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BACKGROUND AND PURPOSE: Traumatic haemorrhage (TH) is the leading cause of potentially preventable deaths that occur during the prehospital phase of care. No effective pharmacological therapeutics are available for critical TH patients yet. Here, we identify terminal complement activation (TCA) as a therapeutic target in combat casualties and evaluate the efficacy of a TCA inhibitor (nomacopan) on organ damage and survival in vivo. EXPERIMENTAL APPROACH: Complement activation products and cytokines were analysed in plasma from 54 combat casualties. The correlations between activated complement pathway(s) and the clinical outcomes in trauma patients were assessed. Nomacopan was administered to rats subjected to lethal TH (blast injury and haemorrhagic shock). Effects of nomacopan on TH were determined using survival rate, organ damage, physiological parameters, and laboratory profiles. KEY RESULTS: Early TCA was associated with systemic inflammatory responses and clinical outcomes in this trauma cohort. Lethal TH in the untreated rats induced early TCA that correlated with the severity of tissue damage and mortality. The addition of nomacopan to a damage-control resuscitation (DCR) protocol significantly inhibited TCA, decreased local and systemic inflammatory responses, improved haemodynamics and metabolism, attenuated tissue and organ damage, and increased survival. CONCLUSION AND IMPLICATIONS: Previous findings of our and other groups revealed that early TCA represents a rational therapeutic target for trauma patients. Nomacopan as a pro-survival and organ-protective drug, could emerge as a promising adjunct to DCR that may significantly reduce the morbidity and mortality in severe TH patients while awaiting transport to critical care facilities.

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BACKGROUND AND OBJECTIVE: Resuscitative Endovascular Balloon Occlusion of Aorta (REBOA) has emerged as a potential life-saving maneuver for the management of non-compressible torso hemorrhage in trauma patients. Complete REBOA (cREBOA) is inherently associated with the burden of ischemia reperfusion injury (IRI) and organ dysfunction. However, the distal organ inflammation and its association with organ injury have been little investigated. This study was conducted to assess these adverse effects of cREBOA following massive hemorrhage in swine. METHODS: Spontaneously breathing and consciously sedated Sinclair pigs were subjected to exponential hemorrhage of 65% total blood volume over 60 minutes. Animals were randomized into 3 groups (n = 7): (1) Positive control (PC) received immediate transfusion of shed blood after hemorrhage, (2) 30min-cREBOA (A30) received Zone 1 cREBOA for 30 minutes, and (3) 60min-cREBOA (A60) given Zone 1 cREBOA for 60 minutes. The A30 and A60 groups were followed by resuscitation with shed blood post-cREBOA and observed for 4h. Metabolic and hemodynamic effects, coagulation parameters, inflammatory and end organ consequences were monitored and assessed. RESULTS: Compared with 30min-cREBOA, 60min-cREBOA resulted in (1) increased IL-6, TNF-α, and IL-1ß in distal organs (kidney, jejunum, and liver) (p < 0.05) and decreased reduced glutathione in kidney and liver (p < 0.05), (2) leukopenia, neutropenia, and coagulopathy (p < 0.05), (3) blood pressure decline (p < 0.05), (4) metabolic acidosis and hyperkalemia (p < 0.05), and (5) histological injury of kidney and jejunum (p < 0.05) as well as higher levels of creatinine, AST, and ALT (p < 0.05). CONCLUSION: 30min-cREBOA seems to be a feasible and effective adjunct in supporting central perfusion during severe hemorrhage. However, prolonged cREBOA (60min) adverse effects such as distal organ inflammation and injury must be taken into serious consideration.

