Complexity of Multi-Channel Electroencephalogram Signal Analysis in Childhood Absence Epilepsy.

Absence epilepsy is an important epileptic syndrome in children. Multiscale entropy (MSE), an entropy-based method to measure dynamic complexity at multiple temporal scales, is helpful to disclose the information of brain connectivity. This study investigated the complexity of electroencephalogram (EEG) signals using MSE in children with absence epilepsy. In this research, EEG signals from 19 channels of the entire brain in 21 children aged 5-12 years with absence epilepsy were analyzed. The EEG signals of pre-ictal (before seizure) and ictal states (during seizure) were analyzed by sample entropy (SamEn) and MSE methods. Variations of complexity index (CI), which was calculated from MSE, from the pre-ictal to the ictal states were also analyzed. The entropy values in the pre-ictal state were significantly higher than those in the ictal state. The MSE revealed more differences in analysis compared to the SamEn. The occurrence of absence seizures decreased the CI in all channels. Changes in CI were also significantly greater in the frontal and central parts of the brain, indicating fronto-central cortical involvement of "cortico-thalamo-cortical network" in the occurrence of generalized spike and wave discharges during absence seizures. Moreover, higher sampling frequency was more sensitive in detecting functional changes in the ictal state. There was significantly higher correlation in ictal states in the same patient in different seizures but there were great differences in CI among different patients, indicating that CI changes were consistent in different absence seizures in the same patient but not from patient to patient. This implies that the brain stays in a homogeneous activation state during the absence seizures. In conclusion, MSE analysis is better than SamEn analysis to analyze complexity of EEG, and CI can be used to investigate the functional brain changes during absence seizures.

Sample entropy analysis of EEG signals via artificial neural networks to model patients' consciousness level based on anesthesiologists experience.

Electroencephalogram (EEG) signals, as it can express the human brain's activities and reflect awareness, have been widely used in many research and medical equipment to build a noninvasive monitoring index to the depth of anesthesia (DOA). Bispectral (BIS) index monitor is one of the famous and important indicators for anesthesiologists primarily using EEG signals when assessing the DOA. In this study, an attempt is made to build a new indicator using EEG signals to provide a more valuable reference to the DOA for clinical researchers. The EEG signals are collected from patients under anesthetic surgery which are filtered using multivariate empirical mode decomposition (MEMD) method and analyzed using sample entropy (SampEn) analysis. The calculated signals from SampEn are utilized to train an artificial neural network (ANN) model through using expert assessment of consciousness level (EACL) which is assessed by experienced anesthesiologists as the target to train, validate, and test the ANN. The results that are achieved using the proposed system are compared to BIS index. The proposed system results show that it is not only having similar characteristic to BIS index but also more close to experienced anesthesiologists which illustrates the consciousness level and reflects the DOA successfully.

Uptake of apoptotic DC converts immature DC into tolerogenic DC that induce differentiation of Foxp3+ Treg.

DC apoptosis has been observed in patients with cancer and sepsis, and defects in DC apoptosis have been implicated in the development of autoimmune diseases. However, the mechanisms of how DC apoptosis affects immune responses, are unclear. In this study, we showed that immature viable DC have the ability to uptake apoptotic DC as well as necrotic DC without it being recognized as an inflammatory event by immature viable DC. However, the specific uptake of apoptotic DC converted immature viable DC into tolerogenic DC, which were resistant to LPS-induced maturation. These tolerogenic DC secreted increased levels of TGF-beta1, which induced differentiation of naïve T cells into Foxp3(+) Treg. Furthermore, induction of Treg differentiation only occurred upon uptake of apoptotic DC and not apoptotic splenocytes by viable DC, indicating that it is specifically the uptake of apoptotic DC that gives viable immature DC the potential to induce Foxp3(+) Treg. Taken together, these findings identify uptake of apoptotic DC by viable immature DC as an immunologically tolerogenic event.

Sterile protection against Plasmodium knowlesi in rhesus monkeys from a malaria vaccine: comparison of heterologous prime boost strategies.

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost.

Caenorhabditis elegans metallothioneins protect against toxicity induced by depleted uranium.

