SMART - Recognising the value of existing practice and introducing recent developments: leaving no stone unturned in the assessment and treatment of the PDOC patient.

Over the last 25 years there have been a number of papers highlighting the issues of high rates of misdiagnosis in prolonged disorders of consciousness (PDOC) (Andrews, K., Murphy, L., Munday, R., & Littlewood, C. (1996). Misdiagnosis of the vegetative state: Retrospective study in a rehabilitation unit. BMJ, 313(7048), 13-16; Childs, N. L., Mercer, W. N., & Childs, H. W. (1993). Accuracy of diagnosis of persistent vegetative state. Neurology, 43(8), 1465-1467). Surprisingly, these rates still remain at the same level despite defined criteria for diagnosis (Schnakers, C., Vanhaudenhuyse, A., Giacino, J., Ventura, M., Boly, M., Majerus, S.,…Laureys, S. (2009). Diagnostic accuracy of the vegetative and minimally conscious state: Clinical consensus versus standardized neurobehavioral assessment. BMC Neurology, 9(35), 1-5; Van Erp, W., Larvrijsen, J., Vos, P., Bor, H., Laureys, S., & Koopmans, R. (2015). The vegetative state: Prevalence, misdiagnosis and treatment limitations. JAMDA, 85, e9-85.e14. doi: 10.1016/j.jamda.2014.10.014 ). This indicates the continued need for careful standardised assessment by skilled assessors to identify all potential meaningful responses and to establish a correct and incontrovertible diagnosis. The Sensory Modality Assessment and Rehabilitation Technique (SMART) is one of three assessments identified for the assessment of PDOC in the Royal College of Physician guidelines (Royal College of Physicians, 2013). The RCP guidelines and recent publications have highlighted and substantiated the value of some of the existing practices and unique features of the SMART. In recognition of the need to keep SMART current, SMART Version 3 is being developed and will be launched shortly. The interim SMART developments will be introduced in this paper and applied to practice through the illustration of a case study. Evidence suggests that SMART is a current and invaluable tool for the clinical and medico-legal assessment and treatment of the PDOC patient.

Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies.

DNA delivery of tumor antigens can activate specific immune attack on cancer cells. However, antigens may be weak, and immune capacity can be compromised. Fusion of genes encoding activating sequences to the tumor antigen sequence facilitates promotion and manipulation of effector pathways. Idiotypic determinants of B-cell tumors, encoded by the variable region genes, are clone-specific tumor antigens. When assembled as single-chain Fv (scFv) alone in a DNA vaccine, immunogenicity is low. Previously, we found that fusion of a sequence from tetanus toxin (fragment C; FrC) promoted anti-idiotypic protection against lymphoma and myeloma. We have now investigated an alternative fusion gene derived from a plant virus, potato virus X coat protein, a primary antigen in humans. When fused to scFv, the self-aggregating protein generates protection against lymphoma and myeloma. In contrast to scFv-FrC, protection against lymphoma is mediated by CD4+ T cells, as is protection against myeloma. Plant viral proteins offer new opportunities to activate immunity against linked T-cell epitopes to attack cancer.

Short-term toxicity studies of loline alkaloids in mice.

Epichloë endophytes have been used successfully in pastoral systems to reduce the impact of insect pests through the expression of secondary metabolites. The use of endophytes could be extended to other plant species, such as cereal crops, where the production of bioactive secondary metabolites would reduce the reliance on pesticides for insect control. The success of this approach is dependent on the selection of an appropriate secondary metabolite target which must not only be effective against insect pests but also be safe for grazing and monogastric animals. The loline alkaloids have been identified as possible target metabolites as they are associated with potent effects on insects and low toxicity to grazing animals. The purpose of the current study was to generate toxicological data on the loline alkaloids in a monogastric system using mice. Male and female mice were fed 415 mg/kg/day total lolines for a 3-week period. The loline treatment caused no statistically significant effect on gross pathology, histology, haematology, blood chemistry, heart rate, blood pressure or motor coordination. Reduced weight gain and food consumption were noted in the loline groups during the initial stages of the experiment. This experiment raises no food safety concerns for the loline alkaloids.

Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species.

