Disulfiram Inhibits Opsonin-Independent Phagocytosis and Migration of Human Long-Lived In Vitro Cultured Phagocytes from Multiple Inflammatory Diseases.

Disulfiram (DSF), an anti-alcoholism medicine, exerts treatment effects in patients suffering from persistent Borreliosis and also exhibits anti-cancer effects through its copper chelating derivatives and induction of oxidative stress in mitochondria. Since chronic/persistent borreliosis is characterized by increased amounts of pro-inflammatory macrophages, this study investigated opsonin-independent phagocytosis, migration, and surface marker expression of in vivo activated and in vitro cultured human monocyte-derived phagocytes (macrophages and dendritic cells) with and without DSF treatment. Phagocytosis of non-opsonized Dynabeads® M-450 and migration of macrophages and dendritic cells were monitored using live cell analyzer Juli™ Br for 24 h, imaging every 3.5 min. To simultaneously monitor phagocyte function, results were analyzed by a newly developed software based on the differential phase contrast images of cells before and after ingestion of Dynabeads. DSF decreased the phagocytic capacities exhibited by in vitro enriched and long-lived phagocytes. Although no chemotactic gradient was applied to the test system, vigorous spontaneous migration was observed. We therefore set up an algorithm to monitor and quantify both phagocytosis and migration simultaneously. DSF not only reduced phagocytosis in a majority of these long-lived phagocytes but also impaired their migration. Despite these selective effects by DSF, we found that DSF reduced the expression densities of surface antigens CD45 and CD14 in all of our long-lived phagocytes. In cells with a high metabolic activity and high mitochondrial contents, DSF led to cell death corresponding to mitochondrial oxidative stress, whereas metabolically inactive phagocytes survived our DSF treatment protocol. In conclusion, DSF affects the viability of metabolically active phagocytes by inducing mitochondrial stress and secondly attenuates phagocytosis and migration in some long-lived phagocytes.

Old and New Biomarkers for Infection, Inflammation, and Autoimmunity in Treatment-Resistant Affective and Schizophrenic Spectrum Disorders.

Affective (AF) and Schizophrenic (SZ) Spectrum disorders manifest with risk factors, involving inflammatory processes linked to infections and autoimmunity. This study searched for novel biomarkers in cerebrospinal fluid (CSF) and peripheral blood. A total of 29 AF and 39 SZ patients with treatment-resistant disease were included. In CSF, the chemokine IL-8 was significantly elevated in AF and SZ patients. IL-8 promotes chemotaxis by neutrophils and may originate from different tissues. S100B, a glia-derived brain damage marker, was higher in CSF from AF than SZ patients. Among the plasma-derived biomarkers, ferritin was elevated in AF and SZ. Soluble CD25, indicating Treg dysfunction, was higher in SZ than in AF patients. Interferon-γ, implying virus-specific immune activation, was positive in selective AF patients, only. Both groups showed elevated expression of immunosuppressive CD33 on monocytes, but higher amounts of CD123+ plasmacytoid dendritic cells were restricted to SZ. In conclusion, chemotactic IL-8 indicates neuronal stress and inflammation in the CSF of both groups. Novel plasma-derived biomarkers such as sCD25 and monocytic CD33 distinguish SZ from AF with an autoimmune phenotype.

Bioassay for Endothelial Damage Mediators Retrieved by Hemoadsorption.

Hemoadsorption devices are used to treat septic shock by adsorbing inflammatory cytokines and as yet incompletely defined danger and pathogen associated molecular patterns. In an ideal case, hemoadsorption results in immediate recovery of microvascular endothelial cells' (mEC) function and rapid recovery from catecholamine-dependency and septic shock. We here tested a single device, which consists of polystyrene-divinylbenzene core particles of 450 µm diameter with a high affinity for hydrophobic compounds. The current study aimed at the proof of concept that endothelial-specific damage mediators are adsorbed and can be recovered from hemoadsorption devices. Because of excellent clinical experience, we tested protein fractions released from a hemoadsorber in a novel endothelial bioassay. Video-based, long-term imaging of mEC proliferation and cell death were evaluated and combined with apoptosis and ATP measurements. Out of a total of 39 fractions recovered from column fractionation, we identified 3 fractions that caused i) inhibition of mEC proliferation, ii) increased cell death and iii) induction of apoptosis in mEC. When adding these 3 fractions to mEC, their ATP contents were reduced. These fractions contained proteins of approximately 15 kDa, and high amounts of nucleic acid, which was at least in part oxidized. The efficacy for endothelial cell damage prevention by hemoadsorption can be addressed by a novel endothelial bioassay and long-term video observation procedures. Protein fractionation of the hemoadsorption devices used is feasible to study and define endothelial damage ligands on a molecular level. The results suggest a significant effect by circulating nucleic acids - bound to an as yet undefined protein, which may constitute a major danger-associated molecular pattern (DAMP) in the exacerbation of inflammation when patients experience septic shock. Hemoadsorption devices may thus limit endothelial damage, through the binding of nucleic acid-bearing aggregates and thus contribute to improved endothelial barrier function.

Vacquinol-1 inducible cell death in glioblastoma multiforme is counter regulated by TRPM7 activity induced by exogenous ATP.

Glioblastomas (GBM) are the most malignant brain tumors in humans and have a very poor prognosis. New therapeutic options are urgently needed. A novel drug, Vacquinol-1 (Vac), a quinolone derivative, displays promising properties by inducing rapid cell death in GBM but not in non-transformed tissues. Features of this type of cell death are compatible with a process termed methuosis. Here we tested Vac on a highly malignant glioma cell line observed by long-term video microscopy. Human dental-pulp stem cells (DPSCs) served as controls. A major finding was that an exogenous ATP concentration of as little as 1 µM counter regulated the Vac-induced cell death. Studies using carvacrol, an inhibitor of transient receptor potential cation channel, subfamily M, member 7 (TRPM7), demonstrated that the ATP-inducible inhibitory effect is likely to be via TRPM7. Exogenous ATP is of relevance in GBM with large necrotic areas. Our results support the use of GBM cultures with different grades of malignancy to address their sensitivity to methuosis. The video-microscopy approach presented here allows decoding of signaling pathways as well as mechanisms of chemotherapeutic resistance by long-term observation. Before implementing Vac as a novel therapeutic drug in GBM, cells from each individual patient need to be assessed for their ATP sensitivity. In summary, the current investigation supports the concept of methuosis, described as non-apoptotic cell death and a promising approach for GBM treatment. Tissue-resident ATP/necrosis may interfere with this cell-death pathway but can be overcome by a natural compound, carvacrol that even penetrates the blood-brain barrier.