A Novel Robot System Integrating Biological and Mechanical Intelligence Based on Dissociated Neural Network-Controlled Closed-Loop Environment.

We propose the architecture of a novel robot system merging biological and artificial intelligence based on a neural controller connected to an external agent. We initially built a framework that connected the dissociated neural network to a mobile robot system to implement a realistic vehicle. The mobile robot system characterized by a camera and two-wheeled robot was designed to execute the target-searching task. We modified a software architecture and developed a home-made stimulation generator to build a bi-directional connection between the biological and the artificial components via simple binomial coding/decoding schemes. In this paper, we utilized a specific hierarchical dissociated neural network for the first time as the neural controller. Based on our work, neural cultures were successfully employed to control an artificial agent resulting in high performance. Surprisingly, under the tetanus stimulus training, the robot performed better and better with the increasement of training cycle because of the short-term plasticity of neural network (a kind of reinforced learning). Comparing to the work previously reported, we adopted an effective experimental proposal (i.e. increasing the training cycle) to make sure of the occurrence of the short-term plasticity, and preliminarily demonstrated that the improvement of the robot's performance could be caused independently by the plasticity development of dissociated neural network. This new framework may provide some possible solutions for the learning abilities of intelligent robots by the engineering application of the plasticity processing of neural networks, also for the development of theoretical inspiration for the next generation neuro-prostheses on the basis of the bi-directional exchange of information within the hierarchical neural networks.

Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins.

A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the beta15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of alpha1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the beta21 and beta37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.

Production and purification of ovine anti-tetanus antibody.

We used the ovine as bioreactor for the production and optimization of anti-tetanus toxin antibody. Four female sheep were immunized with human tetanus vaccine (TT-alum) every two weeks for 16 weeks, after which serum was collected and its titer was estimated by ELISA. The highest titer obtained was 39,000 IU ml-1. To optimize a purification protocol for ovine anti-tetanus toxin, we used four procedures; weak anion (DEAE-Sephadex), weak cation (CM-Sephadex), ammonium sulfate precipitation alone or in combination with caprylic acid. Fifty percent saturation with ammonium sulfate combined with caprylic acid gave us the highest yield of protein with specific activity and the purest Fab product.

Inhibition of collagenase breakdown of equine corneas by tetanus antitoxin, equine serum and acetylcysteine.

OBJECTIVE: To determine whether tetanus antitoxin, equine serum, and acetylcysteine, which are currently used in the treatment of equine corneal ulcer, inhibit the digestion of equine corneal collagen when exposed to collagenase in vitro. ANIMALS STUDIED: Corneas from 40 adult horses. PROCEDURES: Sections of equine corneas were incubated with saline, a solution of bacterial collagenase in saline, bacterial collagenase in saline plus equine tetanus antitoxin, bacterial collagenase in saline plus equine serum, or bacterial collagenase in saline plus acetylcysteine. Each one of the collagenase inhibitors was tested at different concentrations. The degree of corneal collagen digestion was determined by concentrations of hydroxyproline released into the incubation media and/or by weight loss of the cornea. RESULTS: Corneas exposed to collagenase released a significant (0.05 level) large amount of hydroxyproline (43.1 +/- 2.3 microg/mL/100 mg cornea/5 h) and decreased cornea weight by up to 89%. Blood serum (200 microL/mL), purified albumin or globulin fractions of serum, tetanus antitoxin (120 units/mL), and acetylcysteine (20 mg/mL) when used at the highest concentrations blocked collagenase digestive activity by approximately 50%. Dilution of inhibitors decreased corneal protection and linearly increased corneal weight loss. Purified equine serum albumin and globulin fractions were equally effective in protecting corneas. CONCLUSIONS: This experiment indicates that tetanus antitoxin, serum and acetylcysteine equally protected corneas from collagenase digestion, in vitro. However, a clinical trial is needed to establish relative therapeutic value.

Immune complexes are potent inhibitors of interleukin-12 secretion by human monocytes.

We have studied the effect of immune complexes (IC) on interleukin (IL)-12 secretion by human monocytes in vitro. Two experimental models of IC were used. IC formed of tetanus toxoid and polyclonal anti-tetanus toxoid antiserum as well as heat-aggregated human serum IgG almost completely inhibited IL-12 (p70 and p40) secretion induced by interferon-gamma and lipopolysaccharide in human blood-derived monocytes. Neutralizing anti-IL-10 antibodies plus indomethacin restored IL-12 secretion in the presence of IC to a high extent, indicating that IL-10 and prostaglandin (PG) partially mediate the IC-induced inhibition of IL-12 secretion. However, neutralization of tumor necrosis factor (TNF)-alpha by specific antibodies also incompletely restored IL-12 secretion. Indeed, monocytes secrete high levels of TNF-alpha upon stimulation by IC. We found that exogenously added TNF-alpha caused a profound inhibition of monocytic IL-12 secretion in the absence of IC, again mediated via the induction of IL-10 and PG. In summary, IC inhibit IL-12 secretion via TNF-alpha-induced IL-10 and PG synthesis. We conclude that IC, typically appearing in the course of chronic inflammatory processes, may influence the balance between Th1 and Th2 responses and may thus contribute to a deprivation of cell-mediated immune responses.