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OBJECTIVE: Normal iron handling appears to be disrupted in critically ill patients leading to hypoferremia that may contribute to systemic inflammation. Ceruloplasmin (Cp), an acute phase reactant protein that can convert ferrous iron to its less reactive ferric form facilitating binding to ferritin, has ferroxidase activity that is important to iron handling. Genetic absence of Cp decreases iron export resulting in iron accumulation in many organs. The objective of this study was to characterize iron metabolism and Cp activity in burn and non-burn trauma patients to determine if changes in Cp activity are a potential contributor to the observed hypoferremia. MATERIAL AND METHODS: Under Brooke Army Medical Center Institutional Review Board approved protocols, serum or plasma was collected from burn and non-burn trauma patients on admission to the ICU and at times up to 14 days and measured for indices of iron status, Cp protein and oxidase activity and cytokines. RESULTS: Burn patients showed evidence of anemia and normal or elevated ferritin levels. Plasma Cp oxidase activity in burn and trauma patients were markedly lower than controls on admission and increased to control levels by day 3, particularly in burn patients. Plasma cytokines were elevated throughout the 14 days study along with evidence of an oxidative stress. No significant differences in soluble transferrin receptor were noted among groups on admission, but levels in burn patients were lower than controls for the first 5 days after injury. CONCLUSION: This study further established the hypoferremia and inflammation associated with burns and trauma. To our knowledge, this is the first study to show an early decrease in Cp oxidase activity in burn and non-burn trauma patients. The results support the hypothesis that transient loss of Cp activity contributes to hypoferremia and inflammation. Further studies are warranted to determine if decreased Cp activity increases the risk of iron-induced injury following therapeutic interventions such as transfusions with blood that has undergone prolonged storage in trauma resuscitation.

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Single-walled carbon nanotubes (SWCNT) show unique properties find applications in micro devices; electronics to biological systems specially drug delivery and gene therapy. However the manufacture and extensive use of nanotubes raises concern about its safe use and human health. Very few studies have been carried out on toxicity of carbon nanotubes in experimental animals and humans, thus resulted in limiting their use. The extensive toxicological studies using in vitro and in vivo models are necessary and are required to establish safe manufacturing guidelines and also the use of SWCNT. These studies also help the chemists to prepare derivative of SWCNT with less or no toxicity. The present study was undertaken to determine the toxicity exhibited by SWCNT in rat lung epithelial cells as a model system. Lung epithelial cells (LE cells) were cultured with or without SWCNT and reactive oxygen species (ROS) produced were measured by change in fluorescence using dichloro fluorescein (DCF). The results show increased ROS on exposure to SWCNT in a dose and time dependent manner. The decrease in glutathione content suggested the depletion and loss of protective mechanism against ROS in SWCNT treated cells. Use of rotenone, the inhibitor of mitochondrial function have no effect on ROS levels suggested that mitochondria is not involved in SWCNT induced ROS production. Studies carried out on the effect of SWCNT on superoxide dismutase (SOD-1 and SOD-2) levels in LE cells, indicates that these enzyme levels decreased by 24 hours. The increased ROS induced by SWCNT on LE cells decreased by treating the cells with 1 mM of glutathione, N-Acetyl Cysteine, and Vitamin C. These results further prove that SWCNT induces oxidative stress in LE cells and shows loss of antioxidants.

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Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants. Toxicity exhibited by cigarette smoke may be due to combined action of these compounds inducing many cellular processes mediated through reactive oxygen species (ROS). Major player probably nicotine as it is present in tobacco, in higher concentrations. The compounds that induce intracellular oxidative stress recognized as the important agents involved in the damage of biological molecules. Experiments using animal and cell culture model systems suggested that moderately higher concentrations of some forms of ROS like NO and H(2)O(2) can act as signal transducing agents. Nuclear transcription factor kappaB (NF-kappaB) an inducible transcription factor detected in neurons found to be involved in many biological processes such as inflammation, innate immunity, development, apoptosis, and antiapoptosis. Our present study demonstrates that nicotine induces ROS levels in a dose dependent manner in rat mesencephalic cells. Electro mobility shift analysis showed that nicotine activates inducible NF-kappaB by binding to consensus sequence of DNA. Nicotine added to cell culture stimulates the degradation of IkappaB-alpha subunit in 2 h. Further activation of c-Jun terminal kinase indicates that nicotine induces oxidative stress leading to activation of stress dependent NF-kappaB pathway in mesencephalic cells.