To determine the importance of different antioxidative enzymes for the defense status of insulin-producing cells, the effects of stable overexpression of glutathione peroxidase (Gpx), catalase (Cat), or Cu/Zn superoxide dismutase (SOD) in insulin-producing RINm5F cells on the cytotoxicity of hydrogen peroxide (H2O2), hypoxanthine/xanthine oxidase (H/XO), and menadione have been investigated. Single overexpression of Cat or Gpx provided less protection than the combined expression of Cat plus SOD or Cat plus Gpx, while single overexpression of SOD either had no effect on the toxicity of the test compounds or increased it. RINm5F cells were also susceptible to butylalloxan, a lipophilic alloxan derivative that is selectively toxic to pancreatic beta-cells. Overexpression of enzymes, both alone and in combination, did not protect against butylalloxan-induced toxicity while SOD overexpression increased it, as evident from a half maximally effective concentration (EC50) value. The addition of Cat to the culture medium completely prevented the toxic effects of H2O2 and H/XO but had no significant effect on the toxicity of menadione or butylalloxan. Extracellular SOD had no effect on the toxicity of any of the test compounds. The results of this study show the importance of a combination of antioxidant enzymes in protecting against the toxicity of reactive oxygen species. Thus, overexpression of Cat and Gpx, alone or in combination with SOD, by use of molecular biology techniques can protect insulin-producing cells against oxidative damage. This may represent a strategy to protect pancreatic beta-cells against destruction during the development of autoimmune diabetes and emphasizes the importance of optimal antioxidative enzyme equipment for protection against free radical-mediated diseases.

Autoxidation of naphthohydroquinones: effects of metals, chelating agents, and superoxide dismutase.

At neutral pH, 1,4-naphthohydroquinone and 2-methyl-1,4-naphthohydroquinone readily autoxidize to the corresponding quinones. In an unpurified phosphate buffer, the autoxidation of both substances proceeded in a linear fashion after a brief lag phase. Addition of a chelating agent or purification of the buffer decreased the duration of the lag phase of 1,4-naphthohydroquinone autoxidation, but had no effect on the linear rate. In the case of 2-methyl-l,4-naphthohydroquinone, such treatment eliminated the lag phase and greatly increased the linear rate of oxidation. The lag phases and oxidation rates seen in unpurified buffers could be replicated by addition of submicromolar amounts of copper to purified buffer. The effects of low levels of copper were qualitatively similar to those of superoxide dismutase, and it is suggested that the effects of this metal on naphthohydroquinone autoxidation reflects its ability to act as a superoxide dismutase. The relative rates of autoxidation of naphthohydroquinones are important because they may determine the balance between activation and detoxication of naphthoquinones within cells. When measuring such rates, or assessing rates of redox cycling of naphthoquinones, it is important to employ a chelating agent or use highly purified buffers and reagents. Failure to do so may lead to erroneous conclusions concerning the reactivity of naphthohydroquinones and the ability of naphthoquinones to generate "active oxygen" species.

Inhibition of 2,3-dimethyl-1,4-naphthohydroquinone auto-oxidation by copper and by superoxide dismutase.

2,3-Dimethyl-1,4-naphthohydroquinone undergoes auto-oxidation to the corresponding quinone at pH 7.4, with stoichiometric consumption of oxygen and formation of hydrogen peroxide. In an unpurified buffer, the rate of oxidation was low, but it increased nearly 9-fold when trace metals were removed from the buffer by treatment with Chelex resin. A similar increase in rate was achieved by addition of DTPA or bathophenanthroline sulfonate to unpurified buffer, whereas EDTA and desferal were less effective. Addition of copper to purified buffer led to inhibition of oxidation, with a 50% decrease in rate being observed at a metal concentration of 7.1 nM, and it is likely that the low auto-oxidation rate recorded in unpurified buffer was due to copper contamination of the latter. The auto-oxidation of 2,3-dimethyl-1,4-naphthohydroquinone was exceptionally sensitive to inhibition by superoxide dismutase, with a concentration of only 4.5 ng/ml being sufficient for a 50% decrease in rate, and the inhibitory effect of copper may be due to the ability of this metal to catalyse the dismutation of superoxide. Previous studies have shown that the rates of auto-oxidation of 1,4-naphthohydroquinone and 2-methyl-1,4-naphthohydroquinone are influenced by copper contamination of buffer and the present study shows that this is also true for a di-substituted naphthohydroquinone. For accurate assessment of rates of naphthohydroquinone auto-oxidation, it is important that purified buffers or appropriate chelating agents, are employed.

