Kidney injury molecule 1 (KIM-1): a potential biomarker of acute kidney injury and tubulointerstitial injury in patients with ANCA-glomerulonephritis.

Background: Kidney injury molecule 1 (KIM-1) is a transmembrane glycoprotein expressed by proximal tubular cells, recognized as an early, sensitive and specific urinary biomarker for kidney injury. Blood KIM-1 was recently associated with the severity of acute and chronic kidney damage but its value in antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis with glomerulonephritis (ANCA-GN) has not been studied. Thus, we analyzed its expression at ANCA-GN diagnosis and its relationship with clinical presentation, kidney histopathology and early outcomes. Methods: We assessed KIM-1 levels and other pro-inflammatory molecules (C-reactive protein, interleukin-6, tumor necrosis factor α, monocyte chemoattractant protein-1 and pentraxin 3) at ANCA-GN diagnosis and after 6 months in patients included in the Maine-Anjou registry, which gathers data patients from four French Nephrology Centers diagnosed since January 2000. Results: Blood KIM-1 levels were assessed in 54 patients. Levels were elevated at diagnosis and decreased after induction remission therapy. KIM-1 was associated with the severity of renal injury at diagnosis and the need for kidney replacement therapy. In opposition to other pro-inflammatory molecules, KIM-1 correlated with the amount of acute tubular necrosis and interstitial fibrosis/tubular atrophy (IF/TA) on kidney biopsy, but not with interstitial infiltrate or with glomerular involvement. In multivariable analysis, elevated KIM-1 predicted initial estimated glomerular filtration rate (ß = -19, 95% CI -31, -7.6, P = .002). Conclusion: KIM-1 appears as a potential biomarker for acute kidney injury and for tubulointerstitial injury in ANCA-GN. Whether KIM-1 is only a surrogate marker or is a key immune player in ANCA-GN pathogenesis remain to be determined.

A role for ceruloplasmin in the control of human glioblastoma cell responses to radiation.

BACKGROUND: Glioblastoma (GB) is the most common and most aggressive malignant brain tumor. In understanding its resistance to conventional treatments, iron metabolism and related pathways may represent a novel avenue. As for many cancer cells, GB cell growth is dependent on iron, which is tightly involved in red-ox reactions related to radiotherapy effectiveness. From new observations indicating an impact of RX radiations on the expression of ceruloplasmin (CP), an important regulator of iron metabolism, the aim of the present work was to study the functional effects of constitutive expression of CP within GB lines in response to beam radiation depending on the oxygen status (21% O2 versus 3% O2). METHODS AND RESULTS: After analysis of radiation responses (Hoechst staining, LDH release, Caspase 3 activation) in U251-MG and U87-MG human GB cell lines, described as radiosensitive and radioresistant respectively, the expression of 9 iron partners (TFR1, DMT1, FTH1, FTL, MFRN1, MFRN2, FXN, FPN1, CP) were tested by RTqPCR and western blots at 3 and 8 days following 4 Gy irradiation. Among those, only CP was significantly downregulated, both at transcript and protein levels in the two lines, with however, a weaker effect in the U87-MG, observable at 3% O2. To investigate specific role of CP in GB radioresistance, U251-MG and U87-MG cells were modified genetically to obtain CP depleted and overexpressing cells, respectively. Manipulation of CP expression in GB lines demonstrated impact both on cell survival and on activation of DNA repair/damage machinery (γH2AX); specifically high levels of CP led to increased production of reactive oxygen species, as shown by elevated levels of superoxide anion, SOD1 synthesis and cellular Fe2 + . CONCLUSIONS: Taken together, these in vitro results indicate for the first time that CP plays a positive role in the efficiency of radiotherapy on GB cells.

Tailoring PEGylated nanoparticle surface modulates inflammatory response in vascular endothelial cells.

