Combination of urinary fibrinogen ß-chain and tyrosine-phosphorylated proteins for the detection of bladder cancer.

AIM: To evaluate the performance of urinary fibrinogen ß-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.

Gravimetric quantitative validation of botanic impurities in feed.

BACKGROUND: Harmful botanical impurities may contaminate feed and feed materials and be a potential danger to animal or human health, or to the environment. The aim of this study was to establish rapid and sensitive methods that can be used in routine official controls to determine botanical impurities such as Datura stramonium, Ricinus communis, Crotaliaria spp., and Ambrosia spp. in animal feed and raw materials. Claviceps sclerotia were also detected in cereals, due to the similarities of the targets and the analytical procedure. Regulation (EU) 625/2017, which replaces Reg. 2004/882/EC, states that EU member states should conduct official controls in assessed and accredited laboratories and that the analytical methods must be validated before use by considering parameters such as specificity, precision, recovery, and measurement uncertainly. RESULTS AND CONCLUSION: The results demonstrate that all of the methods tested are suitable for the official quantitative analyses required by EU official legislation. © 2020 Society of Chemical Industry.

'In Vitro', 'In Vivo' and 'In Silico' Investigation of the Anticancer Effectiveness of Oxygen-Loaded Chitosan-Shelled Nanodroplets as Potential Drug Vector.

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.

Beta-2-glycoprotein-1 and alpha-1-antitrypsin as urinary markers of renal cancer in von Hippel-Lindau patients.

CONTEXT: Von Hippel-Lindau disease (VHLD) is a rare inherited neoplastic syndrome. Among all the VHLD-associated tumors, clear cell renal cell carcinoma (ccRCC) is the major cause of death. OBJECTIVE: The aim of this paper is the discovery of new non-invasive biomarker for the monitoring of VHLD patients. MATERIALS AND METHODS: We compared the urinary proteome of VHLD patients, ccRCC patients and healthy volunteers. RESULTS: Among all differentially expressed proteins, alpha-1-antitrypsin (A1AT) and APOH (beta-2-glycoprotein-1) are strongly over-abundant only in the urine of VHLD patients with a history of ccRCC. DISCUSSION AND CONCLUSION: A1AT and APOH could be promising non-invasive biomarkers.

LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

Vancomycin-loaded nanobubbles: A new platform for controlled antibiotic delivery against methicillin-resistant Staphylococcus aureus infections.

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.

Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p (72) Syk.

In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i) in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii) the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii) disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv) protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis.

Effects of oxygen tension and dextran-shelled/2H,3H-decafluoropentane-cored oxygen-loaded nanodroplets on secretion of gelatinases and their inhibitors in term human placenta.

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.

Dextran-shelled oxygen-loaded nanodroplets reestablish a normoxia-like pro-angiogenic phenotype and behavior in hypoxic human dermal microvascular endothelium.

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.

Antimicrobial chitosan nanodroplets: new insights for ultrasound-mediated adjuvant treatment of skin infection.

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.

Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing.

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 24h 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.

Oxygen-Loaded Nanodroplets Effectively Abrogate Hypoxia Dysregulating Effects on Secretion of MMP-9 and TIMP-1 by Human Monocytes.

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.

2H,3H-decafluoropentane-based nanodroplets: new perspectives for oxygen delivery to hypoxic cutaneous tissues.

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.

Thalassemic erythrocytes release microparticles loaded with hemichromes by redox activation of p72Syk kinase.

High counts of circulating microparticles, originated from the membrane of abnormal erythrocytes, have been associated with increased thrombotic risk in hemolytic disorders. Our studies indicate that in thalassemia intermedia patients the number of circulating microparticles correlates with the capability of the thalassemic erythrocytes to release microparticles. The microparticles are characteristically loaded with hemichromes formed by denatured α-chains. This finding was substantiated by the positive correlation observed in thalassemia intermedia patients between the amount of hemichromes measured in erythrocytes, their capability to release microparticles and the levels of plasma hemichromes. We observed that hemichromes, following their binding to the cytoplasmic domain of band 3, induce the formation of disulfide band 3 dimers that are subsequently phosphorylated by p72Syk kinase. Phosphorylation of oxidized band 3 appears to be relevant for the formation of large hemichromes/band 3 clusters that, in turn, induce local membrane instability and the release of microparticles. Proteomic analysis of microparticles released from thalassemia intermedia erythrocytes indicated that, besides hemichromes and clustered band 3, the microparticles contain a characteristic set of proteins that includes catalase, heat shock protein 70, peroxiredoxin 2 and carbonic anhydrase. High amounts of immunoglobulins and C3 have also been found to be associated with microparticles, accounting for their intense phagocytosis. The effect of p72Syk kinase inhibitors on the release of microparticles from thalassemia intermedia erythrocytes may indicate new perspectives for controlling the release of circulating microparticles in hemolytic anemias.

