experimental circumstances as follows: Hg, Pb, and Cd. The viability of the cells was then tested after being exposed to Spirulina at doses of 5 at 50 (%v/v) for 24, 48, and 72 h, respectively. SH-SY5Y cells that had been treated with mixtures of heavy metals and Spirulina underwent the same assay. Cell viability is considerably increased by using Spirulina treatments at the prescribed periods and doses. Instead, the same procedure, when applied to SH-SY5Y cells, caused the release of LDH, which is consistent with the reduction in cell viability. We demonstrated for the first time, considering all the available data, that Spirulina 5, 25, and 50 (%v/v) enhanced the number of viable SH-SY5Y cells utilized as a model system for brain cells. Overall, the data from the present study provide a first insight into the promising positive role of Spirulina against the potentially toxic effects of metals.

The Effect of Calcium Ions on hIAPP Channel Activity: Possible Implications in T2DM.

The calcium ion (Ca2+) has been linked to type 2 diabetes mellitus (T2DM), although the role of Ca2+ in this disorder is the subject of intense investigation. Serum Ca2+ dyshomeostasis is associated with the development of insulin resistance, reduced insulin sensitivity, and impaired glucose tolerance. However, the molecular mechanisms involving Ca2+ ions in pancreatic ß-cell loss and subsequently in T2DM remain poorly understood. Implicated in the decline in ß-cell functions are aggregates of human islet amyloid polypeptide (hIAPP), a small peptide secreted by ß-cells that shows a strong tendency to self-aggregate into ß-sheet-rich aggregates that evolve toward the formation of amyloid deposits and mature fibrils. The soluble oligomers of hIAPP can permeabilize the cell membrane by interacting with bilayer lipids. Our study aimed to evaluate the effect of Ca2+ on the ability of the peptide to incorporate and form ion channels in zwitterionic planar lipid membranes (PLMs) composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) and on the aggregation process of hIAPP molecules in solution. Our results may help to clarify the link between Ca2+ ions, hIAPP peptide, and consequently the pathophysiology of T2DM.

The Role of Ion Channels in Functional Gastrointestinal Disorders (FGID): Evidence of Channelopathies and Potential Avenues for Future Research and Therapeutic Targets.

Several gastrointestinal (GI) tract abnormalities, including visceral hypersensitivity, motility, and intestinal permeability alterations, have been implicated in functional GI disorders (FGIDs). Ion channels play a crucial role in all the functions mentioned above. Hormones and natural molecules modulate these channels and represent targets of drugs and bacterial toxins. Mutations and abnormal functional expression of ion channel subunits can lead to diseases called channelopathies. These channelopathies in gastroenterology are gaining a strong interest, and the evidence of co-relationships is increasing. In this review, we describe the correlation status between channelopathies and FGIDs. Different findings are available. Among others, mutations in the ABCC7/CFTR gene have been described as a cause of constipation and diarrhea. Mutations of the SCN5A gene are instead associated with irritable bowel syndrome. In contrast, mutations of the TRPV1 and TRPA genes of the transient receptor potential (TRP) superfamily manifest hypersensitivity and visceral pain in sensory nerves. Recently, mice and humans affected by Cantu syndrome (CS), which is associated with the mutations of the KCNJ8 and ABCC9 genes encoding for the Kir6.1 and SUR2 subunits, showed dysfunction of contractility throughout the intestine and death in the mice after the weaning on solid food. The discovery of a correlation between channelopathies and FIGD opens new avenues for discovering new direct drug targets for specific channelopathies, leading to significant implications for diagnosing and treating functional GI diseases.

Interaction of Quercetin, Cyanidin, and Their O-Glucosides with Planar Lipid Models: Implications for Their Biological Effects.

Flavonoids are specialized metabolites produced by plants, as free aglycones or as glycosylated derivatives, which are particularly endowed with a variety of beneficial health properties. The antioxidant, anti-inflammatory, antimicrobial, anticancer, antifungal, antiviral, anti-Alzheimer's, anti-obesity, antidiabetic, and antihypertensive effects of flavonoids are now known. These bioactive phytochemicals have been shown to act on different molecular targets in cells including the plasma membrane. Due to their polyhydroxylated structure, lipophilicity, and planar conformation, they can either bind at the bilayer interface or interact with the hydrophobic fatty acid tails of the membrane. The interaction of quercetin, cyanidin, and their O-glucosides with planar lipid membranes (PLMs) similar in composition to those of the intestine was monitored using an electrophysiological approach. The obtained results show that the tested flavonoids interact with PLM and form conductive units. The modality of interaction with the lipids of the bilayer and the alteration of the biophysical parameters of PLMs induced by the tested substances provided information on their location in the membrane, helping to elucidate the mechanism of action which underlies some pharmacological properties of flavonoids. To our knowledge, the interaction of quercetin, cyanidin, and their O-glucosides with PLM surrogates of the intestinal membrane has never been previously monitored.

