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Streptococcus pyogenes causes a wide spectrum of diseases varying from mild to life threatening, despite antibiotic treatment. Nanoparticle application could facilitate the foreign pathogen fight by increasing the antimicrobial effectiveness and reducing their adverse effects. Here, we designed and produced erythromycin-loaded chitosan nanodroplets (Ery-NDs), both oxygen-free and oxygen-loaded. All ND formulations were characterized for physico-chemical parameters, drug release kinetics, and tested for biocompatibility with human keratinocytes and for their antibacterial properties or interactions with S. pyogenes. All tested NDs possessed spherical shape, small average diameter, and positive Z potential. A prolonged Ery release kinetic from Ery-NDs was demonstrated, as well as a favorable biocompatibility on human keratinocytes. Confocal microscopy images showed ND uptake and internalization by S. pyogenes starting from 3 h of incubation up to 24 h. According to cell counts, NDs displayed long-term antimicrobial efficacy against streptococci significantly counteracting their proliferation up to 24 h, thanks to the known chitosan antimicrobial properties. Intriguingly, Ery-NDs were generally more effective (104–103 log10 CFU/mL), than free-erythromycin (105 log10 CFU/mL), in the direct killing of streptococci, probably due to Ery-NDs adsorption by bacteria and prolonged release kinetics of erythromycin inside S. pyogenes cells. Based on these findings, NDs and proper Ery-NDs appear to be the most promising and skin-friendly approaches for the topical treatment of streptococcal skin infections allowing wound healing during hypoxia.

Narcisa Mandras,1 Monica Argenziano,2 Mauro Prato,1 Janira Roana,1 Anna Luganini,3 Valeria Allizond,1 Vivian Tullio,1 Nicole Finesso,4 Sara Comini,1 Bruno Emilio Bressan,4 Francesca Pecoraro,4 Giuliana Giribaldi,4 Adriano Troia,5 Roberta Cavalli,2 Anna Maria Cuffini,1,* Giuliana Banche1,* 1Department of Public Health and Pediatric Sciences, University of Torino, Turin, 10126, Italy; 2Department of Drug Science and Technology, University of Torino, Turin, 10125, Italy; 3Department of Life Sciences and Systems Biology, University of Torino, Turin, 10123, Italy; 4Department of Oncology, University of Torino, Turin, 10126, Italy; 5Istituto Nazionale di Ricerca Metrologica, Turin, 10135, Italy*These authors contributed equally to this workCorrespondence: Valeria Allizond, Department of Public Health and Pediatric Sciences, University of Torino, Via Santena 9, Turin, 10126, Italy, Tel +390116705644, Fax +390112365644, Email valeria.allizond@unito.itPurpose: Medium versus low weight (MW vs LW) chitosan-shelled oxygen-loaded nanodroplets (cOLNDs) and oxygen-free nanodroplets (cOFNDs) were comparatively challenged for biocompatibility on human keratinocytes, for antimicrobial activity against four common infectious agents of chronic wounds (CWs) – methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans and C. glabrata – and for their physical interaction with cell walls/membranes.Methods: cNDs were characterized for morphology and physico-chemical properties by microscopy and dynamic light scattering. In vitro oxygen release from cOLNDs was measured through an oximeter. ND biocompatibility and ability to promote wound healing in human normoxic/hypoxic skin cells were challenged by LDH and MTT assays using keratinocytes. ND antimicrobial activity was investigated by monitoring upon incubation with/without MW or LW cOLNDs/cOFNDs either bacteria or yeast growth over time. The mechanical interaction between NDs and microorganisms was also assessed by confocal microscopy.Results: LW cNDs appeared less toxic to keratinocytes than MW cNDs. Based on cell counts, either MW or LW cOLNDs and cOFNDs displayed long-term antimicrobial efficacy against S. pyogenes, C. albicans, and C. glabrata (up to 24 h), whereas a short-term cytostatic effects against MRSA (up to 6 h) was revealed. The internalization of all ND formulations by all four microorganisms, already after 3 h of incubation, was showed, with the only exception to MW cOLNDs/cOFNDs that adhered to MRSA walls without being internalized even after 24 h.Conclusion: cNDs exerted bacteriostatic and fungistatic effects, due to the presence of chitosan in the outer shell and independently of oxygen addition in the inner core. The duration of such effects strictly depends on the characteristics of each microbial species, and not on the molecular weight of chitosan in ND shells. However, LW chitosan was better tolerated by human keratinocytes than MW. For these reasons, the use of LW NDs should be recommended in future research to assess cOLND efficacy for the treatment of infected CWs.Keywords: chitosan nanodroplets, methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes, Candida spp, chronic wounds

Aim: To evaluate the performance of urinary fibrinogen β-chain (FBC) – either alone or associated with urinary tyrosine-phosphorylated proteins (UPY) – as bladder cancer (BCa) diagnostic biomarker. Materials & methods: 164 subjects were tested. Results: Significantly different FBC and UPY levels were found between BCa patients and controls, as well as between low-grade and high-grade cancers. The diagnostic accuracy was 0.84 for FBC and 0.87 for UPY. The combination of FBC and UPY improved the accuracy to 0.91. The addition of clinical variables (age, gender, and smoking habit) to FBC and UPY into a model for BCa prediction significantly improved the accuracy to 0.99. The combination of FBC and UPY adjusted for clinical variables associates with the highest sensitivity and good specificity. Conclusion: Urinary FBC and UPY could be used as biomarkers for BCa diagnosis., Lay abstract This research has developed and validated a highly accurate predictive model for BCa diagnosis based on the combination of two urinary biomarkers, fibrinogen β-chain (FBC), and urinary tyrosine-phosphorylated proteins (UPY), and some clinical variables (age, gender and smoking habit). If the preliminary promising results will be confirmed by external validations and prospective trials in selected clinical scenarios, our model could be transferred to clinical practice for screening of high-risk population., Graphical abstract