Polymer nanoparticles (NPs) are extensively studied as drug delivery systems for various therapeutic indications, including drug and imaging agent delivery to the brain. Despite intensive research, their toxicological profile has yet to be fully characterized. In particular, the more subtle effects of nanomaterials on inflammatory processes have scarcely been investigated. Surface properties of NPs are amongst parameters governing interactions between living cells and NPs. They could considerably influence the toxicity and inflammatory response of the cells exposed to NPs. Polymeric NPs investigated here present a core-shell structure. The core is constituted of hydrophobic poly(lactic acid) (PLA) block and the surface is composed of a shell of hydrophilic block of polyethylene glycol (PEG). The effect of PEG chain length coating on the expression of genes involved in the inflammation response was investigated in two vascular endothelial cell lines (bEnd.3 and HUVEC) by qPCR. Moreover, ROS generation following NP uptake was evaluated. PEGylated NPs induce a mild and transient activation of inflammatory cytokine and chemokine genes. However, differences in PEG chain length did not show any significant effect on cytokine and chemokine gene expression and PEGylated NPs did not trigger ROS generation. The present results could contribute significantly to a deeper understanding of nanomaterial interactions and toxicity with vascular endothelial cells, guiding scientists in material coating choices.

Efficient ferrocifen anticancer drug and Bcl-2 gene therapy using lipid nanocapsules on human melanoma xenograft in mouse.

Metastatic melanoma has been described as a highly aggressive cancer with low sensibility to chemotherapeutic agents. New types of drug, such as metal-based drugs (ferrocifens) have emerged and could represent an alternative for melanoma treatment since they show interesting anticancer potential. Furthermore, molecular analysis has evidenced the role of apoptosis in the low sensibility of melanomas and especially of the key regulator, Bcl-2. The objective of this study was to combine two strategies in the same lipid nanocapsules (LNCs): i) gene therapy to modulate anti-apoptotic proteins by the use of Bcl-2 siRNA, and ii) ferrocifens as a new type of anticancer agent. The efficient gene silencing with LNCs was verified by the specific extinction of Bcl-2 in melanoma cells. The cellular toxicity of ferrocifens (ferrociphenol (FcDiOH) or Ansa-FcDiOH) was demonstrated, showing higher efficacy than dacarbazine. Interestingly, the association of siBcl-2 LNCs with Ansa-FcDiOH demonstrated a significant effect on melanoma cell viability. Moreover, the co-encapsulation of siRNA and ferrocifens was successfully performed into LNCs for animal experiments. A reduction of tumor volume and mass was proved after siBcl-2 LNC treatment and Ansa-FcDiOH LNC treatment, individually (around 25%). Finally, the association of both components into the same LNCs increased the reduction of tumor volume to about 50% compared to the control group. In conclusion, LNCs appeared to provide a promising tool for the co-encapsulation of a metal-based drug and siRNA.

Stress Conditions Induced by Carvacrol and Cinnamaldehyde on Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a major cause of nosocomial infections. The ability of A. baumannii to display various resistance mechanisms against antibiotics has transformed it into a successful nosocomial pathogen. The limited number of antibiotics in development and the disengagement of the pharmaceutical industry have prompted the development of innovative strategies. One of these strategies is the use of essential oils, especially aromatic compounds that are potent antibacterial molecules. Among them, the combination of carvacrol and cinnamaldehyde has already demonstrated antibacterial efficacy against A. baumannii. The aim of this study was to determine the biological effects of these two compounds in A. baumannii, describing their effect on the rRNA and gene regulation under environmental stress conditions. Results demonstrated rRNA degradation by the carvacrol/cinnamaldehyde mixture, and this effect was due to carvacrol. Degradation was conserved after encapsulation of the mixture in lipid nanocapsules. Results showed an upregulation of the genes coding for heat shock proteins, such as groES, groEL, dnaK, clpB, and the catalase katE, after exposure to carvacrol/cinnamaldehyde mixture. The catalase was upregulated after carvacrol exposure wich is related to an oxidative stress. The combination of thiourea (hydroxyl radical scavenger) and carvacrol demonstrated a potent bactericidal effect. These results underline the development of defense strategies of the bacteria by synthesis of reactive oxygen species in response to environmental stress conditions, such as carvacrol.

Effects of mesenchymal stem cell therapy, in association with pharmacologically active microcarriers releasing VEGF, in an ischaemic stroke model in the rat.