Involvement of p38 MAPK in haemozoin-dependent MMP-9 enhancement in human monocytes.

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.

Proteomic identification of Reticulocalbin 1 as potential tumor marker in renal cell carcinoma.

Renal cell carcinoma (RCC) biomarkers are necessary for diagnosis and prognosis. They serve to monitor therapy response and follow-up, as drug targets, and therapy predictors in personalized treatments. Proteomics is a suitable method for biomarker discovery. Here we investigate differential protein expression in RCC, and we evaluate Reticulocalbin 1 (RCN1) use as a new potential marker. Neoplastic and healthy tissue samples were collected from 24 RCC patients during radical nephrectomy. Seven specimens were firstly processed by proteomic analysis (2-DE and MALDI-TOF) and 18 differentially expressed proteins from neoplastic and healthy renal tissues were identified. Among them, RCN1 was over-expressed in all cancer specimens analyzed by proteomics. Consequently RCN1 use as a potential marker was further evaluated in all 24 donors. RCN1 expression was verified by Western blotting (WB) and immunohistochemistry (IHC). WB analysis confirmed RCN1 over-expression in 21 out of 24 tumor specimens, whereas IHC displayed focal or diffuse expression of RCN1 in all 24 RCC tissues. Thus RCN1 appears as a potential marker for clinical approaches. A larger histopathological trial will clarify the prognostic value of RCN1 in RCC. BIOLOGICAL SIGNIFICANCE: The present work aimed at finding new biomarkers for RCC - a life-threatening disease characterized by high incidence in Western countries - by performing differential proteomic analysis of neoplastic and normal renal tissues obtained from a small cohort of RCC patients. Some of the identified proteins have been previously associated to renal cancer however data confirming the possible use of these proteins in clinical practice are not available to date. By IHC we demonstrated that RCN1 could be easily employed in clinical practice, confirming RCN1 over-expression in RCC tissues of all examined patients, and weak protein expression in healthy renal tissues only in correspondence to the renal tubule section. These data indicate a promising role of RCN1 as a possible marker in RCC and indicate the proximal convoluted renal tubule as a putative origin point for RCC. Since IHC staining displayed different grades of intensity in tested tissues, we hypothesized that RCN1 could also be employed as a prognostic marker or as a response predictor for RCC-targeted therapy. To test such a hypothesis, a larger retrospective trial on paraffin-embedded tissues obtained from radical or partial nephrectomy of RCC patients is planned to be performed by our group.

JNK activation is required for TNFα-induced apoptosis in human hepatocarcinoma cells.

BACKGROUND: A frequent distinctive feature of tumors, hepatocellular carcinomas included, is resistance to apoptosis induced by a variety of agents, among which the pleiotropic cytokine tumor necrosis factor-α (TNF). Compared to other cell types, hepatocytes and hepatoma-derived cell lines are poorly susceptible to TNF-induced apoptosis, which is largely ascribed to activation of the prosurvival transcription factor NF-κB and can be overcome by associating TNF to low doses of protein synthesis inhibitors or other drugs. AIMS: This study analyses the molecular mechanisms by which TNF, in combination with cycloheximide (CHX), induces apoptosis in human hepatoma-derived Huh7 cells, focusing on the role played by JNK. METHODS: Huh7 cell cultures were treated with TNF + CHX in the presence or in the absence of the pancaspase inhibitor zVADfmk or of the JNK inhibitor SP600125 as well as after suppression of JNK expression by RNAi. Apoptosis was assessed both by light microscopy and by flow cytometry, JNK and caspase activation by western blotting and/or enzymatic assay. RESULTS: TNF + CHX-induced death of Huh7 cells involved JNK activation since it was partially prevented by suppressing JNK activity or expression. Moreover, apoptosis was significantly reduced also by zVADfmk, while SP600125 and zVADfmk combined totally abrogated cell death in an additive fashion. CONCLUSIONS: These results demonstrate a causal role for JNK and caspases in TNF+CHX-induced apoptosis of Huh7 human hepatoma cells. Therefore, strategies aimed at enhancing both pathways should provide a profitable basis to overcome the resistance of hepatocarcinoma cells to TNF-dependent apoptosis.

