cytochalasin-d and Necrosis

Salmonella is a common foodborne and zoonotic pathogen. Only a few serovars carry a virulence plasmid (pSV), which enhances the pathogenicity of the host. Here, we investigated the pathogenicity roles of the pSVs among wild-type, plasmid-less, and complemented S. Typhimurium, S. Enteritidis S. Choleraesuis in invasion, phagocytosis, and intracellular bacterial survival in human THP-1 cells and cell death patterns by flow cytometry and difference in cell death patterns between pig and human S. Choleraesuis isolates with large pSCVs. Virulence plasmid (pSTV) led to slightly increasing cellular apoptosis for S. Typhimurium; virulence plasmid (pSEV) enhanced apoptosis and necrosis significantly for S. Enteritidis; and pSCV reduced apoptosis significantly for S. Choleraesuis. After complementation, pSTV increased the intracellular survival of pSCV-less Choleraesuis and the cytotoxicity against human THP-1 cells. Using the Cytochalasin D to differentiate the invasion of S. Choleraaesuis and phagocytosis of THP-1 cells determined that pSCV were responsible for invasion and phagocytosis at 0 h and inhibited intracellular replication in THP-1 cells, and pSTV were responsible for invasion and increased intracellular survival for S. Choleraesuis in THP-1 cells. The human isolates with large pSCV induced more cellular apoptosis and necrosis than the pig isolates. In conclusion, human S. Choleraesuis isolates carrying large pSCVs were more adapted to human THP-1 cells for more cell death than pig isolates with large pSCV. The role of pSVs in invasion, phagocytosis, intracellular survival and apoptosis differed among hosted serovars.

Topics: Animals; Apoptosis; Cell Death; Cytochalasin D; DNA Replication; Genes, Bacterial; Host-Pathogen Interactions; Humans; Mice; Microbial Viability; Necrosis; Plasmids; RAW 264.7 Cells; Salmonella enterica; Salmonella enteritidis; Salmonella Infections, Animal; Salmonella typhimurium; Serogroup; Swine; THP-1 Cells; Virulence; Virulence Factors

In crystallopathies, crystals or crystalline particles of environmental and metabolic origin deposit within tissues, induce inflammation, injury and cell death and eventually lead to organ-failure. The NLRP3-inflammasome is involved in mediating crystalline particles-induced inflammation, but pathways leading to cell death are still unknown. Here, we have used broad range of intrinsic and extrinsic crystal- or crystalline particle-sizes and shapes, e.g. calcium phosphate, silica, titanium dioxide, cholesterol, calcium oxalate, and monosodium urate. As kidney is commonly affected by crystallopathies, we used human and murine renal tubular cells as a model system. We showed that all of the analysed crystalline particles induce caspase-independent cell death. Deficiency of MLKL, siRNA knockdown of RIPK3, or inhibitors of necroptosis signaling e.g. RIPK-1 inhibitor necrostatin-1s, RIPK3 inhibitor dabrafenib, and MLKL inhibitor necrosulfonamide, partially protected tubular cells from crystalline particles cytotoxicity. Furthermore, we identify phagocytosis of crystalline particles as an upstream event in their cytotoxicity since a phagocytosis inhibitor, cytochalasin D, prevented their cytotoxicity. Taken together, our data confirmed the involvement of necroptosis as one of the pathways leading to cell death in crystallopathies. Our data identified RIPK-1, RIPK3, and MLKL as molecular targets to limit tissue injury and organ failure in crystallopathies.