Few effective therapeutic interventions are available to limit brain damage and functional deficits after ischaemic stroke. Within this context, mesenchymal stem cell (MSC) therapy carries minimal risks while remaining efficacious through the secretion of trophic, protective, neurogenic and angiogenic factors. The limited survival rate of MSCs restricts their beneficial effects. The usefulness of a three-dimensional support, such as a pharmacologically active microcarrier (PAM), on the survival of MSCs during hypoxia has been shown in vitro, especially when the PAMs were loaded with vascular endothelial growth factor (VEGF). In the present study, the effect of MSCs attached to laminin-PAMs (LM-PAMs), releasing VEGF or not, was evaluated in vivo in a model of transient stroke. The parameters assessed were infarct volume, functional recovery and endogenous cellular reactions. LM-PAMs induced the expression of neuronal markers by MSCs both in vitro and in vivo. Moreover, the prolonged release of VEGF increased angiogenesis around the site of implantation of the LM-PAMs and facilitated the migration of immature neurons towards the ischaemic tissue. Nonetheless, MSCs/LM-PAMs-VEGF failed to improve sensorimotor functions. The use of LM-PAMs to convey MSCs and to deliver growth factors could be an effective strategy to repair the brain damage caused by a stroke.

New PLGA-P188-PLGA matrix enhances TGF-ß3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells.

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest in various clinical applications because it allows cell implantation through minimally invasive surgical procedures. In case of cartilage repair, a tissue engineered construct should provide a support for the cell and allow sustained in situ delivery of bioactive factors capable of inducing cell differentiation into chondrocytes. Pharmacologically active microcarriers (PAMs), made of biodegradable poly(d,l-lactide-co-glycolide acid) (PLGA), are a unique system, which combines these properties in an adaptable and simple microdevice. However, a limitation of such scaffold is low and incomplete protein release that occurs using the hydrophobic PLGA based microspheres. To circumvent this problem, we developed a novel formulation of polymeric PAMs containing a P188 poloxamer, which protects the protein from denaturation and may positively affect chondrogenesis. This poloxamer was added as a free additive for protein complexation and as a component of the scaffold covalently linked to PLGA. This procedure allows getting a more hydrophilic scaffold but also retaining the protective polymer inside the microcarriers during their degradation. The novel PLGA-P188-PLGA PAMs presenting a fibronectin-covered surface allowed enhanced MSC survival and proliferation. When engineered with TGFß3, they allowed the sustained release of 70% of the incorporated TGF-ß3 over time. Importantly, they exerted superior chondrogenic differentiation potential compared to previous FN-PAM-PLGA-TGF-ß3, as shown by an increased expression of specific cartilage markers such as cartilage type II, aggrecan and COMP. Therefore, this microdevice represents an efficient easy-to-handle and injectable tool for cartilage repair.

Invariant NKT cells drive hepatic cytokinic microenvironment favoring efficient granuloma formation and early control of Leishmania donovani infection.

The development of inflammatory granulomas around infected Kupffer cells is necessary for hepatic parasite clearance during visceral leishmaniasis. Invariant NKT (iNKT) cells are predominant T cells in the mouse liver and can synthesize large quantities of IL-4 and IFN-γ, two cytokines involved in granuloma formation. This study analyzed the role of iNKT cells in the hepatic immune response during Leishmania donovani infection, using a murine model of wild-type (WT) and iNKT cell-deficient (Jα18⁻/⁻) C57BL/6 mice sacrificed 15, 30 or 60 days post-infection. We recorded hepatic parasite loads, cytokine expression, and analyzed granulomatous response by immunohistochemistry and hepatic immune cell infiltration by flow cytometry. Whereas WT animals rapidly controlled the infection and developed an inflammatory response associated with a massive influx of iNKT cells observed by flow cytometry, Jα18⁻/⁻ mice had significantly higher parasitic loads on all time points. This lack of control of parasite burden was associated with a delay in granuloma maturation (28.1% of large granulomas at day 60 versus 50.7% in WT). Cytokine transcriptome analysis showed that mRNA of 90/101 genes encoding chemokines, cytokines and their receptors, was underexpressed in Jα18⁻/⁻ mice. Detection of IL-4 and TNF-α by ELISA in liver extracts was also significantly lower in Jα18⁻/⁻ mice. Consistent with flow cytometry analysis, cytokinome profile in WT mice showed a bias of expression towards T cell-chemoattractant chemokines on D15, and displayed a switch towards expression of granulocytes and/or monocytes -chemoattractant chemokines on D60. In Jα18⁻/⁻ mice, the significantly lower expression of CXCL5, MIP-2 and CCL2 mRNA was correlated with a defect in myeloperoxidase positive-cell attraction observed by immunohistochemistry and with a lower granulocyte and monocyte infiltration in the liver, as shown by flow cytometry. These data indicate that iNKT cells play a role in early and sustained pro-inflammatory cytokine response warranting efficient organization of hepatic granulomas and parasite clearance.