Wound healing is a complex process regulated by multiple signals and consisting of several phases known as haemostasis, inflammation, proliferation, and remodelling. Keratinocytes, endothelial cells, macrophages, and fibroblasts are the major cell populations involved in wound healing process. Hypoxia plays a critical role in this process since cells sense and respond to hypoxic conditions by changing gene expression. This study assessed the in vitro expression of 77 genes involved in angiogenesis, metabolism, cell growth, proliferation and apoptosis in human keratinocytes (HaCaT), microvascular endothelial cells (HMEC-1), differentiated macrophages (THP-1), and dermal fibroblasts (HDF). Results indicated that the gene expression profiles induced by hypoxia were cell-type specific. In HMEC-1 and differentiated THP-1, most of the genes modulated by hypoxia encode proteins involved in angiogenesis or belonging to cytokines and growth factors. In HaCaT and HDF, hypoxia mainly affected the expression of genes encoding proteins involved in cell metabolism. This work can help to enlarge the current knowledge about the mechanisms through which a hypoxic environment influences wound healing processes at the molecular level.

Persistent hypoxia is a main clinical feature of chronic wounds. Intriguingly, oxygen-loaded nanodroplets (OLNDs), filled with oxygen-solving 2H,3H-decafluoropentane and shelled with polysaccharides, have been proposed as a promising tool to counteract hypoxia by releasing a clinically relevant oxygen amount in a time-sustained manner. Here, four different types of chitosan (low or medium weight (LW or MW), glycol-(G-), and methylglycol-(MG-) chitosan) were compared as candidate biopolymers for shell manufacturing. The aim of the work was to design OLND formulations with optimized physico-chemical characteristics, efficacy in oxygen release, and biocompatibility. All OLND formulations displayed spherical morphology, cationic surfaces, ≤500 nm diameters (with LW chitosan-shelled OLNDs being the smallest), high stability, good oxygen encapsulation efficiency, and prolonged oxygen release kinetics. Upon cellular internalization, LW, MW, and G-chitosan-shelled nanodroplets did not significantly affect the viability, health, or metabolic activity of human keratinocytes (HaCaT cell line). On the contrary, MG-chitosan-shelled nanodroplets showed very poor biocompatibility. Combining the physico-chemical and the biological results obtained, LW chitosan emerges as the best candidate biopolymer for future OLND application as a skin device to treat chronic wounds.

Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) need to be finely modulated in physiological processes. However, oxygen tension influences MMP/TIMP balances, potentially leading to pathology. Intriguingly, new 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNDs) have proven effective in abrogating hypoxia-dependent dysregulation of MMP and TIMP secretion by single cell populations. This work explored the effects of different oxygen tensions and dextran-shelled OLNDs on MMP/TIMP production in an organized and multicellular tissue (term human placenta). Chorionic villous explants from normal third-trimester pregnancies were incubated with/without OLNDs in 3 or 20% O2. Explants cultured at higher oxygen tension released constitutive proMMP-2, proMMP-9, TIMP-1, and TIMP-2. Hypoxia significantly altered MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios enhancing TIMP-2 and reducing proMMP-2, proMMP-9, and TIMP-1 levels. Intriguingly, OLNDs effectively counteracted the effects of low oxygen tension. Collectively, these data support OLND potential as innovative, nonconventional, and cost-effective tools to counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human tissues.

Background: A noninvasive, highly sensitive and specific urine test is needed for bladder cancer (BC) diagnosis and surveillance in addition to the invasive cystoscopy. We previously described the diagnostic effectiveness of urinary tyrosine-phosphorylated proteins (UPY) and a new assay (UPY-A) for their measurement in a pilot study. The aim of this work was to evaluate the performances of the UPY-A using an independent cohort of 262 subjects. Methods: Urinary tyrosine-phosphorylated proteins were measured by UPY-A test. The area under ROC curve, cutoff, sensitivity, specificity and predictive values of UPY-A were determined. The association of UPY levels with tumour staging, grading, recurrence and progression risk was analysed by Kruskal–Wallis and Wilcoxon's test. To test the probability to be a case if positive at the UPY-A, a logistic test adjusted for possible confounding factor was used. Results: Results showed a significant difference of UPY levels between patients with BC vs healthy controls. For the best cutoff value, 261.26 Standard Units (SU), the sensitivity of the assay was 80.43% and the specificity was 78.82%. A statistically significant difference was found in the levels of UPY at different BC stages and grades between Ta and T1 and with different risk of recurrence and progression. A statistically significant increased risk for BC at UPY-A ⩾261.26 SU was observed. Conclusions: The present study supplies important information on the diagnostic characteristics of UPY-A revealing remarkable performances for early stages and allowing its potential use for different applications encompassing the screening of high-risk subjects, primary diagnosis and posttreatment surveillance.