Depleted uranium (DU) is a dense and heavy metal used in armor, ammunition, radiation shielding, and counterbalances. The military usage has led to growing public concern regarding the health effects of DU. In this study, we used the nematode, Caenorhabditis elegans, to evaluate the toxicity of DU and its effects in knockout strains of metallothioneins (MTs), which are small thiol-rich proteins that have numerous functions, such as metal sequestration, transport, and detoxification. We examined nematode viability, the accumulation of uranium, changes in MT gene expression by quantitative reverse transcription-PCR, and the induction of green fluorescent protein under the control of the MT promoters, following exposure to DU. Our results indicate that (1) DU causes toxicity in a dose-dependent manner; (2) MTs are protective against DU exposure; and (3) nematode death by DU is not solely a reflection of intracellular uranium concentration. (4) Furthermore, only one of the isoforms of MTs, metallothionein-1 (mtl-1), appears to be important for uranium accumulation in C. elegans. These findings suggest that these highly homologous proteins may have subtle functional differences and indicate that MTs mediate the response to DU.

Toxicity studies on depleted uranium in primary rat cortical neurons and in Caenorhabditis elegans: what have we learned?

Depleted uranium (DU) is the major by-product of the uranium enrichment process for its more radioactive isotopes, retaining approximately 60% of its natural radioactivity. Given its properties as a pyrophoric and dense metal, it has been extensively used in armor and ammunitions. Questions have been raised regarding the possible neurotoxic effects of DU in humans based on follow-up studies in Gulf War veterans, where a decrease in neurocognitive behavior in a small population was noted. Additional studies in rodents indicated that DU readily traverses the blood-brain barrier, accumulates in specific brain regions, and results in increased oxidative stress, altered electrophysiological profiles, and sensorimotor deficits. This review summarizes the toxic potential of DU with emphasis on studies on thiol metabolite levels, high-energy phosphate levels, and isoprostane levels in primary rat cortical neurons. Studies in Caenorhabditis elegans detail the role of metallothioneins, small thiol-rich proteins, in protecting against DU exposure. In addition, recent studies also demonstrate that only one of the two forms, metallothionein-1, is important in the accumulation of uranium in worms.

A study of airborne chrysotile concentrations associated with handling, unpacking, and repacking boxes of automobile clutch discs.

Although automotive friction products (brakes and manual clutches) historically contained chrysotile asbestos, industrial hygiene surveys and epidemiologic studies of auto mechanics have consistently shown that these workers are not at an increased risk of developing asbestos-related diseases. Airborne asbestos levels during brake repair and brake parts handling have been well-characterized, but the potential exposure to airborne asbestos fibers during the handling of clutch parts has not been examined. In this study, breathing zone samples on the lapel of a volunteer worker (n=100) and area samples at bystander (n=50), remote area (n=25), and ambient (n=9) locations collected during the stacking, unpacking, and repacking of boxes of asbestos-containing clutches, and the subsequent cleanup and clothes handling, were analyzed by phase contrast microscopy (PCM) and transmission electron microscopy (TEM). In addition, fiber morphology and size distribution was evaluated using X-ray diffraction, polarized light microscopy, and ISO analytical methods. It was observed that the (1) airborne asbestos concentrations increased with the number of boxes unpacked and repacked, (2) repetitive stacking of unopened boxes of clutches resulted in higher asbestos concentrations than unpacking and repacking the boxes of clutches, (3) cleanup and clothes handling tasks yielded very low asbestos concentrations. Fiber size and morphology analyses showed that amphibole fibers were not detected in the clutches and that the vast majority (>95%) of the airborne chrysotile fibers were less than 20 microm in length. Applying the ratio of asbestos fibers:total fibers (including non-asbestos) as determined by TEM to the PCM results, it was found that 30-min average airborne chrysotile concentrations (PCM adjusted) were 0.026+/-0.004 f/cc or 0.100+/-0.017 f/cc for a worker unpacking and repacking 1 or 2 boxes of clutches, respectively. The 30-min PCM adjusted average airborne asbestos concentrations at bystander locations ranged from 0.002+/-0.001 f/cc and 0.004+/-0.002 f/cc when 1 or 2 boxes of clutches were handled, respectively. Estimated 8-h TWA asbestos exposures for a worker handling 1 or 2 boxes of clutches over a workday ranged from 0.002 to 0.006 f/cc. The 30-min PCM adjusted average airborne asbestos concentration for a worker continuously stacking unopened boxes of clutches was 0.212+/-0.014 f/cc; the 8-h TWA was 0.013 f/cc. Additionally, 30-min PCM adjusted average airborne asbestos concentrations following cleanup and clothing handling were 0.002+/-0.001 f/cc and 0.002+/-0.002 f/cc, respectively, both resulting in estimated 8-h TWA asbestos exposures of 0.0001 f/cc. The results of this study indicate that the handling, unpacking, and repacking of clutches, and the subsequent cleanup and clothes handling by a worker within a short-term period or over the entire workday, result in exposures below the historical and current short-term and 8-h occupational exposure limits for asbestos.

