Vertebrates achieve internal homeostasis during infection or injury by balancing the activities of proinflammatory and anti-inflammatory pathways. Endotoxin (lipopolysaccharide), produced by all gram-negative bacteria, activates macrophages to release cytokines that are potentially lethal. The central nervous system regulates systemic inflammatory responses to endotoxin through humoral mechanisms. Activation of afferent vagus nerve fibres by endotoxin or cytokines stimulates hypothalamic-pituitary-adrenal anti-inflammatory responses. However, comparatively little is known about the role of efferent vagus nerve signalling in modulating inflammation. Here, we describe a previously unrecognized, parasympathetic anti-inflammatory pathway by which the brain modulates systemic inflammatory responses to endotoxin. Acetylcholine, the principle vagal neurotransmitter, significantly attenuated the release of cytokines (tumour necrosis factor (TNF), interleukin (IL)-1beta, IL-6 and IL-18), but not the anti-inflammatory cytokine IL-10, in lipopolysaccharide-stimulated human macrophage cultures. Direct electrical stimulation of the peripheral vagus nerve in vivo during lethal endotoxaemia in rats inhibited TNF synthesis in liver, attenuated peak serum TNF amounts, and prevented the development of shock.

Numerous pathologies may involve toxic side effects of free heme and hemederived iron. Deficiency of the hemecatabolizing enzyme, heme oxygenase-1 (HO-1), in both a human patient and transgenic knockout mice leads to an abundance of circulating heme and damage to vascular endothelium. Although heme can be directly cytotoxic, the present investigations examine the possibility that hemoglobin-derived heme and iron might be indirectly toxic through the generation of oxidized forms of low-density lipoprotein (LDL). In support, hemoglobin in plasma, when oxidized to methemoglobin by oxidants such as leukocyte-derived reactive oxygen, causes oxidative modification of LDL. Heme, released from methemoglobin, catalyzes the oxidation of LDL, which in turn induces endothelial cytolysis primarily caused by lipid hydroperoxides. Exposure of endothelium to sublethal concentrations of this oxidized LDL leads to induction of both HO-1 and ferritin. Similar endothelial cytotoxicity was caused by LDL isolated from plasma of an HO-1-deficient child. Spectral analysis of the child's plasma revealed a substantial oxidation of plasma hemoglobin to methemoglobin. Iron accumulated in the HO-1-deficient child's LDL and several independent assays revealed oxidative modification of the LDL. We conclude that hemoglobin, when oxidized in plasma, can be indirectly cytotoxic through the generation of oxidized LDL by released heme and that, in response, the intracellular defense-HO-1 and ferritin-is induced. These results may be relevant to a variety of disorders-such as renal failure associated with intravascular hemolysis, hemorrhagic injury to the central nervous system, and, perhaps, atherogenesis-in which hemoglobin-derived heme may promote the formation of fatty acid hydroperoxides. IntroductionAerobic organisms are well endowed with enzymatic oxidant defense systems, which provide protection against activated oxygen species. Damage caused by reactive oxygen can be greatly amplified by "free" redox active iron. 1-2 For example, iron-rich Staphylococcus aureus is 3 orders of magnitude more susceptible to killing by hydrogen peroxide than are iron-poor staphylococci. 3 Conversely, depletion of cellular iron powerfully protects eukaryotic and prokaryotic cells against oxidant challenge. 4-5 One abundant source of potentially toxic iron is heme, and both exogenous and endogenous heme can synergistically enhance oxidantmediated cellular damage. [6][7][8][9][10] Heme, a ubiquitous iron-containing compound, is quite hydrophobic, readily enters cell membranes, and greatly increases cellular susceptibility to oxidant-mediated killing. 8 Heme also acts as a catalyst for the oxidation of low-density lipoprotein (LDL), generating products toxic to endothelium. 9,[11][12] The toxic effects of heme may be important in a number of pathologies. These include not only acute conditions such as intravascular hemolysis (which can lead to renal failure) but also more insidious processes such as atherogenesis in which intralesional deposits of iron (perhaps ...

Iron-derived reactive oxygen species are implicated in the pathogenesis of various vascular disorders including atherosclerosis, vasculitis, and reperfusion injury.

BackgroundDespite intensive research efforts, reports of cellular responses to nanomaterials are often inconsistent and even contradictory. Additionally, relationships between the responding cell type and nanomaterial properties are not well understood. Using three model cell lines representing different physiological compartments and nanomaterials of different compositions and sizes, we have systematically investigated the influence of nanomaterial properties on the degrees and pathways of cytotoxicity. In this study, we selected nanomaterials of different compositions (TiO2 and SiO2 nanoparticles, and multi-wall carbon nanotubes [MWCNTs]) with differing size (MWCNTs of different diameters < 8 nm, 20-30 nm, > 50 nm; but same length 0.5-2 μm) to analyze the effects of composition and size on toxicity to 3T3 fibroblasts, RAW 264.7 macrophages, and telomerase-immortalized (hT) bronchiolar epithelial cells.ResultsFollowing characterization of nanomaterial properties in PBS and serum containing solutions, cells were exposed to nanomaterials of differing compositions and sizes, with cytotoxicity monitored through reduction in mitochondrial activity. In addition to cytotoxicity, the cellular response to nanomaterials was characterized by quantifying generation of reactive oxygen species, lysosomal membrane destabilization and mitochondrial permeability. The effect of these responses on cellular fate - apoptosis or necrosis - was then analyzed. Nanomaterial toxicity was variable based on exposed cell type and dependent on nanomaterial composition and size. In addition, nanomaterial exposure led to cell type dependent intracellular responses resulting in unique breakdown of cellular functions for each nanomaterial: cell combination.ConclusionsNanomaterials induce cell specific responses resulting in variable toxicity and subsequent cell fate based on the type of exposed cell. Our results indicate that the composition and size of nanomaterials as well as the target cell type are critical determinants of intracellular responses, degree of cytotoxicity and potential mechanisms of toxicity.