USDA: progress toward in vitro tests and other trends.

The Animal and Plant Health Inspection Service of the United States Department of Agriculture has demonstrated a commitment toward replacement, reduction and refinement of animal use in the development and control of biological products. This presentation describes some specific approaches with which APHIS has reduced the number of animals used in testing by replacing host or laboratory animal potency tests with validated in vitro tests, reduced the number of animals required for tests by allowing sequential use of animals for tests of immunologically distinct entities, and replaced host or laboratory animal challenge studies with serological tests. It also describes APHIS' plans to reduce pain and suffering of animals by allowing euthanasia when death from causes unrelated to the test is expected. Finally, it reports on refinements in extraneous agent testing, which began when host animal tests were replaced with an in vitro test method and continued when the in vitro test was replaced with a more sensitive status of these approaches is discussed in the context of APHIS' current regulatory framework.

Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin.

Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations. Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin. When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M. When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively. In all cases the lectins behaved as competitive antagonists. In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes. Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin. The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetanus toxin-induced protein kinase C activation and elevated serotonin levels in the perinatal rat brain.

A single intracerebral injection of tetanus toxin (TeTox) is able to produce a time-dependent translocation of Ca2(+)-phosphatidylserine-dependent protein kinase C (PKC) in close-to-term rat brain. TeTox-triggered translocation of PKC is dose- and time-dependent, can be prevented by tetanus antitoxin, and does not occur upon administration of toxin fragments B and C. TeTox-triggered PKC translocation is accompanied by a time-dependent increase in brain serotonin (5-HT). Increase of brain 5-HT is independent of monoamine oxidase inhibition by pargyline. Phorbol ester and TeTox cause a significant increase in serotonin while H-7, a kinase inhibitor, does not affect serotonin levels but abolishes the effect of TeTox. Gangliosides prevent TeTox-triggered 5-HT increase. The data are consistent with the possibility that TeTox acts effectively on the serotonergic innervation, presumably in conjunction with PKC to cause accumulation of serotonin.

[Heat stability of potency of bacterial vaccines and the titer of tetanus antitoxin of Polish production]. / Stabilnosc cieplna mocy szczepionek bakteryjnych oraz miano antytoksyny tezcowej produkcji krajowej.

Described the stability of potency vaccines (DTP, BCG) and immunoglobulins (human's and animal's) at storage and experimental temperatures. Thermal degradation rate and design of loss of potency in time have been determined by Arrhenius equation. Our results were similar to WHO data from preparations which have been made in another countries.

[Effect of tetanus toxin on K+ and Na+ concentration in synaptosomes]. / Vliianie stolbniachnogo toksina na soderzhanie K+ i Na+ v sinaptosomakh.

A study was made of the effect of tetanus toxin (TT) in doses of 20-1000 MLD (for rats) on synaptosomes isolated from the rat brain cortex. TT produced a decrease in K+ content in synaptosomes. The effect depended on the dose of TT (I50=22 MLD). The content of Na+ remained unchanged. The action of TT depended on the time and the maximal effect was seen upon 60 minutes of incubation. TT inactivated by boiling or by antitoxin did not affect K+ content in synaptosomes. An increase of K+ concentration up to 17 mM in an incubation medium led to an increase in K+ content in TT-poisoned synaptosomes but not in the control ones. Possible changes in membrane electrogenesis in nerve terminals caused by TT are discussed.

Resistance induced by concanavalin A and phytohaemagglutinin P against tetanus toxin in mice.

