Chronic Administration of Non-Constitutive Proteasome Inhibitor Modulates Long-Term Potentiation and Glutamate Signaling-Related Gene Expression in Murine Hippocampus.

Proteasomes degrade most intracellular proteins. Several different forms of proteasomes are known. Little is known about the role of specific proteasome forms in the central nervous system (CNS). Inhibitors targeting different proteasome forms are used in clinical practice and were shown to modulate long-term potentiation (LTP) in hippocampal slices of untreated animals. Here, to address the role of non-constitutive proteasomes in hippocampal synaptic plasticity and reveal the consequences of their continuous inhibition, we studied the effect of chronic administration of the non-constitutive proteasome inhibitor ONX-0914 on the LTP induced by two different protocols: tetanic stimulation and theta-burst stimulation (TBS). Both the tetanus- and TBS-evoked potentiation contribute to the different forms of hippocampal-dependent memory and learning. Field-excitatory postsynaptic potentials (fEPSPs) in hippocampal slices from control animals and animals treated with DMSO or ONX-0914 were compared. LTP induced by the TBS was not affected by ONX-0914 administration; however, chronic injections of ONX-0914 led to a decrease in fEPSP slopes after tetanic stimulation. The observed effects correlated with differential expression of genes involved in synaptic plasticity, glutaminergic synapse, and synaptic signaling. Obtained results indicate that non-constitutive proteasomes are likely involved in the tetanus-evoked LTP, but not the LTP occurring after TBS, supporting the relevance and complexity of the role of specific proteasomes in synaptic plasticity, memory, and learning.

Tetanus and tetanus neurotoxin: From peripheral uptake to central nervous tissue targets.

Tetanus is a deadly but preventable disease caused by a protein neurotoxin produced by Clostridium tetani. Spores of C. tetani may contaminate a necrotic wound and germinate into a vegetative bacterium that releases a toxin, termed tetanus neurotoxin (TeNT). TeNT enters the general circulation, binds to peripheral motor neurons and sensory neurons, and is transported retroaxonally to the spinal cord. It then enters inhibitory interneurons and blocks the release of glycine or GABA causing a spastic paralysis. This review attempts to correlate the metalloprotease activity of TeNT and its trafficking and localization into the vertebrate body to the nature and sequence of appearance of the symptoms of tetanus.

[PROPERTIES OF INDIVIDUAL CONTRACTILE RESPONSES WITHIN TETANUS OF RAT SLOW MUSCLE UNDER CONDITIONS OF MODULATION OF SARCOPLASMIC RETICULUM Ca²âº RELEASE].

During direct stimulation of m. Soleus by trains of 5, 10 and 50 stimuli with a frequency of 20 Hz in control experiments (n = 16) a biphasic change was observed in the amplitude of the last contractile responses (LCRN) depending on N, where N is the number of individual contractile responses within the te- tanus. Thus, an initial decrease of LCRN amplitude (up to 54 ± 8 % for LCR5) was replaced by their subsequent growth (up 218 ± 14 % for LCR5o) associated with a significant shortening of their half-relaxation time relative to the initial response (to 44 ± 8 % for LCR50). Caffeine at concentrations of 5 mM (n = 6) and 10 mM (n = 4), at the background of developing characteristic stationary contracture respon- ses, increased LCR5 depression during the initial inhibitory phase (31 ± 8 % and 15 ± 4 %, respectively). The subsequent growth of LCRN amplitude was significantly lower than in the control (114 ± 18 % and 46 ± 9 % for LCR50 at 5 and 10 mM caffeine, respectively). LCR50 half-relaxation time during the action of both caffeine concentrations remained still considerably shorter than the individual responses recorded both in the presence of caffeine and in control. In contrast to the control and caffeine effects, LCR5 and 10 (to 143 ± 14 %) than was observed in the control muscle. Additionally, dantrolene enhanced muscle relaxation at rest. Caffeine (10 mM), at the background of dantrolene, restored the dynamics of changes of amplitude time characteristics of the last contractile responses to values close to the control. The time-amplitude characteristics of the extracellular AP recorded in individual muscle fibers in m. Soleus did not change significantly during tetanic stimulation under protocol similar to that used for mechanografical experiments. These data can be interpreted to support the previously suggested theory about the participation of <> as an additional mechanism of excitation-contraction coupling in skeletal muscle under conditions of tetanic stimulation [1, 2].