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Carbon nanotubes are now becoming an important material for use in day to day life because of their unique physical properties. The toxicological impact of these materials has not yet been studied in detail, thereby limiting their use. In the present study, the toxicity of single-walled carbon nanotubes (SWCNT) was assessed in human keratinocyte cells. The results show increased oxidative stress and inhibition of cell proliferation in response to treatment of keratinocytes with SWCNT particles. In addition, the signaling mechanism in keratinocytes upon exposure to SWCNT particles was investigated. Results from the study suggest that SWCNT particles activate NF-kappaB in a dose-dependent manner in human keratinocytes. Further, the mechanism of activation of NF-kappaB was due to the activation of stress-related kinases by SWCNT particles in keratinocytes. In conclusion, these studies show the mechanism of toxicity induced by SWCNT particles.

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Degeneration of dopaminergic neurons is one of the major features of Parkinson's disease. Many redox-active metals such as iron and manganese have been implicated in neuronal degeneration characterized by symptoms resembling Parkinson's disease. Even though, arsenic, which is another redox-active metal, has been shown to affect the central monoaminergic systems, but its potential in causing dopaminergic cell degeneration has not been fully known. Hence, the present study was designed to investigate arsenic signaling especially that is mediated by reactive oxygen species and its effect on early transcription factors in dopamine producing mesencephalic cell line 1RB3AN27. These mesencephalic cells were treated with low concentrations of sodium arsenite (0.1, 0.5, 1, 5, and 10 microM) and incubated for different periods of time (0-4 h). Arsenite was cytotoxic at 5 and 10 microM concentrations only after 72-h incubation period. Arsenite, in a dose-dependent manner, induced generation of reactive oxygen species (ROS) and activation of early transcription factors such as nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) as shown by electro mobility shift assay. Incubation of antioxidants, either N-acetyl-L-cysteine (50 microM) or alpha-tocopherol (50 microM) with 1 microM arsenite, suppressed ROS generation. Arsenite at 1 microM concentration was sufficient for maximal activation of NF-kappaB and AP-1 activation. Time kinetics studies showed maximal activation of NF-kappaB by 1 microM concentration of arsenite was seen at 120 min and correlated with complete degradation of Ikappa Balpha at 60 min. Similarly, maximal activation of AP-1 by 1 microM concentration of arsenite occurred at 120 min. N-acetyl-L-cysteine at 50 microM concentration inhibited arsenite-induced NF-kappa B and AP-1. In addition, arsenite was shown to induce phosphorylation of extracellular signal regulated kinase (ERK) 1/2 at concentrations of 1 microM and above. These results suggest that arsenite, at low and subcytoxic concentrations, appears to induce oxidative stress leading to activation of early transcription factors whereas addition of antioxidant inhibited the activation of these factors.

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Low-level exposure of manganese (Mn2+) induces adverse neurological effects. We have previously shown that low-level Mn2+ exposure induces cell death in dopaminergic neurons, in which the redox-sensitive transcription factor nuclear factor kappa-B (NF-kappaB) and associated mitogen-activated protein kinase (MAPK) were stimulated. In continuation of this work, we evaluated the activator protein-1 (AP-1). DNA binding activity and N-terminal c-Jun kinase (JNK) activation due to low-level exposure to manganese. Rat catecholaminergic-rich pheochromocytoma (PC12) cells were exposed to low concentrations of Mn2+ (0-10 microM) for 120 min and examined changes in AP-1 DNA binding activity by electrophoretic mobility shift assay (EMSA). Mn(2+)-exposed cells produced a eightfold increase in AP-1 DNA binding activity compared to untreated controls. This significant increase was seen within 60 min after Mn2+ exposure. In addition, Mn2+ activated N-terminal c-Jun kinase, suggesting that the JNK pathway is involved in Mn(2+)-induced AP-1 activation. Thus, JNK activation may in turn stimulate AP-1 via c-Jun phosphorylation. Since the transcription factors NF-kappaB and AP-1 have been shown to play an essential role in oxidative stress related toxicity, we suggest that induction of the signaling factor AP-1 during Mn2+ exposure in the present study may have a significant cytotoxic effect on neuronal function.

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