Purpose: Chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLN) are a new class of nanodevices to effectively deliver anti-cancer drugs to tumoral cells. This study investigated their antitumoral effects ‘per se’, using a mathematical model validated on experimental data. Methods: OLN were prepared and characterized either in vitro or in vivo. TUBO cells, established from a lobular carcinoma of a BALB-neuT mouse, were investigated following 48 h of incubation in the absence/presence of different concentrations of OLN. OLN internalization, cell viability, necrosis, apoptosis, cell cycle and reactive oxygen species (ROS) production were checked as described in the Method section. In vivo tumor growth was evaluated after subcutaneous transplant in BALB/c mice of TUBO cells either without treatment or after 24 h incubation with 10% v/v OLN. Results: OLN showed sizes of about 350 nm and a positive surface charge (45 mV). Dose-dependent TUBO cell death through ROS-triggered apoptosis following OLN internalization was detected. A mathematical model predicting the effects of OLN uptake was validated on both in vitro and in vivo results. Conclusions: Due to their intrinsic toxicity OLN might be considered an adjuvant tool suitable to deliver their therapeutic cargo intracellularly and may be proposed as promising combined delivery system.

BAPGEAPSIINRA; The faba bean (Vicia faba L.) is a potential source of proteins for poultry, mainly for laying hens whose protein requirements are lower than those of other birds such as growing broilers and turkeys. However, this feedstuff contains anti-nutritional factors, that is, vicine (V) and convicine (C) that are already known to reduce laying hen performance. The aim of the experiment reported here was to evaluate the effects of a wide range of dietary V and C concentrations in laying hens. Two trials were performed with laying hens fed diets including 20% or 25% of faba bean genotypes highly contrasting in V+C content. In Trial 1, faba beans from two tannin-containing cultivars, but with high or low V+C content were dehulled in order to eliminate the tannin effect. In addition to the contrasting levels of V+C in the two cultivars, two intermediate levels of V+C were obtained by mixing the two cultivars (70/30 and 30/70). In Trial 2, two isogenic zero-tannin faba bean genotypes with high or low V+C content were used. In both trials, a classical corn–soybean diet was also offered to control hens. Each experimental diet was given to 48 laying hens for 140 (Trial 1) or 89 (Trial 2) days. Laying performance and egg quality were measured. The redox sensitivity of red blood cells (RBCs) was assessed by measuring hemolysis and reduced glutathione (GSH) concentration in these cells. Egg weight was significantly reduced by the diets containing the highest concentrations of V+C (P

Asbestos exposure increases the risk of asbestosis and malignant mesothelioma (MM). Both fibrosis and cancer have been correlated with the Epithelial to Mesenchymal Transition (EMT)&mdash, an event involved in fibrotic development and cancer progression. During EMT, epithelial cells acquire a mesenchymal phenotype by modulating some proteins. Different factors can induce EMT, but Transforming Growth Factor &beta, (TGF-&beta, ) plays a crucial role in promoting EMT. In this work, we verified if EMT could be associated with MM development. We explored EMT in human mesothelial cells (MeT-5A) exposed to chrysotile asbestos: we demonstrated that asbestos induces EMT in MeT-5A cells by downregulating epithelial markers E-cadherin, &beta, catenin, and occludin, and contemporarily, by upregulating mesenchymal markers fibronectin, &alpha, SMA, and vimentin, thus promoting EMT. In these cells, this mechanism is mediated by increased TGF-&beta, secretion, which in turn downregulates E-cadherin and increases fibronectin. These events are reverted in the presence of TGF-&beta, antibody, via a Small Mother Against Decapentaplegic (SMAD)-dependent pathway and its downstream effectors, such as Zinc finger protein SNAI1 (SNAIL-1), Twist-related protein (Twist), and Zinc Finger E-Box Binding Homeobox 1 (ZEB-1), which downregulate the E-cadherin gene. Since SNAIL-1, Twist, and ZEB-1 have been shown to be overexpressed in MM, these genes could be considered possible predictive or diagnostic markers of MM development.

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming growth factor-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

The lipid moiety of natural haemozoin (nHZ, malarial pigment) was previously shown to enhance expression and release of human monocyte matrix metalloproteinase-9 (MMP-9), and a major role for 15-(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a nHZ lipoperoxidation product, was proposed. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen-activated protein kinases (MAPKs). Results showed that nHZ promoted either early or late p38 MAPK phosphorylation; however, nHZ did not modify basal phosphorylation/expression ratios of extracellular signal-regulated kinase-1/2 and c-jun N-terminal kinase-1/2. 15-HETE mimicked nHZ effects on p38 MAPK, whereas lipid-free synthetic (s)HZ and delipidized (d)HZ did not. Consistently, both nHZ and 15-HETE also promoted phosphorylation of MAPK-activated protein kinase-2, a known p38 MAPK substrate; such an effect was abolished by SB203580, a synthetic p38 MAPK inhibitor. SB203580 also abrogated nHZ-dependent and 15-HETE-dependent enhancement of MMP-9 mRNA and protein (latent and activated forms) levels in cell lysates and supernatants. Collectively, these data suggest that in human monocytes, nHZ and 15-HETE upregulate MMP-9 expression and secretion through activation of p38 MAPK pathway. The present work provides new evidence on mechanisms underlying MMP-9 deregulation in malaria, which might be helpful to design new specific drugs for adjuvant therapy in complicated malaria.