Induction of multi-antigen multi-stage immune responses against Plasmodium falciparum in rhesus monkeys, in the absence of antigen interference, with heterologous DNA prime/poxvirus boost immunization.

The present study has evaluated the immunogenicity of single or multiple Plasmodium falciparum (Pf) antigens administered in a DNA prime/poxvirus boost regimen with or without the poloxamer CRL1005 in rhesus monkeys. Animals were primed with PfCSP plasmid DNA or a mixture of PfCSP, PfSSP2/TRAP, PfLSA1, PfAMA1 and PfMSP1-42 (CSLAM) DNA vaccines in PBS or formulated with CRL1005, and subsequently boosted with ALVAC-Pf7, a canarypox virus expressing the CSLAM antigens. Cell-mediated immune responses were evaluated by IFN-gamma ELIspot and intracellular cytokine staining, using recombinant proteins and overlapping synthetic peptides. Antigen-specific and parasite-specific antibody responses were evaluated by ELISA and IFAT, respectively. Immune responses to all components of the multi-antigen mixture were demonstrated following immunization with either DNA/PBS or DNA/CRL1005, and no antigen interference was observed in animals receiving CSLAM as compared to PfCSP alone. These data support the down-selection of the CSLAM antigen combination. CRL1005 formulation had no apparent effect on vaccine-induced T cell or antibody responses, either before or after viral boost. In high responder monkeys, CD4+IL-2+ responses were more predominant than CD8+ T cell responses. Furthermore, CD8+ IFN-gamma responses were detected only in the presence of detectable CD4+ T cell responses. Overall, this study demonstrates the potential for multivalent Pf vaccines based on rational antigen selection and combination, and suggests that further formulation development to increase the immunogenicity of DNA encoded antigens is warranted.

Protection of rhesus monkeys by a DNA prime/poxvirus boost malaria vaccine depends on optimal DNA priming and inclusion of blood stage antigens.

BACKGROUND: We have previously described a four antigen malaria vaccine consisting of DNA plasmids boosted by recombinant poxviruses which protects a high percentage of rhesus monkeys against Plasmodium knowlesi (Pk) malaria. This is a multi-stage vaccine that includes two pre-erythrocytic antigens, PkCSP and PkSSP2(TRAP), and two erythrocytic antigens, PkAMA-1 and PkMSP-1(42kD). The present study reports three further experiments where we investigate the effects of DNA dose, timing, and formulation. We also compare vaccines utilizing only the pre-erythrocytic antigens with the four antigen vaccine. METHODOLOGY: In three experiments, rhesus monkeys were immunized with malaria vaccines using DNA plasmid injections followed by boosting with poxvirus vaccine. A variety of parameters were tested, including formulation of DNA on poly-lactic co-glycolide (PLG) particles, varying the number of DNA injections and the amount of DNA, varying the interval between the last DNA injection to the poxvirus boost from 7 to 21 weeks, and using vaccines with from one to four malaria antigens. Monkeys were challenged with Pk sporozoites given i.v. 2 to 4 weeks after the poxvirus injection, and parasitemia was measured by daily Giemsa stained blood films. Immune responses in venous blood samples taken after each vaccine injection were measured by ELIspot production of interferon-gamma, and by ELISA. CONCLUSIONS: 1) the number of DNA injections, the formulation of the DNA plasmids, and the interval between the last DNA injection and the poxvirus injection are critical to vaccine efficacy. However, the total dose used for DNA priming is not as important; 2) the blood stage antigens PkAMA-1 and PkMSP-1 were able to protect against high parasitemias as part of a genetic vaccine where antigen folding is not well defined; 3) immunization with PkSSP2 DNA inhibited immune responses to PkCSP DNA even when vaccinations were given into separate legs; and 4) in a counter-intuitive result, higher interferon-gamma ELIspot responses to the PkCSP antigen correlated with earlier appearance of parasites in the blood, despite the fact that PkCSP vaccines had a protective effect.

Down-regulation of early ionotrophic glutamate receptor subunit developmental expression as a mechanism for observed plasticity deficits following gestational exposure to benzo(a)pyrene.