SummaryAlthough "biocompatible" polymeric elastomers are generally nontoxic, nonimmunogenic, and chemically inert, implants made of these materials may trigger acute and chronic inflammatory responses. Early interactions between implants and inflammatory cells are probably mediated by a layer of host proteins on the material surface. To evaluate the importance of this protein layer, we studied acute inflammatory responses of mice to samples of polyester terephthalate film (PET) that were implanted intraperitoneally for short periods. Material preincubated with albumin is "passivated;' accumulating very few adherent neutrophils or macrophages, whereas uncoated or plasma-coated PET attracts large numbers ofphagocytes. Neither IgG adsorption nor surface complement activation is necessary for this acute inflammation; phagocyte accumulation on uncoated implants is normal in hypogammaglobulinemic mice and in severely hypocomplementemic mice. Rather, spontaneous adsorption of fibrinogen appears to be critical : (a) PET coated with serum or hypofibrinogenemic plasma attracts as few phagocytes as does albumin-coated material; (b) in contrast, PET preincubated with serum or hypofibrinogenemic plasma containing physiologic amounts of fibrinogen elicits "normal" phagocyte recruitment; (c) most importantly, hypofibrinogenemic mice do not mount an inflammatory response to implanted PET unless the material is coated with fibrinogen or the animals are injected with fibrinogen before implantation. Thus, spontaneous adsorption of fibrinogen appears to initiate the acute inflammatory response to an implanted polymer, suggesting an interesting nexus between two major iatrogenic effects of biomaterials : clotting and inflammation .

Oxidized low density lipoprotein (LDL), formed in vivo from presently unknown reactions, may play a role in atherogenesis. In vitro, transition metals such as iron and copper will facilitate LDL oxidation, but these metals are unlikely to exist in free form in normal body fluids. We have explored the possibility that LDL oxidation may be promoted by heme, a physiologically ubiquitous, hydrophobic, iron-containing compound. Indeed, during several-hour incubation, heme caused extensive oxidative modification of LDL; however, such modification requires only minutes in the presence of small amounts of HjO 2 or preformed lipid hydroperoxides within the LDL. Oxidative interactions between heme, LDL, and peroxides lead to degradation of the heme ring and consequent release of heme iron, which further accelerates heme degradation. Coupled (evidently iron-catalyzed) heme degradation and LDL oxidation are both effectively inhibited by hydrophobic antioxidants and iron chelators. That such hemin-induced LDL oxidation may be involved in atherogenesis is supported by the finding that LDL oxidized by hemin is extremely cytotoxic to cultured aortic endothelial cells. Overall, these investigations not only lend support to the idea that LDL oxidation by physiological substances such as heme may play a role in the process of atherogenesis but also may have broader implications, as similar oxidative reactions between heme and unsaturated fatty acids may occur consequent to hemorrhagic injury. (Arteriosclerosis and Thrombosis 1991;ll:1700-1711)

For unknown reasons, humans infected with the bacterium Bordetella pertussis are exceptionally vulnerable to secondary infections. Bordetella species elaborate a soluble, heat-stable, and highly active adenylate cyclase. This enzyme is internalized by phagocytic cells and catalyzes the unregulated formation of adenosine 3',5'-monophosphate (cyclic AMP), thereby disrupting normal cellular function. This unusual phenomenon may explain Bordetella-induced aphylaxis and may prove to be useful for investigating a variety of cyclic AMP-governed processes.

6-Phosphofructo-1-kinase, a rate-limiting enzyme of glycolysis, is activated in neoplastic cells by fructose-2,6-bisphosphate (Fru-2,6-BP), a product of four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes (PFKFB1-4). The inducible PFKFB3 isozyme is constitutively expressed by neoplastic cells and required for the high glycolytic rate and anchorage-independent growth of ras-transformed cells. We report herein the computational identification of a small-molecule inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), which suppresses glycolytic flux and is cytostatic to neoplastic cells. 3PO inhibits recombinant PFKFB3 activity, suppresses glucose uptake, and decreases the intracellular concentration of Fru-2,6-BP, lactate, ATP, NAD + , and NADH. 3PO markedly attenuates the proliferation of several human malignant hematopoietic and adenocarcinoma cell lines (IC 50 , 1.4-24 Mmol/L) and is selectively cytostatic to rastransformed human bronchial epithelial cells relative to normal human bronchial epithelial cells. The PFKFB3 enzyme is an essential molecular target of 3PO because transformed cells are rendered resistant to 3PO by ectopic expression of PFKFB3 and sensitive to 3PO by heterozygotic genomic deletion of PFKFB3. Importantly, i.p. administration of 3PO (0.07 mg/g) to tumor-bearing mice markedly reduces the intracellular concentration of Fru-2,6-BP, glucose uptake, and growth of established tumors in vivo. Taken together, these data support the clinical development of 3PO and other PFKFB3 inhibitors as chemotherapeutic agents.