Vancomycin (Vm) currently represents the gold standard against methicillin-resistant Staphylococcus aureus (MRSA) infections. However, it is associated with low oral bioavailability, formulation stability issues, and severe side effects upon systemic administration. These drawbacks could be overcome by Vm topical administration if properly encapsulated in a nanocarrier. Intriguingly, nanobubbles (NBs) are responsive to physical external stimuli such as ultrasound (US), promoting drug delivery. In this work, perfluoropentane (PFP)-cored NBs were loaded with Vm by coupling to the outer dextran sulfate shell. Vm-loaded NBs (VmLNBs) displayed ∼300nm sizes, anionic surfaces and good drug encapsulation efficiency. In vitro, VmLNBs showed prolonged drug release kinetics, not accompanied by cytotoxicity on human keratinocytes. Interestingly, VmLNBs were generally more effective than Vm alone in MRSA killing, with VmLNB antibacterial activity being more sustained over time as a result of prolonged drug release profile. Besides, VmLNBs were not internalized by staphylococci, opposite to Vm solution. Further US association promoted drug delivery from VmLNBs through an in vitro model of porcine skin. Taken together, these results support the hypothesis that proper Vm encapsulation in US-responsive NBs might be a promising strategy for the topical treatment of MRSA wound infections.

Severe falciparum malaria is characterized by the sequestration of infected erythrocytes and leukocyte recruitment in the microvasculature, resulting in impaired blood flow and metabolic disturbances. Which parasite products cause chemokine production, thus contributing to the strong host inflammatory response and cellular recruitment are not well characterized. Here, we studied haemozoin (Hz), the end-product of haem, a ferriprotoporphyrin-IX crystal bound to host and parasite lipids, DNA, and proteins. We found that natural Hz isolated from Plasmodium falciparum cultures induces CXCL8 and CCL5 production in human dermal microvascular endothelial cells (HMEC-1) in a time-dependent manner. This up-regulation is not caused by haem but rather by Hz-generated lipoperoxidation products (15-HETE) and fibrinogen associated to Hz, and is, at least in part, triggered by the activation of NF-κB, as it was significantly inhibited by artemisinin and other NF-κB pathway inhibitors.

Background: Chrysotile asbestos accounts for > 90% of the asbestos used worldwide, and exposure is associated with asbestosis (asbestos-related fibrosis) and other malignancies; however, the molecular mechanisms involved are not fully understood. A common pathogenic mechanism for these malignancies is represented by epithelial–mesenchymal transition (EMT), through which epithelial cells undergo a morphological transformation to assume a mesenchymal phenotype. In the present work, we propose that chrysotile asbestos induces EMT through a mechanism involving a signaling pathway mediated by tranforming growth factor beta (TGF-β). Objectives: We investigated the role of chrysotile asbestos in inducing EMT in order to elucidate the molecular mechanisms involved in this event. Methods: Human bronchial epithelial cells (BEAS-2B) were incubated with 1 μg/cm2 chrysotile asbestos for ≤ 72 hr, and several markers of EMT were investigated. Experiments with specific inhibitors for TGF-β, glycogen synthase kinase–3β (GSK-3β), and Akt were performed to confirm their involvement in asbestos-induced EMT. Real-time polymerase chain reaction (PCR), Western blotting, and gelatin zymography were performed to detect mRNA and protein level changes for these markers. Results: Chrysotile asbestos activated a TGF-β–mediated signaling pathway, implicating the contributions of Akt, GSK-3β, and SNAIL-1. The activation of this pathway in BEAS-2B cells was associated with a decrease in epithelial markers (E-cadherin and β-catenin) and an increase in mesenchymal markers (α-smooth muscle actin, vimentin, metalloproteinases, and fibronectin). Conclusions: Our findings suggest that chrysotile asbestos induces EMT, a common event in asbestos-related diseases, at least in part by eliciting the TGF-β–mediated Akt/GSK-3β/SNAIL-1 pathway. Citation: Gulino GR, Polimeni M, Prato M, Gazzano E, Kopecka J, Colombatto S, Ghigo D, Aldieri E. 2016. Effects of chrysotile exposure in human bronchial epithelial cells: insights into the pathogenic mechanisms of asbestos-related diseases. Environ Health Perspect 124:776–784; http://dx.doi.org/10.1289/ehp.1409627

Summary Severe malaria, including cerebral malaria (CM), is characterized by the sequestration of parasitized erythrocytes in the microvessels after cytoadher- ence to endothelial cells. Products of parasite origin, such as haemozoin (HZ), contribute to the pathogenesis of severe malaria by interfering with host inflammatory response. In human monocytes, HZ enhanced the levels of matrix metalloproteinase-9 (MMP-9), a protease involved in neuroinflammation. Here the effects of HZ on the regulation of MMPs by the human microvascular endothelial cell line HMEC-1 were investigated. Cells treated with natural (n)HZ appeared elongated instead of polygonal, and formed microtubule-like vessels on synthetic basement membrane. nHZ enhanced total gelatinolytic activity by inducing proMMP-9 and MMP-9 without affecting basal MMP-2. The level of the endogenous tissue inhibitor of MMP-9 (TIMP-1) was not altered by nHZ, while TIMP-2, the MMP-2 inhibitor, was enhanced. Addi- tionally, nHZ induced MMP-1 and MMP-3, two enzymes sequentially involved in collagenolysis and proMMP-9 proteolytic activation. Lipid-free HZ did not reproduce nHZ effects. Present data suggest that the lipid moiety of HZ alters the MMP/ TIMP balances and promotes the proteolytic activa- tion of proMMP-9 in HMEC-1, thereby enhancing total gelatinolytic activity, cell activation and inflam- mation. These findings might help understanding the mechanisms of blood brain barrier damage during CM.