Combined Dopamine and Grape Seed Extract-Loaded Solid Lipid Nanoparticles: Nasal Mucosa Permeation, and Uptake by Olfactory Ensheathing Cells and Neuronal SH-SY5Y Cells.

We have already formulated solid lipid nanoparticles (SLNs) in which the combination of the neurotransmitter dopamine (DA) and the antioxidant grape-seed-derived proanthocyanidins (grape seed extract, GSE) was supposed to be favorable for Parkinson's disease (PD) treatment. In fact, GSE supply would reduce the PD-related oxidative stress in a synergic effect with DA. Herein, two different methods of DA/GSE loading were studied, namely, coadministration in the aqueous phase of DA and GSE, and the other approach consisting of a physical adsorption of GSE onto preformed DA containing SLNs. Mean diameter of DA coencapsulating GSE SLNs was 187 ± 4 nm vs. 287 ± 15 nm of GSE adsorbing DA-SLNs. TEM microphotographs evidenced low-contrast spheroidal particles, irrespective of the SLN type. Moreover, Franz diffusion cell experiments confirmed the permeation of DA from both SLNs through the porcine nasal mucosa. Furthermore, fluorescent SLNs also underwent cell-uptake studies by using flow cytometry in olfactory ensheathing cells and neuronal SH-SY5Y cells, evidencing higher uptake when GSE was coencapsulated rather than adsorbed onto the particles.

Antioxidant activity and protective effect of the outer scales hydroalcoholic extract of Allium cepa L. var. Tropea on toxicity damage induced by Cadmium in Caco-2 cells.

The characterization of bioactive metabolites and the protective effects against Cadmium (Cd) of the hydroalcoholic extract from Allium cepa var. Tropea (Tropea red onion) in human Caco-2 colon adenocarcinoma cells and in vitro antioxidant effects were investigated. Tropea red onion extract showed high levels of bioactive compounds and a strong activity as radical scavenger and inhibitor of lipid peroxidation. The most abundant specialized metabolites were quercetin derivatives. In addition, the extract inhibited NO release in a dose-dependent manner. Exposure of Cd treated human Caco-2 cells to the Tropea red onion extract resulted in a higher cytoprotection with a significant falloff of cells damage produced by Cd. The effect of Tropea red onion evaluated with the MTT assay and LDH test, was time-dependent and more evident after 24h of treatment. Caco-2 cells treated with the Tropea red onion extract and CdCl2 at 24h showed a significant cytoprotection at concentrations equal to 50 µg/mL + 25 µM and 100 µg/mL + 25 µM while at 72h at a concentration of 25 µg/mL + 25 µM. We observed with MTT and LDH assays that treatment of cells with Tropea red onion extract at 24h was able to significantly prevent Cd-induced cytotoxicity alone.

Residual Levels of Mercury, Cadmium, Lead and Arsenic in Some Commercially Key Species from Italian Coasts (Adriatic Sea): Focus on Human Health.

This study provides information on the potential human health risk of Hg, Cd, Pb and As exposure from consumption of two fish species (Umbrina cirrosa and Sciaena umbra) in the general population and in high-level fish consumers. The concentrations did not show significant differences between the two species, and no fish length element level-body-length relationship was observed, except for Hg. The average metal(loid) levels, irrespective of species, varied in the following ranges: Hg = 0.18-0.19, Cd = 0.07-0.10, Pb = 0.10-0.12, As = 0.59-0.69 µg g-1 w.w. The concentrations remained below the maximum permissible limits (MPLs) for human consumption, except for Cd. The estimated intakes of Hg, Cd and Pb in both consumption scenarios were lower than the respective PTWI/PTMIs, as well as those of inorganic As, which were even lower than the BMDL01. The non-carcinogenic risk (THQ) did not reveal any concerns, except for Hg. The lifetime health cancer risk (ILCR) suggested hazard exclusively from Cd, although for high-level fish consumers, even the ILCR of inorganic As was, in some cases, above the acceptable range. Continuous monitoring of metal(loid) levels in these fish is strongly recommended because the results demonstrate the occurrence of potential health risks, especially in high-level fish consumers, due to the presence of Hg and Cd.

Inclisiran in lipid management: A Literature overview and future perspectives.

