Design, Characterization, and Biological Activities of Erythromycin-Loaded Nanodroplets to Counteract Infected Chronic Wounds Due to Streptococcus pyogenes.

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.

Comparative Evaluation of Different Chitosan Species and Derivatives as Candidate Biomaterials for Oxygen-Loaded Nanodroplet Formulations to Treat Chronic Wounds.

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.

MMP23B expression and protein levels in blood and urine are associated with bladder cancer.

Urothelial bladder cancer (UBC) represents a public health problem because of its high incidence/relapse rates. At present, there are no suitable biomarkers for early diagnosis or relapse/progression prognosis. To improve diagnostic accuracy and overcome the disadvantages of current diagnostic strategies, the detection of UBC biomarkers in easily accessible biofluids, such as urine, represents a promising approach compared with painful biopsies. We investigated the levels of MMP23 genes (microarray and qPCR) and protein (western blot and enzyme-linked immunosorbent assay) in a set of samples (blood, plasma and urine) from patients with UBC and controls as biomarkers for this cancer. MMP23B and its pseudogene MMP23A resulted downregulated in blood cells from UBC compared with controls (66 cases, 70 controls; adjusted P-value = 0.02 and 0.03, respectively). In contrast, MMP23B protein levels in plasma (53 UBC, 49 controls) and urine (59 UBC, 57 controls) increased in cases, being statistically significant in urine. MMP23B dosage observed in urine samples was related to both tumor risk classification and grading. As the lack of correlation between mRNA and protein levels could be due to a posttranscriptional regulation mediated by microRNAs (miRNAs), we investigated the expression of urinary miRNAs targeting MMP23B. Five miRNAs resulted differentially expressed between cases and controls. We reported the first evidence of MMP23B secretion in plasma and urine, suggesting a role of this poorly characterized metalloproteinase in UBC as a potential non-invasive biomarker for this cancer. Further analyses are needed to elucidate the mechanism of regulation of MMP23B expression by miRNAs.

'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.

Complement Activation Correlates With Disease Severity and Contributes to Cytokine Responses in Plasmodium falciparum Malaria.

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.

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.

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.

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.

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.

Antibacterial and Antifungal Efficacy of Medium and Low Weight Chitosan-Shelled Nanodroplets for the Treatment of Infected Chronic Wounds.

Purpose: 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.

Antimicrobial oxygen-loaded nanobubbles as promising tools to promote wound healing in hypoxic human keratinocytes.

Chronic wounds (CWs) are typically characterized by persistent hypoxia, exacerbated inflammation, and impaired skin tissue remodeling. Additionally, CWs are often worsened by microbial infections. Oxygen-loaded nanobubbles (OLNBs), displaying a peculiar structure based on oxygen-solving perfluorocarbons such as perfluoropentane in the inner core and polysaccharydes including chitosan in the outer shell, have proven effective in delivering oxygen to hypoxic tissues. Antimicrobial properties have been largely reported for chitosan. In the present work chitosan/perfluoropentane OLNBs were challenged for biocompatibility with human skin cells and ability to promote wound healing processes, as well as for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. After cellular internalization, OLNBs were not toxic to human keratinocytes (HaCaT), whereas oxygen-free NBs (OFNBs) slightly affected their viability. Hypoxia-dependent inhibition of keratinocyte migratory ability after scratch was fully reversed by OLNBs, but not OFNBs. Both OLNBs and OFNBs exerted chitosan-induced short-term bacteriostatic activity against MRSA (up to 6 h) and long-term fungistatic activity against C. albicans (up to 24 h). Short-term antibacterial activity associated with NB prolonged adhesion to MRSA cell wall (up to 24 h) while long-term antifungal activity followed NB early internalization by C. albicans (already after 3 h of incubation). Taken altogether, these data support chitosan-shelled and perfluoropentane-cored OLNB potential as innovative, promising, non-toxic, and cost-effective antimicrobial devices promoting repair processes to be used for treatment of MRSA- and C. albicans-infected CWs.

Evidence of abnormal tyrosine phosphorylated proteins in the urine of patients with bladder cancer: the road toward a new diagnostic tool?

PURPOSE: Since changes in protein phosphorylation are a common feature of cancer cells, we analyzed phosphoproteins in the tissue and urine of patients with bladder cancer and assessed the diagnostic relevance of abnormally phosphorylated proteins as tumor markers. MATERIALS AND METHODS: Enrolled in this study were 66 patients and 82 healthy volunteers. From the first 14 patients with bladder cancer we obtained samples of malignant and normal bladder tissue. All patients and volunteers provided a urine sample. Protein extracts of tissue specimens were separated by 2-dimensional gel electrophoresis for comparative analysis of neoplastic and normal tissue. Phosphoproteins were studied by Western blot and characterized by mass spectrometry. Urine samples were analyzed by 1-dimensional gel electrophoresis. Phosphoproteins were measured by affinity dot blotting. RESULTS: Profound changes in the pattern of protein tyrosine phosphorylation were consistently, reproducibly observed in bladder cancer tissues. A total of 24 phosphorylated proteins were differentially expressed in cancer tissue and identified by mass spectrometry. Phosphoproteins were fairly stable in urine samples, leading to accumulation. Urinary tyrosine phosphoproteins showed the most remarkable changes in patients with cancer with an approximately 5-fold increase compared to levels in healthy controls. CONCLUSIONS: To our knowledge we investigated for the first time the diagnostic potential of tissue and urinary tyrosine phosphoproteins for bladder carcinoma. Results indicate that phosphorylated proteins may represent a new, valuable class of urinary biomarkers for bladder cancer.