Haemozoin (HZ, malarial pigment) is a crystalline ferriprotoporphyrin IX polymer derived from undigested host haemoglobin haem, present in late stages of Plasmodium falciparum-parasitized RBCs and in residual bodies shed after schizogony. It was shown previously that phagocytosed HZ or HZ-containing trophozoites increased monocyte matrix metalloproteinase-9 (MMP-9) activity and enhanced production of MMP-9-related cytokines TNF and IL-1beta. Here we show that in human monocytes the HZ/trophozoite phagocytosis effects and their recapitulation by 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem catalysis, were mediated via activation of NF-κB transcription pathway. After phagocytosis of HZ/trophozoites or treatment with 15-HETE, the NF-κB complex migrated to the nuclear fraction while the inhibitory cytosolic IκBalpha protein was phosphorylated and degraded. All HZ/trophozoite/15-HETE effects on MMP-9 activity and TNF/IL-1beta production were abrogated by quercetin, artemisinin and parthenolide, inhibitors of IκBalpha phosphorylation and subsequent degradation, NF-κB nuclear translocation, and NF-κB-p65 binding to DNA respectively. In conclusion, enhanced activation of MMP-9, and release of pro-inflammatory cytokines TNF and IL-1beta, a triad of effects involved in malaria pathogenesis, elicited in human monocytes by trophozoite and HZ phagocytosis and recapitulated by 15-HETE, appear to be causally connected to persisting activation of the NF-κB system.

ObjectiveMore than 2 billion people are at risk of malaria, which primarily affects poor populations in tropical and subtropical areas, including Southern Asia. As malaria incidence has been reduced strongly in some parts of endemic regions by combinations of interventions, including artemisinin-based therapies and insecticide-treated bed nets, a new goal has been established recently by charity foundations which support research on malaria: the worldwide eradication of the pathology. Doing away with control approaches which have been applied for the last 50 years and more focus on elimination objectives will deeply change priorities in the area of malaria treatment, chemoprevention, vector control, vaccine research and health system assessment. In this review, actual knowledge on pathogenesis and pharmacology is discussed, and new drugs, vaccines and insecticides are described.

Com o avanço da meliponicultura, a utilização das abelhas sem ferrão, assim como de seus subprodutos, tem abrangido novas áreas, como a polinização de culturas agrícolas. Assim, a demanda pelo aumento do número de colônias tem sido constante, porém devido ao pouco conhecimento sobre a biologia reprodutiva dessas abelhas, há dificuldade de produção de colônias em larga escala, acarretando uma séria limitação quanto à utilização comercial desses polinizadores. Buscamos com este trabalho, oferecer ferramentas que possibilitem a multiplicação de colônias de abelhas sem ferrão em grande quantidade em um curto período de tempo. Nas abelhas indígenas sem ferrão (exceto no gênero Melipona e nos casos onde ocorrem rainhas-miniatura) a quantidade de alimento ingerido pelas larvas fêmeas é o fator responsável pela diferenciação das castas, pois as larvas que se tornam rainhas ingerem mais alimento que as larvas de futuras operárias, não havendo diferença qualitativa entre o alimento fornecido às larvas que originarão ambas as castas. De acordo com este modelo de determinação de castas, buscamos estabelecer a produção in vitro de rainhas em Tetragonisca angustula oferecendo maior quantidade de alimento às larvas de operárias, induzindo seu desenvolvimento em rainhas, que após a emergência foram introduzidas em mini-colônias órfãs para verificação da sua viabilidade (fecundação natural e postura de ovos). Em condições naturais as larvas de T. angustula que se tornam rainhas e operárias recebem respectivamente 55 µL e 8 µL de alimento em média. Assim, para a produção in vitro de rainhas oferecemos 55 µL de alimento a larvas de operárias coletadas em estágio pré-alimentação de ninhos naturais e conseguimos uma taxa de sobrevivência de até 51% e de 19% na obtenção de rainhas fisiogástricas, configurando um avanço em relação à taxa natural de emergência de rainhas, que foi de 0,21%. Das mini-colônias onde foram introduzidas rainhas virgens, 41% tiveram sucesso e se tornaram colônias perenes. A utilização de larvas de operárias na produção in vitro de rainhas é possível devido ao fato de as larvas serem totipotentes, assim como a utilização de alimento coletado de células de cria de operárias/machos, pois os resultados das análises comparativas do conteúdo protéico e de aminoácidos totais e livres dos alimentos contidos em células de cria de operárias/machos e células reais mostraram não haver diferenças significativas. A utilização de alimento larval de Scaptotrigona aff. depilis na criação in vitro de rainhas de T. angustula mostrou a possibilidade de produção de rainhas viáveis com esta nova técnica embora os perfis protéicos dos alimentos larvais de ambas as espécies sejam diferentes. Os experimentos de produção in vitro de rainhas com diferentes quantidades de alimento oferecido às larvas mostraram a existência de uma quantidade limite de alimento entre 35 µL e 45 µL acima da qual todos os indivíduos se tornam rainhas e abaixo da qual todos se tornam operárias, não havendo a ocorrência de indivíduos intermediários (intercastas). Introduzindo as rainhas produzidas in vitro em mini-colônias órfãs, conseguimos o estabelecimento de 16 colônias perenes a partir de seis colônias doadoras de material no período de seis meses, o que configura um avanço de 33% em relação às técnicas tradicionais de multiplicação de colônias que conseguiriam formar, no máximo, 12 colônias no mesmo período. A verificação da freqüência de produção de sexuados nas colônias naturais ao longo do ano mostrou que os machos são produzidos sazonalmente com alta taxa no período de fevereiro a abril, e embora as rainhas possuam uma produção baixa e homogênea ao longo do ano, concluímos que aquelas produzidas no período onde ocorre maior disponibilidade de machos possuem maiores chances de serem fecundadas. Assim, foi extremamente importante sincronizar a produção in vitro de rainhas com o período de maior disponibilidade de machos, uma vez que a fecundação destas ocorreu naturalmente. A produção in vitro de rainhas e a multiplicação de colônias nas abelhas sem ferrão se tornam ferramentas importantes para as técnicas de manejo que visam obter colônias em larga escala, com emprego na polinização, meliponicultura e conservação. With the improvement of meliponiculture, the use of stingless bees, as well as their byproducts, has comprised new areas, such as the pollination of crops. Thus, the requirement by increasing the number of colonies has been constant, but due to poor knowledge about reproductive biology of these bees, have been difficult produce colonies on a large scale, what causes serious limitations on the commercial use of these pollinators. Our goals were to offer tools that allowed the multiplication of stingless bees colonies in large quantities in a short time. In stingless bees (except in the genus Melipona and in cases where there are miniature-queens) the amount of food ingested by female larvae is the responsible feature for caste differentiation, because the larvae that will become queens must to ingest more food than the larvae of future workers, and there is no qualitative difference between the food provided to larvae of both castes. According to this model of caste determination, we aimed at to establish the in vitro production of queens in Tetragonisca angustula, offering a large quantity of larval food to the workers larvae, thus inducing their development into queens that after rearing were introduced in orphaned mini-colonies to verify their feasibility (natural mating and egg laying). Under natural conditions the larvae of T. angustula that become queens and workers receive respectively, 55 µL and 8 µL of food on average. Thus, for the in vitro production of queens we offered 55 µL of food to workers larvae collected in pre-feeding stage from natural nests and we were able to get a survival rate up to 51 % and 19% in obtaining viable queens, which is an improvement compared to 0,21% of rearing queens observed in natural hives. About 41% of mini-colonies, where viable queens were introduced were successful and have become perennial colonies. The use of workers larvae on in vitro production of queens is possible because the larvae are totipotent, as well as the use of food collected from workers/males brood cells, because the results of comparative analysis of protein content and total and free amino acids of food stored in worker/males brood cells and royal cells showed no significant differences. The use of larval food from Scaptotrigona aff. depilis on in vitro rearing of T. angustula queens showed the possibility of producing viable queens, with this new technique, although the protein profiles of larval food of both species were different. The experiments of in vitro production of queens with different amounts of food offered to the larvae showed the existence of a threshold quantity of food between 35 mL and 45 mL above which all individuals become queens and below which all individuals become workers, without the occurrence of intermediate individuals (intercaste). Inserting the queens produced in vitro in orphaned mini-colonies, we were able to establish 16 perennial colonies as from six colonies donors of material in six months, constituting an increase of 33% compared to traditional techniques of multiplication of colonies which would form up only 12 colonies in the same period. Verification of frequency of sexuals reared in natural colonies over the year showed that males are produced seasonally with high rate in the period from February to April, and although the queens have a low and homogeneous occurrence over the year, we concluded that the queens produced during the period where there is greater availability of males, have higher chances of being fertilized. Thus, was extremely important synchronize the in vitro production of queens with the period where there is greater availability of males, since fertilization of these queens occurred naturally. The in vitro production of queens and multiplication of colonies in stingless bees become important tools for handling techniques that aim to obtain colonies on a large scale, with employment in pollination, meliponiculture and conservation.