Toxicity of thiols and disulphides: involvement of free-radical species.

Sulphur is essential to life, and thiols and disulphides play essential roles in cellular biochemistry. Such compounds are also widely distributed in the food of man and his domestic animals, and they are extensively used in industry. However, many thiols and disulphides have been shown to be toxic. Aliphatic, aromatic, and heterocyclic compounds of this type are haemolytic agents in animals while aminothiols have been shown to induce many cytotoxic effects in vitro and the epidithiodioxopiperazine mycotoxin, sporidesmin, is a potent hepatotoxic agent. Structure-activity relationships among these compounds and factors which modulate their harmful effects are consistent with a toxic mechanism involving redox cycling between the thiol and the corresponding disulphide. Thiyl radicals and "active oxygen" species are formed in this process, and it is suggested that these substances are responsible for initiating the tissue damage provoked by thiols and disulphides.

Effect of superoxide dismutase, catalase, chelating agents, and free radical scavengers on the toxicity of alloxan to isolated pancreatic islets in vitro.

The effect of superoxide dismutase, catalase, metal-chelating agents and hydroxyl radical scavengers on the toxicity of alloxan to isolated ob/ob mouse pancreatic islets in vitro has been compared with the reported ability of such substances to protect against alloxan diabetes in vivo. Superoxide dismutase and catalase protected beta-cells of isolated pancreatic islets against alloxan cytotoxicity, as did the hydroxyl radical scavengers dimethyl sulfoxide (DMSO) and butanol. However, 1,3-dimethylurea and thiourea, that are recognised as effective hydroxyl radical scavengers and that protect animals against the diabetogenic effects of alloxan, were without effect. Similarly, desferrioxamine, that inhibits hydroxyl radical formation from alloxan in chemically defined systems, did not protect against alloxan toxicity. Diethylenetriamine pentaacetic acid, which does not inhibit hydroxyl radical formation from alloxan, also gave no significant protection. The results indicate a role for superoxide radical and hydrogen peroxide in the mechanism of toxicity of alloxan but do not support the involvement of the hydroxyl radical in this process. Alternative explanations must be sought for the ability of hydroxyl radical scavengers and metal-chelating agents to protect against alloxan toxicity in vivo.

Toxicity of 2,3-dialkyl-1,4-naphthoquinones in rats: comparison with cytotoxicity in vitro.

The short-term toxicities of 2-methyl-1,4-naphthoquinone and a series of 2,3-dialkyl-1,4-naphthoquinones have been determined in rats and compared with their ability to cause oxidative damage to erythrocytes in vitro. In accord with previous results, 2-methyl-1,4-naphthoquinone caused marked oxidative damage to erythrocytes in vitro and haemolytic anaemia in rats. The dialkylnaphthoquinones were also haemolytic agents in vivo, with 2,3-dimethyl-1,4-naphthoquinone being particularly active. Unlike the monoalkyl derivative, however, these substances caused little or no damage to red cells in vitro. The in vivo toxicity of dialkylnaphthoquinones cannot, therefore, be predicted on the basis of in vitro cytotoxicity tests.

Comparative toxicity of alkyl-1,4-naphthoquinones in rats: relationship to free radical production in vitro.

2-Methyl-1,4-naphthoquinone causes haemolysis in vivo. This toxic effect is believed to result from oxidative damage to erythrocytes by "active oxygen" species formed via one-electron reduction of the naphthoquinone by oxyhaemoglobin. In the present investigation, seven 2-alkyl-1,4-naphtoquinones have been studied with regard to their haemolytic activity in rats, their ability to cause oxidative damage in erythrocytes in vitro, and their reactivity toward oxyhaemoglobin. A close correlation was observed between the in vivo and in vitro parameters, suggesting that the proposed mechanism of toxicity of 2-methyl-1,4-naphthoquinone is correct and is also applicable to other alkylnaphthoquinones.