Role of the NF-κB transcription pathway in the haemozoin- and 15-HETE-mediated activation of matrix metalloproteinase-9 in human adherent monocytes.

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.

Transforming Growth Factor-ß and Oxidative Stress in Cancer: A Crosstalk in Driving Tumor Transformation.

Cancer metabolism involves different changes at a cellular level, and altered metabolic pathways have been demonstrated to be heavily involved in tumorigenesis and invasiveness. A crucial role for oxidative stress in cancer initiation and progression has been demonstrated; redox imbalance, due to aberrant reactive oxygen species (ROS) production or deregulated efficacy of antioxidant systems (superoxide dismutase, catalase, GSH), contributes to tumor initiation and progression of several types of cancer. ROS may modulate cancer cell metabolism by acting as secondary messengers in the signaling pathways (NF-kB, HIF-1α) involved in cellular proliferation and metastasis. It is known that ROS mediate many of the effects of transforming growth factor ß (TGF-ß), a key cytokine central in tumorigenesis and cancer progression, which in turn can modulate ROS production and the related antioxidant system activity. Thus, ROS synergize with TGF-ß in cancer cell metabolism by increasing the redox imbalance in cancer cells and by inducing the epithelial mesenchymal transition (EMT), a crucial event associated with tumor invasiveness and metastases. Taken as a whole, this review is addressed to better understanding this crosstalk between TGF-ß and oxidative stress in cancer cell metabolism, in the attempt to improve the pharmacological and therapeutic approach against cancer.

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.

Analysis of changes in tyrosine and serine phosphorylation of red cell membrane proteins induced by P. falciparum growth.

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti-phosphotyrosine and anti-phosphoserine antibodies following 2-DE in conjunction with double channel laser-induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans-membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.

Involvement of inflammatory chemokines in survival of human monocytes fed with malarial pigment.

Hemozoin (HZ)-fed monocytes are exposed to strong oxidative stress, releasing large amounts of peroxidation derivatives with subsequent impairment of numerous functions and overproduction of proinflammatory cytokines. However, the histopathology at autopsy of tissues from patients with severe malaria showed abundant HZ in Kupffer cells and other tissue macrophages, suggesting that functional impairment and cytokine production are not accompanied by cell death. The aim of the present study was to clarify the role of HZ in cell survival, focusing on the qualitative and temporal expression patterns of proinflammatory and antiapoptotic molecules. Immunocytochemical and flow cytometric analyses showed that the long-term viability of human monocytes was unaffected by HZ. Short-term analysis by macroarray of a complete panel of cytokines and real-time reverse transcription (RT)-PCR experiments showed that HZ immediately induced interleukin-1ß (IL-1ß) gene expression, followed by transcription of eight additional chemokines (IL-8, epithelial cell-derived neutrophil-activating peptide 78 [ENA-78], growth-regulated oncogene α [GROα], GROß, GROγ, macrophage inflammatory protein 1α [MIP-1α], MIP-1ß, and monocyte chemoattractant protein 1 [MCP-1]), two cytokines (tumor necrosis factor alpha [TNF-α] and IL-1receptor antagonist [IL-1RA]), and the cytokine/chemokine-related proteolytic enzyme matrix metalloproteinase 9 (MMP-9). Furthermore, real-time RT-PCR showed that 15-HETE, a potent lipoperoxidation derivative generated by HZ through heme catalysis, recapitulated the effects of HZ on the expression of four of the chemokines. Intermediate-term investigation by Western blotting showed that HZ increased expression of HSP27, a chemokine-related protein with antiapoptotic properties. Taken together, the present data suggest that apoptosis of HZ-fed monocytes is prevented through a cascade involving 15-HETE-mediated upregulation of IL-1ß transcription, rapidly sustained by chemokine, TNF-α, MMP-9, and IL-1RA transcription and upregulation of HSP27 protein expression.

Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells.

Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. Diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS(3) (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results of the present study suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.

Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells.

BACKGROUND: Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. METHODS: Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70-2/70-3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. RESULTS: In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs. Protein expression of antioxidant enzymes was also increased in oxidatively-stressed trophozoites. CONCLUSION: Results indicated that mRNA expression of parasite antioxidant enzymes and HSPs was co-ordinated and stage-dependent. Secondly, both systems were redox-responsive and showed remarkably increased and co-ordinated expression in oxidatively-stressed parasites and in parasites growing in antioxidant blunted G6PD-deficient RBCs. Lastly, as important anti-malarials either increase oxidant stress or impair antioxidant defense, results may encourage the inclusion of anti-HSP molecules in anti-malarial combined drugs.