ABSTRACT Background: Chronic wounds, characterized by hypoxia, inflammation and impaired tissue remodeling, are often worsened by bacterial/fungal infections. Intriguingly, chitosan-shelled/decafluoropentane-cored oxygen-loaded nanodroplets (OLNs) have proven effective in delivering oxygen to hypoxic tissues. Aim: The present work aimed at investigating nanodroplet antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) or Candida albicans, toxicity on human keratinocytes (HaCaT) and ultrasound (US)-triggered transdermal delivery. Materials & methods: Nanodroplet antibacterial/antifungal properties, human cytotoxicity, and US-triggered transdermal delivery were measured through microbiological, biochemical, and sonophoresis assays, respectively. Results: OLNs and oxygen-free nanodroplets (OFNs) displayed short- or long-term cytostatic activity against MRSA or Candida albicans, respectively. OLNs were not toxic to keratinocytes, whereas OFNs slightly affected cell viability. Complementary US treatment promoted OLN transdermal delivery. Conclusion: As such, US-activated chitosan-shelled OLNs appear as promising, nonconventional and innovative tools for adjuvant treatment of infected chronic wounds.

In chronic wounds, hypoxia seriously undermines tissue repair processes by altering the balances between pro-angiogenic proteolytic enzymes (matrix metalloproteinases, MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) released from surrounding cells. Recently, we have shown that in human monocytes hypoxia reduces MMP-9 and increases TIMP-1 without affecting TIMP-2 secretion, whereas in human keratinocytes it reduces MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. Provided that the phenotype of the cellular environment is better understood, chronic wounds might be targeted by new oxygenating compounds such as chitosan- or dextran-shelled and 2H,3H-decafluoropentane-cored oxygen-loaded nanodroplets (OLNs). Here, we investigated the effects of hypoxia and dextran-shelled OLNs on the pro-angiogenic phenotype and behavior of human dermal microvascular endothelium (HMEC-1 cell line), another cell population playing key roles during wound healing. Normoxic HMEC-1 constitutively released MMP-2, TIMP-1 and TIMP-2 proteins, but not MMP-9. Hypoxia enhanced MMP-2 and reduced TIMP-1 secretion, without affecting TIMP-2 levels, and compromised cell ability to migrate and invade the extracellular matrix. When taken up by HMEC-1, nontoxic OLNs abrogated the effects of hypoxia, restoring normoxic MMP/TIMP levels and promoting cell migration, matrix invasion, and formation of microvessels. These effects were specifically dependent on time-sustained oxygen diffusion from OLN core, since they were not achieved by oxygen-free nanodroplets or oxygen-saturated solution. Collectively, these data provide new information on the effects of hypoxia on dermal endothelium and support the hypothesis that OLNs might be used as effective adjuvant tools to promote chronic wound healing processes.