Fos and Jun potentiate individual release sites and mobilize the reserve synaptic vesicle pool at the Drosophila larval motor synapse.

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP- and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites. Electrophysiological and optical measurements of vesicle dynamics demonstrate that enhanced neurotransmitter release is accompanied by an increase in the actively cycling synaptic vesicle pool at the expense of the reserve pool. Finally, the observation that AP-1 mediated enhancement eliminates tetanus-induced forms of presynaptic potentiation suggests: (i) that reserve-pool mobilization is required for tetanus-induced short-term synaptic plasticity; and (ii) that long-term synaptic plasticity may, in some instances, be accomplished by stable recruitment of mechanisms that normally underlie short-term synaptic change.

Vav independently regulates synaptic growth and plasticity through distinct actin-based processes.

Modulation of presynaptic actin dynamics is fundamental to synaptic growth and functional plasticity; yet the underlying molecular and cellular mechanisms remain largely unknown. At Drosophila NMJs, the presynaptic Rac1-SCAR pathway mediates BMP-induced receptor macropinocytosis to inhibit BMP growth signaling. Here, we show that the Rho-type GEF Vav acts upstream of Rac1 to inhibit synaptic growth through macropinocytosis. We also present evidence that Vav-Rac1-SCAR signaling has additional roles in tetanus-induced synaptic plasticity. Presynaptic inactivation of Vav signaling pathway components, but not regulators of macropinocytosis, impairs post-tetanic potentiation (PTP) and enhances synaptic depression depending on external Ca2+ concentration. Interfering with the Vav-Rac1-SCAR pathway also impairs mobilization of reserve pool (RP) vesicles required for tetanus-induced synaptic plasticity. Finally, treatment with an F-actin-stabilizing drug completely restores RP mobilization and plasticity defects in Vav mutants. We propose that actin-regulatory Vav-Rac1-SCAR signaling independently regulates structural and functional presynaptic plasticity by driving macropinocytosis and RP mobilization, respectively.

Detection of Tetanus Antibody Applying a Cu-Zn-In-S/ZnS Quantum Dot-Based Lateral Flow Immunoassay.

Lateral flow test strip (LFTS) enables rapid, portable, and low-cost point-of-care testing (POCT) diagnosis. Quantum dots (QDs), which are fluorescent semiconductor nanocrystals with distinctive and unique photophysical properties, have become promising candidates to serve as labels for LFTS with improved sensitivity. Here, by using QDs as a signal reporter, we report a fluorescent LFTS for detection of tetanus antibody. This LFTS possess a high sensitivity for tetanus antibody, with a detection limit of 0.001 IU/mL. This assay was also applied for detection of tetanus antibody in human serum. More importantly, these strips can retain their specificity and sensitivity for at least 4 months when they are stored at 4 °C.

Molecular Methods for Identification of Clostridium tetani by Targeting Neurotoxin.

Tetanus is a potentially fatal muscle spasm disease. It is an important public health problem, especially in rural/tribal areas of developing countries. Tetanus toxin, a neurotoxin (tetanospasmin ), is the most important virulence factor that plays a key role in the pathogenicity of tetanus . Confirmation of virulence by confirming the production of tetanospasmin by infecting species forms the most important part in the diagnosis of tetanus . Various molecular methods have been devised for confirmation of diagnosis by targeting different genes. The most common molecular methods are tetanospasmin producing (TetX) gene-targeted methods using TetX-specific primers. Here, we describe various molecular methods targeting TetX gene such as polymerase chain reaction, pulsed-field gel electrophoresis, Southern blotting, loop-mediated isothermal amplification assay, etc. to confirm the virulence of Cl. tetani.

Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

Tetanus toxin as a neurobiological tool to study mechanisms of neuronal cell death in the mammalian brain.

Tetanus toxin is a potent clostridial neurotoxin responsible for causing spastic paralysis in humans, often accompanied by seizures and death. The tetanic syndrome is believed to originate from a disinhibitory action of the toxin in the CNS. To produce its effects, tetanus toxin undergoes retrograde, intra-axonal transport to the CNS, where it blocks preferentially the release of gamma-aminobutyric acid and glycine, two inhibitory neurotransmitters. These effects stem from the cleavage of synaptobrevin, a constitutive small-vesicle protein, by tetanus toxin, whose zinc-dependent metalloprotease characteristics recently have been recognized. Blockade of inhibitory transmission produces a predominance of excitatory amino acid neurotransmission, which is responsible for the neurodegenerative effect caused by tetanus toxin after intrahippocampal injection in rats. In fact, hippocampal damage can effectively be prevented by reduction of glutamate-mediated excitatory transmission, thus suggesting that unopposed excitation may be the underlying mechanism for neuronal cell death.