Topics: Animals; Apoptosis; Calcium Oxalate; Calcium Phosphates; Cell Line; Cholesterol; Crystallization; Cytochalasin D; Epithelial Cells; Gene Expression Regulation; Humans; Imidazoles; Indoles; Inflammasomes; Kidney Tubules; Mice; Necrosis; Oximes; Particle Size; Particulate Matter; Phagocytosis; Primary Cell Culture; Protein Kinases; Receptor-Interacting Protein Serine-Threonine Kinases; Signal Transduction; Silicon Dioxide; Titanium; Uric Acid

Graphene oxide (GO) has been a focus of research in the fields of electronics, energy, and biomedicine, including drug delivery. Thus, single- and multi-layered GO (SLGO and MLGO) have been produced and investigated. However, little information on their toxicity and biocompatibility is available. In the present study, we performed a comprehensive study of the size- and dose-dependent toxicity of GOs in the presence or absence of Pluronic F-127 on THP-1 cells by examining their viability, membrane integrity, levels of cytokine and ROS production, phagocytosis, and cytometric apoptosis. Moreover, as an extended study, a toxicity evaluation in the acute and chronic phases was performed in mice via intravenous injection of the materials. GOs exhibited dose- and size-dependent toxicity. Interestingly, SLGO induced ROS production to a lesser extent than MLGO. Cytometric analysis indicated that SLGO induced necrosis and apoptosis to a lesser degree than MLGO. In addition, cell damage and IL-1β production were influenced by phagocytosis. A histological animal study revealed that GOs of various sizes induced acute and chronic damage to the lung and kidney in the presence or absence of Pluronic F-127. These results will facilitate studies of GO prior to its biomedical application.

Topics: Animals; Apoptosis; Chemokine CCL2; Cytochalasin D; Dose-Response Relationship, Drug; Graphite; Humans; Interleukin-1beta; Kidney; L-Lactate Dehydrogenase; Lung; Mice; Necrosis; Oxides; Phagocytosis; Poloxamer; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; THP-1 Cells; Transforming Growth Factor beta

Essential cytoskeletal functions of macrophages are migration, phagocytosis of foreign materials, and intracellular transport and digestion The influence of fine and ultrafine test particles (UFP), such as TiO(2), elemental carbon, commercial carbon black, diesel exhaust particulate matter, and urban dust (UrbD), on cytoskeleton-related functions of macrophages, such as phagocytosis, phagosome transport mechanisms, and mechanical cytoskeletal integrity, were studied by flow cytometry and by cytomagnetometry. Additionally, necrosis and apoptosis caused by the test particles was detected. The diameter of the test particles ranged from 12 to 220 nm and the Brunauer-Emmet-Teller specific surface area ranged from 6 to 600 m(2)/g. Primary alveolar macrophages from beagle dogs (BD-AM), obtained by bronchoalveolar lavage, were used as well as macrophages originating from the cell line J774A.1. For cytomagnetometry studies, spherical 1.8-microm ferromagnetic particles served as probes for cytoskeletal functions and were incubated together with the macrophages 24 h prior to UFP exposure. Macrophages were exposed in vitro with 10-320 microg UFP/ml/10(6) cells up to 24 h. In all experiments, J774A.1 macrophages were more sensitive than BD-AM to UFP exposure. Cytoskeletal dysfunctions evaluated by cytomagnetometry were an impaired phagosome transport and an increased cytoskeletal stiffness and occurred at concentrations of 100 microg UFP/ml/10(6) cells and above, in both BD-AM and J774A.1. Only fine TiO(2) did not show any effect. Urban dust (standard reference material 1649a) and diesel exhaust particles (DEP, standard reference material 1650) caused comparable cytoskeletal dysfunctions to elemental carbon with high specific surface area. Cytoskeletal dysfunctions induced by DEP or UrbD could be reduced after washing the particles by dichloromethane. UFP caused an impaired phagocytosis of 1-microm diameter fluorescent latex beads, inhibited cell proliferation, and decreased cell viability. All recorded cytotoxic parameters showed only weak correlations with the specific surface area or the total number of UFP, which can result from the different types of particles and different surface compositions. UFP cause cytoskeletal toxicity in vitro in macrophages, which can cause cellular dysfunctions, such as impaired proliferation, impaired phagocytic activity, and retarded intracellular transport processes as well as increased cell stiffness and can result in impaired defe

Topics: Air Pollutants; Animals; Apoptosis; Carbon; Cell Division; Colchicine; Cytochalasin D; Cytoskeleton; Dogs; Flow Cytometry; Macrophages, Alveolar; Mice; Microspheres; Necrosis; Nocodazole; Particle Size; Phagocytosis; Surface Properties