The impact of complement activation and its possible relation to cytokine responses during malaria pathology was investigated in plasma samples from patients with confirmed Plasmodium falciparum malaria and in human whole-blood specimens stimulated with malaria-relevant agents ex vivo. Complement was significantly activated in the malaria cohort, compared with healthy controls, and was positively correlated with disease severity and with certain cytokines, in particular interleukin 8 (IL-8)/CXCL8. This was confirmed in ex vivo-stimulated blood specimens, in which complement inhibition significantly reduced IL-8/CXCL8 release. P. falciparum malaria is associated with systemic complement activation and complement-dependent release of inflammatory cytokines, of which IL-8/CXCL8 is particularly prominent.

Plasmodium parasites are able to detoxify high amounts of heme in human red blood cells through biocrystallization into malarial pigment hemozoin (Hz), which is avidly phagocytosed by human monocytes and neutrophils. As a consequence of phagocytosis, the phenotype of these cells is dramatically altered and their functions are seriously impaired. Hz induces quite immediately an enhanced release of lysozyme, stored in the so-called gelatinase granules, and promotes complete degranulation at later time points. Hz-dependent lysozyme release has been recently investigated in a series of recent studies. Among several proinflammatory molecules, TNF-α, IL-1β, and MIP-1α/CCL3 have been indicated as potential soluble mediators of Hz-dependent lysozyme upregulation. The mechanisms of signal transduction underlying such an enhancement have also been investigated, suggesting a potential role for p38 mitogen-activated protein kinase and nuclear factor-kappaB transcriptional pathways. Finally, a major role in lysozyme release has been suggested for the lipid moiety of Hz. Interestingly, 15-HETE, a major lipoperoxidation product of Hz, was strongly supported to be the main actor in these events.

Background : In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. Objective : To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. Methods : HaCaT cells were treated for 24 h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. Results : Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. Conclusion : Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds.

Monocytes play a key role in the inflammatory stage of the healing process. To allow monocyte migration to injured tissues, the balances between secreted matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) must be finely modulated. However, a reduction of blood supply and local oxygen tension can modify the phenotype of immune cells. Intriguingly, hypoxia might be targeted by new effective oxygenating devices such as 2H,3H-decafluoropentane- (DFP-) based oxygen-loaded nanodroplets (OLNs). Here, hypoxia effects on gelatinase/TIMP release from human peripheral monocytes were investigated, and the therapeutic potential of dextran-shelled OLNs was evaluated. Normoxic monocytes constitutively released ~500 ng/mL MMP-9, ~1.3 ng/mL TIMP-1, and ~0.6 ng/mL TIMP-2 proteins. MMP-2 was not detected. After 24 hours, hypoxia significantly altered MMP-9/TIMP-1 balance by reducing MMP-9 and increasing TIMP-1, without affecting TIMP-2 secretion. Interestingly OLNs, not displaying toxicity to human monocytes after cell internalization, effectively counteracted hypoxia, restoring a normoxia-like MMP-9/TIMP-1 ratio. The action of OLNs was specifically dependent on time-sustained oxygen diffusion up to 24 h from their DFP-based core. Therefore, OLNs appear as innovative, nonconventional, cost-effective, and nontoxic therapeutic tools, to be potentially employed to restore the physiological invasive phenotype of immune cells in hypoxia-associated inflammation.

Perfluoropentane (PFP)-based oxygen-loaded nanobubbles (OLNBs) were previously proposed as adjuvant therapeutic tools for pathologies of different etiology sharing hypoxia as a common feature, including cancer, infection, and autoimmunity. Here we introduce a new platform of oxygen nanocarriers, based on 2H,3H-decafluoropentane (DFP) as core fluorocarbon. These new nanocarriers have been named oxygen-loaded nanodroplets (OLNDs) since DFP is liquid at body temperature, unlike gaseous PFP. Dextran-shelled OLNDs, available either in liquid or gel formulations, display spherical morphology, ~600 nm diameters, anionic charge, good oxygen carrying capacity, and no toxic effects on human keratinocytes after cell internalization. In vitro OLNDs result more effective in releasing oxygen to hypoxic environments than former OLNBs, as demonstrated by analysis through oxymetry. In vivo, OLNDs effectively enhance oxy-hemoglobin levels, as emerged from investigation by photoacoustic imaging. Interestingly, ultrasound (US) treatment further improves transdermal oxygen release from OLNDs. Taken together, these data suggest that US-activated, DFP-based OLNDs might be innovative, suitable and cost-effective devices to topically treat hypoxia-associated pathologies of the cutaneous tissues.

Diagnostic and prognostic biomarkers are used to predict the progress and outcome of several chronic diseases, including neoplastic and noncommunicable diseases. Recently, biomarker research has been exploited to accurately discriminate the severe forms of malaria, since no biological tests to confirm the diagnosis of severe malaria and its complications are available so far. In this context, human lysozyme appears as a good candidate. Since its discovery almost one century ago, several easy-to-handle and cost-effective methods to determine lysozyme activity in various samples (plasma, urine, and tissue sections) have been developed. Interestingly, results from two small prospective studies enrolling healthy volunteers and malaria patients from Sudan showed a significant correlation between disease severity and lysozyme plasma levels. These promising data encourage further studies employing larger cohorts of patients from different areas endemic for malaria. Intriguingly, an old molecule such as human lysozyme might reveal itself as a new and effective biomarker for malaria severity.