(R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) fails to block long-term potentiation under urethane anaesthesia in vivo.

The effects of the metabotropic glutamate receptor antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) on the induction of long-term potentiation (LTP) in the dentate gyrus were examined under urethane anaesthesia in vivo. In experiment 1, bilateral intraventricular infusion of either 20 mM or 200 mM (R,S)-MCPG (5 microl each side) failed to block LTP in the perforant path-granule cell projection, relative to vehicle-infused controls; 30 mM D-AP5 (5 microl each side) infused in the same way as MCPG completely blocked LTP. Experiment 2, in which the contralateral perforant path-dentate gyrus pathway was used as a non-tetanized control, revealed that slight baseline changes induced by MCPG infusion were transient; again no block of LTP was obtained. The efficacy of mGluR blockade was confirmed in experiment 3, in which MCPG antagonized an increase in spontaneous activity induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). In experiment 4, significant depotentiation was induced by low frequency stimulation (5 Hz for 1 min) given 2 min after high frequency tetanization, but MCPG remained ineffective in blocking LTP after a second tetanus. In experiment 5, increasing the period of low frequency stimulation from 1 to 10 min produced greater depotentiation, but still did not unmask an MCPG-sensitive component of LTP. These experiments fail to support a role for mGluRs in the induction of LTP in the dentate gyrus under urethane anaesthesia in vivo, nor do they support the idea that a metabotropic switch controlling sensitivity to MCPG is reset by depotentiation.

The metabolic response and problems with nutritional support in acute tetanus.

Thirteen patients with severe tetanus were studied in the first and second week of illness in order to assess metabolic changes and nutritional requirements. Nine required muscular paralysis and subsequent assisted ventilation in order to control spasms. Symptoms and signs of sympathetic overactivity were especially common in the latter groups with a fourfold increase in urinary adrenaline and noradrenaline excretion. Other hormonal and metabolic abnormalities included hyperglycemia, mildly elevated insulin concentrations with no significant rise in cortisol, and glucagon. Evidence of excessive protein catabolism was obtained particularly during the second week of illness, mean urinary nitrogen excretion being 20.5 +/- 13.8 g/d. Maintenance of nutrient homeostasis proved impossible with conventional enteral-feeding techniques: high-density feeds exacerbated hyperglycemia and diarrhea; low-density feeds were unable to maintain nitrogen balance (-12.34 g/d in ventilated patients, second week). The results indicate that loss of lean body mass is inevitable in such patients unless the metabolic response can be suppressed or more aggressive forms of nutritional support (eg, total parenteral nutrition including sufficient insulin to maintain normoglycemia) are employed.

Local tetanus in rats; concentration of amino acids as studied in spinal cord segments, spinal roots, and dorsal root ganglia.

1. The effect of tetanus toxin injected into one gastrocnemius muscle on the steady state concentration of several amino acids was investigated in spinal cord half segments, spinal roots and dorsal root ganglia of rats. Care was taken to ensure a symmetrical afferent input to the spinal cord and to localize the segment with the highest concentration of tetanus toxin. 2. In the spinal cord segments containing the highest concentration of tetanus toxin the steady state concentration of glycine was higher on the side of the tetanus than on the contralateral control side. Results obtained after intravenous injection of 14C-glycine do not indicate that the higher concentration of glycine on the side of the tetanus was due to a higher uptake of glycine. 3. The results obtained in the spinal cord contradict previous findings of other authors but lend support to the prevailing concept about the action of tetanus toxin in local and general tetanus.

Intraaxonal and extraaxonal transport of 125I-tetanus toxin in early local tetanus.

The distribution of radioactivity in the sciatic nerve, the spinal ganglia, the ventral roots and the spinal cord was studied by means of histoautoradiography after injection of 125I-labelled tetanus toxin into gastrocnemius muscles of cats. In the sciatic nerve the major part of the radioactivity was found in the epineurium, but some axons also contained radioactivity. In the ventral root the radioactivity was strictly confined to a few axons; no radioactivity was found in other parts of the ventral root. In the spinal cord the radioactivity was confined to a few motoneurones where it was found in the soma as well as in the dendrites. Transient cooling of the ventral roots prevented the ascent of radioactivity into the spinal cord. Colchicine and vinblastine, after local application to the sciatic nerve, reduced the amount of radioactivity found in the ventral roots and in the spinal cord. However, the same effect was also obtained but to a lesser degree with lumicolchicine. It is concluded that the intraaxonal compartment is involved in the neural ascent of tetanus toxin into the spinal cord.