The focus of this study was to characterize the impact of gestational exposure to benzo(a)pyrene [B(a)P] on modulation of glutamate receptor subunit expression that is critical for the maintenance of synaptic plasticity mechanisms during hippocampal or cortical development in offspring. Previous studies have demonstrated that hippocampal and/or cortical synaptic plasticity (as measured by long-term potentiation and S1-cortex spontaneous/evoked neuronal activity) and learning behavior (as measured by fixed-ratio performance operant testing) is significantly impaired in polycyclic aromatic or halogenated aromatic hydrocarbon-exposed offspring as compared to controls. These previous studies have also revealed that brain to body weight ratios are greater in exposed offspring relative to controls indicative of intrauterine growth retardation which has been shown to manifest as low birth weight in offspring. Recent epidemiological studies have identified an effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 years of life among inner-city children [Perera FP, Rauh V, Whyatt RM, Tsai WY, Tang D, Diaz D, et al. Effect of prenatal exposure to airborne polycyclic aromatic hydrocarbons on neurodevelopment in the first 3 years of life among inner-city children. Environ Health Perspect 2006;114:1287-92]. The present study utilizes a well-characterized animal model to test the hypothesis that gestational exposure to B(a)P causes dysregulation of developmental ionotropic glutamate receptor subunit expression, namely the N-methyl-d-aspartate receptor (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR) both critical to the expression of synaptic plasticity mechanisms. To mechanistically ascertain the basis of B(a)P-induced plasticity perturbations, timed pregnant Long-Evans rats were exposed in an oral subacute exposure regimen to 0, 25 and 150mug/kg BW B(a)P on gestation days 14-17. The first sub-hypothesis tested whether gestational exposure to B(a)P would result in significant disposition in offspring. The second sub-hypothesis tested whether gestational exposure to B(a)P would result in down-regulation of early developmental expression of NMDA and AMPA receptor subunits in the hippocampus of offspring as well as in primary neuronal cultures. The results of these studies revealed significant: (1) disposition to the hippocampus and cortex, (2) down-regulation of developmental glutamate receptor mRNA and protein subunit expression and (3) voltage-dependent decreases in the amplitude of inward currents at negative potentials in B(a)P-treated cortical neuronal membranes. These results suggest that plasticity and behavioral deficits produced as a result of gestational B(a)P exposure are at least, in part, a result of down-regulation of early developmental glutamate receptor subunit expression and function at a time when excitatory synapses are being formed for the first time in the developing central nervous system. The results also predict that in B(a)P-exposed offspring with reduced early glutamate receptor subunit expression, a parallel deficit in behaviors that depend on normal hippocampal or cortical functioning will be observed and that these deficits will be present throughout life.

Neurotoxic potential of depleted uranium effects in primary cortical neuron cultures and in Caenorhabditis elegans.

Depleted uranium (DU) is an extremely dense metal that is used in radiation shielding, counterbalances, armor, and ammunition. In light of the public concerns about exposure to DU and its potential role in Gulf War Syndrome (GWS), this study evaluated the neurotoxic potential of DU using focused studies on primary rat cortical neurons and the nematode Caenorhabditis elegans. We examined cell viability, cellular energy metabolism, thiol metabolite oxidation, and lipid peroxidation following exposure of cultured neurons to DU, in the form of uranyl acetate. We concurrently evaluated the neurotoxicity of uranyl acetate in C. elegans using various neuronal-green fluourescent protein reporter strains to visualize neurodegeneration. Our studies indicate that uranyl acetate has low cytotoxic potential, and uranium exposure does not result in significant changes in cellular energy metabolism, thiol metabolite oxidation, or lipid peroxidation. Furthermore, our C. elegans studies do not show any significant neurodegeneration following uranyl acetate exposure. Together, these studies suggest that DU, in the form of uranyl acetate, has low neurotoxic potential. These findings should alleviate the some of public concerns regarding DU as an etiologic agent of neurodegenerative conditions associated with GWS.

Neurotoxicity of depleted uranium: reasons for increased concern.

Depleted uranium (DU) is a byproduct of the enrichment process of uranium for its more radioactive isotopes to be used in nuclear energy. Because DU is pyrophoric and a dense metal with unique features when combined in alloys, it is used by the military in armor and ammunitions. There has been significant public concern regarding the use of DU by such armed forces, and it has been hypothesized to play a role in Gulf War syndrome. In light of experimental evidence from cell cultures, rats, and humans, there is justification for such concern. However, there are limited data on the neurotoxicity of DU. This review reports on uranium uses and its published health effects, with a major focus on in vitro and in vivo studies that escalate concerns that exposure to DU might be associated with neurotoxic health sequelae.

Analysis of cellular, transgenic and human models of Huntington's disease reveals tyrosine hydroxylase alterations and substantia nigra neuropathology.