Malaria is a global disease that clinically affects more than two hundred million people annually. Despite the availability of effective antimalarials, mortality rates associated with severe complications are high. Hepatopathy is frequently observed in patients with severe malarial disease and its pathogenesis is poorly understood. Previously, we observed high amounts of hemozoin or malaria pigment in livers from infected mice. In this study, we investigated whether hemozoin is associated with liver injury in different mouse malaria models. C57BL/6J mice infected with the rodent parasites Plasmodium berghei ANKA, P. berghei NK65 or P. chabaudi AS had elevated serum liver enzymes without severe histological changes in the liver, in line with the observations in most patients. Furthermore, liver enzymes were significantly higher in serum of P. chabaudi AS-infected mice compared to mice infected with the P. berghei parasite strains and a strong positive correlation was found between hepatic hemozoin levels, hepatocyte damage and inflammation in the liver with P. chabaudi AS. The observed liver injury was only marginally influenced by the genetic background of the host, since similar serum liver enzyme levels were measured in infected C57BL/6J and BALB/c mice. Intravenous injection of P. falciparum-derived hemozoin in malaria-free C57BL/6J mice induced inflammatory gene transcription in the liver, suggesting that hemozoin may be involved in the pathogenesis of malaria hepatopathy by inducing inflammation. ispartof: PLoS One vol:9 issue:11 ispartof: location:United States status: published

Malaria is a parasitic disease caused by Plasmodium protozoan parasites and transmitted by Anopheles mosquitoes. The disease is diffused in tropical areas, where it is associated with high morbidity and mortality. P. falciparum is the most dangerous species, mainly affecting young children. The parasite cycle occurs both in humans (asexual stages) and in mosquitoes (sexual stages). In humans, Plasmodium grows and multiplies within red blood cells using hemoglobin as essential source of nutrients and energy. However, this process generates toxic heme that the parasite aggregates into an insoluble inert biocrystal called hemozoin. This molecule sequesters in various organs (liver, spleen, and brain), potentially contributing to the development of malaria immunopathogenesis. Uncomplicated falciparum malaria clinical frame ranges from asymptomatic infection to classic symptoms such as fever, chills, sweating, headache, and muscle aches. However, malaria can also evolve into severe life-threatening complications, including cerebral malaria, severe anemia, respiratory distress, and acute renal failure.

In human adherent monocytes matrix metalloproteinase-9 (MMP-9) expression and secretion is upregulated by the lipid moiety of natural haemozoin (nHz, malarial pigment). A role for 15-(S, R)-hydroxy-6, 8, 11, 13-eicosatetraenoic acid (15-HETE), a nHZ lipoperoxidation product, has been suggested. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen-activated protein kinases (MAPKs). Either early or late p38 MAPK phosphorylation was induced by nHz, which did not modify basal phosphorylation and expression ratios of extracellular signal-regulated kinase-1/2 and c-jun N-terminal kinase-1/2. 15-HETE mimicked nHZ effects on p38 MAPK. Lipid-free synthetic (s)HZ and delipidized (d)HZ did not. Both nHZ and 15-HETE promoted the phosphorylation of MAPK-activated protein kinase-2, a known substrate of p38 MAPK. Such an effect was abolished by SB203580 (synthetic p38 MAPK inhibitor). SB203580 also abrogated the nHZ-dependent and 15-HETE-dependent enhancement of MMP-9 mRNA and protein levels in cell lysates and supernatants. These data suggest that nHZ and 15-HETE upregulate MMP-9 expression and secretion through the activation of p38 MAPK pathway in human adherent monocytes. This work provides new evidence on the mechanisms underlying MMP-9 deregulation in malaria. These data might help to design new specific drugs for adjuvant therapy in complicated malaria.

Ultrasound (US)-activated perfluoropentane-cored oxygen-loaded nanobubbles (OLNBs) were recently proposed as adjuvant therapeutic tools for pathologies of different etiology sharing hypoxia as a common feature (e.g. diabetes-associated chronic wounds, anaerobic infections, cancer). Here we introduce a new platform of oxygen nanocarriers, constituted of 2H,3H-decafluoropentane (DFP) as core fluorocarbon and chitosan as shell polysaccharide, and available either in liquid or gel formulations. Such oxygen-loaded nanodroplets (OLNDs) display spherical morphology, ∼700 nm diameters, cationic surfaces, good oxygen carrying capacity (without singlet oxygen generation after sterilization by ultraviolet-C rays), and no toxic effects on human keratinocytes. In vitro, OLNDs are more effective in releasing oxygen to hypoxic environments than former OLNBs, either with or without complementary US administration (f = 1 MHz; P = 5 W). In vivo, sonication of topically applied OLNDs appears essential to allow significant and time-sustained oxygen release. Taken together, the present data suggest that US-activated chitosan-shelled/DFP-cored OLNDs might be innovative, suitable and cost-effective devices to treat several hypoxia-associated pathologies of the cutaneous tissues.

Malaria is a parasitic disease confined mostly to the tropical areas, caused by Plasmodium parasites and transmitted by Anopheles mosquitoes. In 2010, nearly 655.000 human deaths, mainly of children ≤5 years of age, were registered among more than 200 million cases worldwide of clinical malaria; the vast majority of cases occurred in the African Region (81%) and South-East Asia (13%), and 91% of them were due to P. falciparum, the most viru‐ lent among Plasmodia strains (WHO, 2011a).