[New orientations in the treatment of proclaimed tetanus: use of continuous peridural anesthesia]. / Nuovi orientamenti nel trattamento del tetano conclamato: uso della peridurale continua

Notes on the typical infectious and toxis picture associated with tetatus and the physiopathological aspects of the disease are followed by the presentation of continuous C8-L2 peridural anaesthesia, giving antalgic relief and partial motor block, as a primary form of therapeutic management. A classic two catheter technique is employed and fractioned doses are used so as to exploit the action of the local anaesthetic absorbed by the patient over several days. Sedation of the orthosympathetic leads to reduced catecholamine production, hypometabolism and cardiac sedation. Analgesia and relaxation are achieved by blocking the local and general spasms typical of the disease. Success with the method in serious cases is mentioned and its use in all forms is recommended.

Changes in blood chemistry and liver histopathology of rabbits during experimental tetanus toxicity.

Changes in blood chemistry, especially blood glucose, were studied in rabbits developing tetanus after injection of tetanus toxin. Blood glucose levels increased significantly above control values after the animals developed tetanus. The increase in glucose level paralleled the depletion of liver glycogen, detected by means of PAS staining. The observed changes were not affected by adrenergic receptor block or catecholamine depletion by reserpine, suggesting non-mediation of the sympathetic nervous system in the response. A direct action of the toxin on the liver, initiating glycogen depletion, is being postulated.

[Changes in histamine and serotonin metabolism in experimental tetanus]. / K voprosu ob izmenenii metabolizma gistamina i serotonina pri éksperimental'nom stolbniake

A gradual increase in histamine content with simultaneous elevation in the histaminase activity were observed in many tissues of guinea-pigs and cats in dynamics of tetanic intoxication. A universal accumulation of serotonin at early steps of intoxication with subsequent decrease in its content were also found in development of severe generalized tetanus. At the same time, in several tissues the inhibition of glycolysis and impairment in electrolyte balance were observed. Artificial pulmonary ventilation was accompanied only by partial compensation in impairment of the histamine and electrolytes metabolism. The metabolic impairments studied possess the complex genesis, they are not related solely to the effect of hypoxic factor and may form the basis for impairment in the excitability of various structures, for alteration in their energy provision and for operation in dynamics of tetanic intoxication.

A randomized phase II trial of multiepitope vaccination with melanoma peptides for cytotoxic T cells and helper T cells for patients with metastatic melanoma (E1602).

PURPOSE: This multicenter randomized trial was designed to evaluate whether melanoma helper peptides augment cytotoxic T lymphocyte (CTL) responses to a melanoma vaccine and improve clinical outcome in patients with advanced melanoma. EXPERIMENTAL DESIGN: One hundred seventy-five patients with measurable stage IV melanoma were enrolled into 4 treatment groups, vaccinated with 12 MHC class I-restricted melanoma peptides to stimulate CTL (12 MP, group A), plus a tetanus peptide (group B), or a mixture of 6 melanoma helper peptides (6 MHP, group C) to stimulate helper T lymphocytes (HTL), or with 6 melanoma helper peptide (6 MHP) alone (group D), in incomplete Freund's adjuvant plus granulocyte macrophage colony-stimulating factor. CTL responses were assessed using an in vitro-stimulated IFN-γ ELIspot assay, and HTL responses were assessed using a proliferation assay. RESULTS: In groups A to D, respectively, CTL response rates to 12 melanoma peptides were 43%, 47%, 28%, and 5%, and HTL response rates to 6 MHP were in 3%, 0%, 40%, and 41%. Best clinical response was partial response in 7 of 148 evaluable patients (4.7%) without significant difference among study arms. Median overall survival (OS) was 11.8 months. Immune response to 6 MHP was significantly associated with both clinical response (P = 0.036) and OS (P = 0.004). CONCLUSION: Each vaccine regimen was immunogenic, but MHPs did not augment CTL responses to 12 melanoma peptides. The association of survival and immune response to 6 MHP supports further investigation of helper peptide vaccines. For patients with advanced melanoma, multipeptide vaccines should be studied in combination with other potentially synergistic active therapies.