Oxygen carriers affect kidney immunogenicity during ex-vivo machine perfusion

Normothermic ex-vivo machine perfusion provides a powerful tool to improve donor kidney preservation and a route for the delivery of pharmacological or gene therapeutic interventions prior to transplantation. However, perfusion at normothermic temperatures requires adequate tissue oxygenation to meet the physiological metabolic demand. For this purpose, the addition of appropriate oxygen carriers (OCs) to the perfusion solution is essential to ensure a sufficient oxygen supply and reduce the risk for tissue injury due to hypoxia. It is crucial that the selected OCs preserve the integrity and low immunogenicity of the graft. In this study, the effect of two OCs on the organ's integrity and immunogenicity was evaluated. Porcine kidneys were perfused ex-vivo for four hours using perfusion solutions supplemented with red blood cells (RBCs) as conventional OC, perfluorocarbon (PFC)-based OC, or Hemarina-M101 (M101), a lugworm hemoglobin-based OC named HEMO2life®, recently approved in Europe (i.e., CE obtained in October 2022). Perfusions with all OCs led to decreased lactate levels. Additionally, none of the OCs negatively affected renal morphology as determined by histological analyses. Remarkably, all OCs improved the perfusion solution by reducing the expression of pro-inflammatory mediators (IL-6, IL-8, TNFα) and adhesion molecules (ICAM-1) on both transcript and protein level, suggesting a beneficial effect of the OCs in maintaining the low immunogenicity of the graft. Thus, PFC-based OCs and M101 may constitute a promising alternative to RBCs during normothermic ex-vivo kidney perfusion

Functional cross talk between CXCR4 and PDGFR on glioblastoma cells is essential for migration.

Glioblastoma (GBM) is the most common and aggressive form of brain tumor, characterized by high migratory behavior and infiltration in brain parenchyma which render classic therapeutic approach ineffective. The migratory behaviour of GBM cells could be conditioned by a number of tissue- and glioma-derived cytokines and growth factors. Although the pro-migratory action of CXCL12 on GBM cells in vitro and in vivo is recognized, the molecular mechanisms involved are not clearly identified. In fact the signaling pathways involved in the pro-migratory action of CXCL12 may differ in individual glioblastoma and integrate with those resulting from abnormal expression and activation of growth factor receptors. In this study we investigated whether some of the receptor tyrosine kinases commonly expressed in GBM cells could cooperate with CXCL12/CXCR4 in their migratory behavior. Our results show a functional cross-talk between CXCR4 and PDGFR which appears to be essential for GBM chemotaxis

KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo

Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca 2+ -dependent K + channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H- pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue. © 2013 Macmillan Publishers Limited All rights reserved

CXCL12-induced glioblastoma cell migration requires intermediate conductance Ca2+-activated K+ channel activity

The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance Ca(2+)-activated K (IK(Ca)) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the IK(Ca) channel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) or IK(Ca) channel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced cell migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates IK(Ca) channels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the IK(Ca) channels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce Ca(2+) mobilization and IK(Ca) channel activation but only CXCL12 induces a long-term upregulation of the IK(Ca) channel activity. Furthermore, the Ca(2+)-chelating agent BAPTA-AM abolished the CXCL12-induced, but not the EGF-induced, glioblastoma cell chemotaxis. In addition, we demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, whereas the phosphoinositol-3 kinase (PI3K) pathway is not involved. In contrast, EGF-induced glioblastoma migration requires both ERK1/2 and PI3K activity. All together these findings suggest that the efficacy of glioblastoma invasiveness might be related to an array of nonoverlapping mechanisms activated by different chemotactic agents

Polyamine-polycarboxylate metal complexes with different biological effectiveness as nitric oxide scavengers. Clues for drug design

The synthesis of the Fe(III), Co(II), Mn(II), and Ru(III) complexes with two polyamine-polycarboxylate ligands, N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (H(3)L1) and ethylene bisglycol tetraacetic acid (H(4)L2) is reported. Potentiometric studies showed that these ligands form stable complexes in aqueous solution and no metal release occurs, thus accounting for their low toxicity in cultured RAW 264.7 macrophages. X-ray characterization of the [Co(L1)](-) complex showed that binding sites are available at the metal for NO binding. Efficiency of these compounds to bind NO was studied by UV-vis spectrophotometry. Then their NO-scavenging properties were assayed in a cell-free system under physiological conditions, using S-nitroso-N-acetyl-D,L-penicillamine (SNAP) as NO source. The L1 complexes caused the most effective reduction of free NO, [Mn(L1)](-) being the most efficient. Conversely, in NOS 11 induced RAW 264.7 macrophages, the Ru(III) and Co(II) complexes with L2 were the most effective compounds. [Ru(L2)](-) also afforded significant protection against lipopolysaccharide-induced endotoxic shock in the mouse in vivo