The effect of concanavalin A (ConA), phytohaemagglutinin P (PHA) and Limulus polyphemus haemocyanin (LPH) on the lethal activity of tetanus toxin (TT) is reported. C3H mice treated s.c. with ConA or PHA but not with LPH from 48 h before to 12 h after s.c. TT challenge showed a significant increase in median survival time compared to control mice inoculated with toxin alone. This protective effect was also obtained when PHA or ConA was administered by the i.p. route, TT being injected s.c. In further studies, mice treated with ConA or PHA by different routes (s.c., i.p. or i.v.) were challenged s.c. with graded minimal lethal doses of TT, with or without i.p. administration of horse antitetanus serum (HATS) 24 h after toxin inoculation. The mice treated with ConA or PHA + HATS showed a significantly increased survival rate and a higher percentage of cured mice with respect to control animals treated with lectins alone. In contrast, the mice challenged with TT and treated with HATS alone did not show any increased survival with respect to untreated controls. Sera from ConA- or PHA-treated mice were unable to neutralize the TT. Immune depression in mice by total-body irradiation (400 R) did not abolish the protective activity of the lectins. These results show that in vivo treatment of mice with ConA or PHA but not with LPH can protect against the lethal effects of TT.

At least three sequential steps are involved in the tetanus toxin-induced block of neuromuscular transmission.

Tetanus toxin causes a block of the neuromuscular transmission. The kinetic aspects of the block were studied in vitro on the mouse phrenic nerve-hemidiaphragm exposed to toxin (1 microgram/ml). 1. The toxin action on the nerve ending involves three sequential steps: binding, "translocation" and paralysis. 2. Diffusion and binding of tetanus toxin molecules to the presynaptic membrane is complete in about 60 min. The binding step is irreversible, independent of transmitter release and of the temperature. Tetanus antitoxin, however, inactivates the bound toxin molecules. 3. After a second step which is probably due to a "translocation" of the toxin molecules into or through the presynaptic membrane the antitoxin molecules are now ineffective to prevent the toxin-induced inhibition of transmitter release. This so called "translocation" step requires transmitter release and therefore depends strongly on the frequency of nerve stimulation. 4. The paralytic step does not depend on the transmitter release. It, however, depends strongly on temperature with a break in the Arrhenius-plot around 33 degrees C which suggests the involvement of a phase transition rather than of an enzymatic activity of the toxin.

[Effect of tetanic anatoxin and carbonic acid level in blood on biosynthesis of antibodies in rats]. / Vliianie stolbniachnogo anatoksina i urovnia uglekisloty v krovi na obrazovanie antitel u krys.

The paper deals with dynamics of antibody formation in rats. Tetanic anatoxin independently of doses and methods of administration manifests weak immunogenic properties. A complete Freind adjuvant stimulates considerably the biosynthesis of antitetanic antibodies in the primary and secondary immune responses. Electrophoresis of tetanic anatoxin polyacrylamide gel permits isolating two protein fractions, one of them migrates in the immunoglobulin G zone and its mass constitutes 73%, the other-more low-molecular and contains 27% of protein. Dynamics of the acid-base blood state indices and of antibody biosynthesis is studied on models of chronic compensated alkalosis and metabolic acidosis in rats. It is established that during the experiment, simultaneously with an insignificant increase in the total carbonic acid concentration blood, there occurs a 25-60% increase in the titres of antitetanic antibodies as compared to the control. In rats with acidosis there occurs a simultaneously decrease in the intensity of antibody biosynthesis as compared to the control (43-54%) and in the total CO2 content in blood.

[The microbiological cancer test--further studies with experimental mouse tumor tetanus and wound tetanus]. / Der mikrobiologische Krebstest - weiterführende Untersuchungen mit dem experimentellen Tumor-Tetanus und Wund-Tetanus der Maus.

A re-evaluation of the specificity of the tumour-tetanus assay of the mouse was performed by analysing the wound-tetanus assay under comparable test conditions. This was achieved by injecting 1 X 10(6) viable Ehrlich carcinoma cells admixed with 1 X 10(2) tetanus spores subcutaneously, in a 0.1 ml dose volume or, 1 X 10(2) tetanus spores suspended in 5% CaCl2 solution, respectively. By comparison, these two groups of mice developed about the same tetanus mortality rates, however, following tetanus antitoxin therapy with 3 doses of 100 IU each day on days 0, 4 and 7 after infection, clinical signs of late tetanus exclusively belonged to tumour bearing animals. This typical tetanus behaviour may be explained by a spatial-temporal association between growing tetanus rods and proliferating cells of warm-blooded animals. In this manner tumour tissue can be differed from wound granulomatous tissue by way of permanently cloning stem cells.

Toxin-neutralizing effect of antibody against subtilisin-digested tetanus toxin.

A form of systemic tetanus with atypical symptoms was observed in mice injected in the left thigh with a mixture of tetanus toxin and antibody produced in guinea pigs against a fragment of toxin obtained from a subtilisin digest of the crystallized toxin. The mice did not show typical symptoms of the local tetanus such as convulsions or spastic paralysis of the injected limb.