Primary and secondary prevention protocols aim at reducing the plasma levels of lipids - with particular reference to low-density lipoprotein cholesterol (LDL-C) plasma concentrations - in order to improve the overall survival and reduce the occurrence of major adverse cardiovascular events. The use of statins has been widely considered as the first-line approach in lipids management as they can dramatically impact on the cardiovascular risk profile of individuals. The introduction of ezetimibe and proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors overcame the adverse effects of statins and ameliorate the achievement of the target lipids levels. Indeed, advances in therapies promote the use of specific molecules - i.e. short strands of RNA named small-interfering RNAs (siRNAs) - to suppress the transcription of genes related to lipids metabolism. Recently, the inclisiran has been developed: this is a siRNA able to block the mRNA of the PCSK9 gene. About 50% reduction in low-density lipoprotein cholesterol levels have been observed in randomized controlled trials with inclisiran. The aim of this review was to summarize the literature regarding inclisiran and its possible role in the general management of patients with lipid disorders and/or in primary/secondary prevention protocols.

Polychlorinated dioxins, furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) in food from Italy: Estimates of dietaryintake and assessment.

Dietary intake of polychlorinated dioxins and furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) from various foods (fish and seafood, meat and meat-based products, milk and dairy products, hen eggs, olive oil and fats) was investigated for various sex/age groups of the Italian population. The concentrations of PCDD/Fs and dl-PCBs and their contribution to total TEQ values varied depending on food matrix. Fish (0.50 pg WHO-TEQ/g wet weight) and seafood (0.16 pg WHO-TEQ/g wet weight) showed the highest mean concentrations of PCDD/Fs plus dl-PCBs, followed by meat (1.70 pg WHO-TEQ/g lipid weight), meat based products (1.03 pg WHO-TEQ/g lipid weight), milk and dairy products (0.78 pg WHO-TEQ/g lipid weight), hen eggs (0.71 pg WHO-TEQ/g lipid weight), fats (0.27 pg WHO-TEQ/g lipid weight) and olive oil (0.09 pg WHO-TEQ/g lipid weight). In all samples WHO-TEQ PCDD/F plus dl-PCB concentrations fulfilled the European Union food law, except in pork loin samples (1.39 pg WHO-TEQ/g lipid weight). Differences in exposure depending on the sex/age groups (children > teenagers > adults > elders) and hypotheses considered (lower bound and upper bound) were encountered. Non-cancer risk values showed a low exposure. Carcinogenicity risk results revealed that highly exposed individuals were distributed over all sex/age groups, even though the proportion of individuals exceeding the safe limit was higher in children. These data once again underline the importance of trying to control the levels of these contaminants in fishery products, particularly in fish, who represents one of the main exposure sources for consumers. PRACTICAL APPLICATION: This paper may help the consumer in making food choices to minimize the exposure risk to dioxins, furans and PCBs.

Olive Tree in Circular Economy as a Source of Secondary Metabolites Active for Human and Animal Health Beyond Oxidative Stress and Inflammation.

Extra-virgin olive oil (EVOO) contains many bioactive compounds with multiple biological activities that make it one of the most important functional foods. Both the constituents of the lipid fraction and that of the unsaponifiable fraction show a clear action in reducing oxidative stress by acting on various body components, at concentrations established by the European Food Safety Authority's claims. In addition to the main product obtained by the mechanical pressing of the fruit, i.e., the EVOO, the residual by-products of the process also contain significant amounts of antioxidant molecules, thus potentially making the Olea europea L. an excellent example of the circular economy. In fact, the olive mill wastewaters, the leaves, the pomace, and the pits discharged from the EVOO production process are partially recycled in the nutraceutical and cosmeceutical fields also because of their antioxidant effect. This work presents an overview of the biological activities of these by-products, as shown by in vitro and in vivo assays, and also from clinical trials, as well as their main formulations currently available on the market.

Evidence of cadmium and mercury involvement in the Aß42 aggregation process.

Aß42 is a small peptide formed from 42 aminoacids that presents a great propensity to aggregate until it forms fibrils. Aß42 aggregation and fibrillation are very complex processes whose molecular mechanisms seem to depend on characteristics intrinsic to the peptide molecule, as well as extrinsic factors. Peptide concentration, mean pH and several substances, including metal ions, are principal extrinsic factors for the oligomerization process. Different metals affect the aggregation of the Aß42 molecule, and their toxicity favours the misfolding and aggregation of the peptide. In this study, we evaluate the effect of different concentrations of Cd2+ and Hg2+ on the Aß42 peptide in solution by different methods. The toxicity of Aß42 was evaluated with the MTT assay, while the aggregation process was monitored by single-channel measurements, electrophoresis and western blot. Cd2+ and Hg2+ seem to favour the formation of high-molecular-weight aggregates, to decrease ion channel turnover inside the membrane and to significantly increase Aß42 toxicity.