Role of 15-hydroxyeicosatetraenoic acid in hemozoin-induced lysozyme release from human adherent monocytes.

Natural hemozoin (nHZ), a lipid-bound ferriprotoporphyrin IX crystal produced by Plasmodium parasites after hemoglobin catabolism, seriously compromises the functions of human monocytes, and 15-hydroxyeicosatetraenoic acid (15-HETE) and 4-hydroxynonenal (4-HNE), two nHZ lipoperoxidation products, have been related to such a functional impairment. nHZ was recently shown to promote inflammation-mediated lysozyme release from human monocytes through p38 mitogen-activated protein kinase- (MAPK)- and nuclear factor (NF)-κB-dependent mechanisms. This study aimed at identifying the molecule of nHZ lipid moiety that was responsible for these effects. Results showed that 15-HETE mimicked nHZ effects on lysozyme release, whereas 4-HNE did not. 15-HETE-enhanced lysozyme release was abrogated by anti-TNF-α and anti-IL-1ß-blocking antibodies and mimicked by recombinant cytokines; on the contrary, MIP-1α/CCL3 was not involved as a soluble mediator of 15-HETE effects. Moreover, 15-HETE early activated p38 MAPK and NF-κB pathways by inducing p38 MAPK phosphorylation; cytosolic I-κBα phosphorylation and degradation; NF-κB nuclear translocation and DNA-binding. Inhibition of both routes through chemical inhibitors (SB203580, quercetin, artemisinin, and parthenolide) prevented 15-HETE-dependent lysozyme release. Collectively, these data suggest that 15-HETE plays a major role in nHZ-enhanced monocyte degranulation.

Haemozoin induces early cytokine-mediated lysozyme release from human monocytes through p38 MAPK- and NF-kappaB-dependent mechanisms.

Malarial pigment (natural haemozoin, HZ) is a ferriprotoporphyrin IX crystal produced by Plasmodium parasites after haemoglobin catabolism. HZ-fed human monocytes are functionally compromised, releasing increased amounts of pro-inflammatory molecules, including cytokines, chemokines and cytokine-related proteolytic enzyme Matrix Metalloproteinase-9 (MMP-9), whose role in complicated malaria has been recently suggested. In a previous work HZ was shown to induce through TNFalpha production the release of monocytic lysozyme, an enzyme stored in gelatinase granules with MMP-9. Here, the underlying mechanisms were investigated. Results showed that HZ lipid moiety promoted early but not late lysozyme release. HZ-dependent lysozyme induction was abrogated by anti-TNFalpha/IL-1 beta/MIP-1 alpha blocking antibodies and mimicked by recombinant cytokines. Moreover, HZ early activated either p38 MAPK or NF-kappaB pathways by inducing: p38 MAPK phosphorylation; cytosolic I-kappaB alpha phosphorylation and degradation; NF-kappaB nuclear translocation and DNA-binding. Inhibition of both routes through selected molecules (SB203580, quercetin, artemisinin, parthenolide) prevented HZ-dependent lysozyme release. These data suggest that HZ-triggered overproduction of TNFalpha, IL-1 beta and MIP-1 alpha mediates induction of lysozyme release from human monocytes through activation of p38 MAPK and NF-kappaB pathways, providing new evidence on mechanisms underlying the HZ-enhanced monocyte degranulation in falciparum malaria and the potential role for lysozyme as a new affordable marker in severe malaria.

Characterization of the protein ubiquitination response induced by Doxorubicin.

Doxorubicin is commonly considered to exert its anti-tumor activity by triggering apoptosis of cancer cells through DNA damage. Recent reports have shown that Doxorubicin elicits a marked heat shock response, and that either inhibition or silencing of heat shock proteins enhance the Doxorubicin apoptotic effect in neuroblastoma cells. In order to investigate whether Doxorubicin may also act through protein modification, we performed a proteomic analysis of ubiquitinated proteins. Here we show that nanomolar Doxorubicin treatment of neuroblastoma cells caused: (a) dose-dependent over-ubiquitination of a specific set of proteins in the absence of measurable inhibition of proteasome, (b) protein ubiquination patterns similar to those with Bortezomib, a proteasome inhibitor, (c) depletion and loss of activity of ubiquitinated enzymes such as lactate dehydrogenase and α-enolase, and (d) a decrease in HSP27 solubility, probably a consequence of its binding to denatured proteins. These data strongly reinforce the hypothesis that Doxorubicin may also exert its effect by damaging proteins.