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disorder that is pathologically characterized by a striatal-specific degeneration. Aberrant dopamine neurotransmission has been proposed as a mechanism underlying the movement disorder of HD. We report that the enzymatic activity of tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine biosynthesis, is decreased in a transgenic mouse model of HD. In addition, mutant huntingtin was found to disrupt transcription of TH and dopamine beta-hydroxylase (DbetaH) promoter reporter constructs. In situ hybridization revealed extensive loss of TH mRNA and decreased dopaminergic cell size in human HD substantia nigra. TH-immunoreactive protein was reduced in human grade 4 HD substantia nigra by 32% compared to age-matched controls. These findings implicate abnormalities in dopamine neurotransmission in HD and may provide new insights into targets for pharmacotherapy.

Immobilization of Chinese herbal medicine onto the surface-modified calcium hydrogenphosphate.

To accelerate the healing of bone defects or for healing to take place, it is often necessary to fill them with suitable substance. Various artificial materials defects have been developed. Among these, calcium phosphates and bioactive glass have been proven to be biocompatibile and bioactive materials that can chemically bond with bone, and have been successfully used clinically for repair of bone defects and augmentation of osseous tissue. However, those bioceramics have only the property of osteoconduction without any osteoinduction. Many ligands have been physicochemically absorbed onto substrates to enhance cell-substrate interactions. Although widely developed, they are still limited to use in long-term implantation because of their half-life period. Thus, some interfacial modification will be required for enhancing the efficacy of the delivery system. These models involve the immobilization of biologically active ligands of natural and synthetic origin onto various substrates to produce an interface with stronger chemical bond between ligand and substrate. The advantage of covalently immobilizing a ligand is that a chemical bond is present to prevent ligand or medicine from desorption. In our study, a two-step chemical immobilization was performed to surface-modified calcium hydrogenphosphate powders. The first was to modify the surface of calcium hydrogen-phosphate (CHP) with a coupling agent of hexanmethylene diisocyanate (HMDI). CHP surface modified by HMDI is abbreviated as MCHP. The linkage between CHP and HMDI will be characterized by FTIR. The second step was to immobilize chemically Gusuibu onto MCHP. Moreover, the sorption and desorption of Gusuibu was evaluated and quantitatively analyzed by spectrophotometer and HPLC. Bioceramic CHP was surface-modified by a two-step chemical immobilization. First, the surface of calcium hydrogen-phosphate (CHP) was successfully modified with coupling agent of hexanmethylene diisocyanate (HMDI). The first step was also activated the surface of CHP to induce primary amine terminator. The reaction of this functional group with Gusuibu was the second step. We confirmed simultaneously that Gusuibu could be immobilized chemically onto the surface of MCHP. Although some immobilized Gusuibu was also released rapidly at the first 12h, the degree of the released Gusuibu was lower than both by Gusuibu-adsorbing MCHP and Gusuibu-adsorbing CHP.

Inhibition of tryptophan hydroxylase activity and decreased 5-HT1A receptor binding in a mouse model of Huntington's disease.

The pathogenic mechanisms of the mutant huntingtin protein that cause Huntington's disease (HD) are unknown. Previous studies have reported significant decreases in the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brains of the R6/2 transgenic mouse model of HD. In an attempt to elucidate the cause of these neurochemical perturbations in HD, the protein levels and enzymatic activity of tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, were determined. Enzyme activity was measured in brainstem homogenates from 4-, 8-, and 12-week-old R6/2 mice and compared with aged-matched wild-type control mice. We observed a 62% decrease in brainstem TPH activity (p = 0.009) in 4-week-old R6/2 mice, well before the onset of behavioral symptoms. In addition, significant decreases in TPH activity were also observed at 8 and 12 weeks of age (61%, p = 0.02 and 86%, p = 0.005, respectively). In the 12-week-old-mice, no change in immunoreactive TPH was observed. In vitro binding showed that TPH does not bind to exon 1 of huntingtin in a polyglutamine-dependent manner. Specifically, glutathione-S-transferase huntingtin exon 1 proteins with 20, 32 or 53 polyglutamines did not interact with radiolabeled tryptophan hydroxylase. Therefore, the inhibition of TPH activity does not appear to result from a direct huntingtin/TPH interaction. Receptor binding analyses for the 5-HT1A receptor in 12-week-old R6/2 mice revealed significant reductions in 8-OH-[3H]DPAT binding in several hippocampal and cortical regions. These results demonstrate that the serotonergic system in the R6/2 mice is severely disrupted in both presymptomatic and symptomatic mice. The presymptomatic inhibition of TPH activity in the R6/2 mice may help explain the functional consequences of HD and provide insights into new targets for pharmacotherapy.