Emerging high-risk ST101 and ST307 carbapenem-resistant Klebsiella pneumoniae clones from bloodstream infections in Southern Italy.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is an urgent public health issue in Italy. This pattern of resistance is due mainly to dissemination of carbapenemase genes. Molecular characterization of carbapenem-resistant Klebsiella pneumoniae (CR-KP) strains was performed over a three-year period. In-depth analysis was performed on a subset of emerging CR-KP ST101 and ST307 clones. METHODS: A prospective study was performed on 691 patients with CR-KP bloodstream infections hospitalized in 19 hospitals located in three large provinces in Southern Italy. Carbapenemase genes were identified via genotyping methods. Multi-locus sequence typing (MLST) and Whole Genome Sequencing (WGS) were carried out on ST101 and ST307 isolates. RESULTS: Among the CR-KP isolates, blaKPC was found in 95.6%, blaVIM was found in 3.5%, blaNDM was found in 0.1% and blaOXA-48 was found in 0.1%. The blaKPC-3 variant was identified in all 104 characterized KPC-KP isolates. MLST of 231 representative isolates revealed ST512 in 45.5%, ST101 in 20.3% and ST307 in 18.2% of the isolates. cgMLST of ST307 and ST101 isolates revealed presence of more than one beta-lactam resistance gene. Amino acid substitution in the chromosomal colistin-resistance gene pmrB was found in two ST101 isolates. CONCLUSIONS: ST512 is widespread in Southern Italy, but ST101 and ST307 are emerging since they were found in a significant proportion of cases. Aggressive infection control measures and a continuous monitoring of these high-risk clones are necessary to avoid rapid spread of CR-KP, especially in hospital settings.

HB-EGF-EGFR Signaling in Bone Marrow Endothelial Cells Mediates Angiogenesis Associated with Multiple Myeloma.

Epidermal growth factor receptor (EGFR) and its ligand heparin-binding EGF-like growth factor (HB-EGF) sustain endothelial cell proliferation and angiogenesis in solid tumors, but little is known about the role of HB-EGF-EGFR signaling in bone marrow angiogenesis and multiple myeloma (MM) progression. We found that bone marrow endothelial cells from patients with MM express high levels of EGFR and HB-EGF, compared with cells from patients with monoclonal gammopathy of undetermined significance, and that overexpressed HB-EGF stimulates EGFR expression in an autocrine loop. We also found that levels of EGFR and HB-EGF parallel MM plasma cell number, and that HB-EGF is a potent inducer of angiogenesis in vitro and in vivo. Moreover, blockade of HB-EGF-EGFR signaling, by an anti-HB-EGF neutralizing antibody or the EGFR inhibitor erlotinib, limited the angiogenic potential of bone marrow endothelial cells and hampered tumor growth in an MM xenograft mouse model. These results identify HB-EGF-EGFR signaling as a potential target of anti-angiogenic therapy, and encourage the clinical investigation of EGFR inhibitors in combination with conventional cytotoxic drugs as a new therapeutic strategy for MM.

High Public-Health Impact in an Influenza-B-Mismatch Season in Southern Italy, 2017-2018.

BACKGROUND: Yearly influenza epidemics have considerable effects on public health worldwide. The 2017-2018 influenza season in Italy was of greater severity than previous seasons. The aim of this study was to describe the 2017-2018 influenza season in Southern Italy and the molecular characteristics of the circulating viral strains. METHODS: The incidence of influenza-like illness (ILI) was analysed. Nasopharyngeal swabs collected from patients with ILI from week 46/2017 to week 17/2018 were tested to identify influenza A viruses (IAV) and influenza B viruses (IBV). Sequencing and phylogenetic analysis of haemagglutinin genes were also performed on 73 positive samples (35 IBV, 36 IAV H1, and 2 IAV H3 strains). RESULTS: During the 2017-2018 season, the peak incidence was 14.32 cases per 1,000 inhabitants. IBV strains were identified in 71.0% of cases. The 35 characterised IBV strains belonged to Yamagata lineage clade 3, the 36 A/H1N1pdm09 strains clustered with the genetic subgroup 6B.1, and the 2 A/H3N2 strains clustered with the genetic subgroup 3C.2a. Intensive-care unit (ICU) admission was required in 50 cases of acute respiratory distress syndrome (ARDS). Among the >64-year age group, 18 out of 26 ICU-ARDS cases (69.2%) were caused by IBV, and 14 of these (77.8%) were B/Yamagata lineage. CONCLUSIONS: The 2017-2018 influenza season was one of the most severe in a decade in Southern Italy. IBV mismatch between the trivalent vaccine and the circulating strains occurred. The high number of ICU-ARDS cases caused by B/Yamagata strains in the >64-year age group suggests that further data on the effectiveness of the available influenza vaccines are needed to determine the best way to protect the elderly against both IBV lineages.